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1. Su  YA, He  P, Clewell  DB,     ( 1992 )

Characterization of the tet(M) determinant of Tn916: evidence for regulation by transcription attenuation.

Antimicrobial agents and chemotherapy 36 (4)
PMID : 1323953  :   DOI  :   10.1128/aac.36.4.769     PMC  :   PMC189400    
Abstract >>
The nucleotide sequence of the tetracycline resistance determinant tet(M), located on conjugative transposon Tn916 of Enterococcus faecalis, was determined and found to encode a 72,486-dalton protein exhibiting a high degree of homology with other tet(M) determinants. A short open reading frame corresponding to a 28-amino-acid peptide and containing a number of inverted repeat sequences was noted immediately upstream of tet(M), suggesting that regulation might occur by a mechanism involving transcriptional attenuation. Transcription analyses found this to indeed be the case, showing that the expression of tet(M) resulted from an extension of a small transcript representing the upstream leader region into the resistance determinant. Exposure of cells to tetracycline resulted in a significant increase in the amount of tet(M) transcription; this increase could be explained on the basis of increased transcriptional read-through from the upstream transcript. A model suggesting how transcriptional attenuation might operate in this system is presented.
KeywordMeSH Terms
DNA Transposable Elements
Transcription, Genetic
2. Nilsen  T, Nes  IF, Holo  H,     ( 2003 )

Enterolysin A, a cell wall-degrading bacteriocin from Enterococcus faecalis LMG 2333.

Applied and environmental microbiology 69 (5)
PMID : 12732574  :   DOI  :   10.1128/aem.69.5.2975-2984.2003     PMC  :   PMC154489    
Abstract >>
A novel antimicrobial protein, designated enterolysin A, was purified from an Enterococcus faecalis LMG 2333 culture. Enterolysin A inhibits growth of selected enterococci, pediococci, lactococci, and lactobacilli. Antimicrobial activity was initially detected only on solid media, but by growing the bacteria in a fermentor under optimized production conditions (MRS broth with 4% [wt/vol] glucose, pH 6.5, and a temperature between 25 and 35 degrees C), the bacteriocin activity was increased to 5,120 bacteriocin units ml(-1). Enterolysin A production was regulated by pH, and activity was first detected in the transition between the logarithmic and stationary growth phases. Killing of sensitive bacteria by enterolysin A showed a dose-response behavior, and the bacteriocin has a bacteriolytic mode of action. Enterolysin A was purified, and the primary structure was determined by combined amino acid and DNA sequencing. This bacteriocin is translated as a 343-amino-acid preprotein with an sec-dependent signal peptide of 27 amino acids, which is followed by a sequence corresponding to the N-terminal part of the purified protein. Mature enterolysin A consists of 316 amino acids and has a calculated molecular weight of 34,501, and the theoretical pI is 9.24. The N terminus of enterolysin A is homologous to the catalytic domains of different cell wall-degrading proteins with modular structures. These include lysostaphin, ALE-1, zoocin A, and LytM, which are all endopeptidases belonging to the M37 protease family. The N-terminal part of enterolysin A is linked by a threonine-proline-rich region to a putative C-terminal recognition domain, which shows significant sequence identity to two bacteriophage lysins.
KeywordMeSH Terms
3. Dina  J, Malbruny  B, Leclercq  R,     ( 2003 )

Nonsense mutations in the lsa-like gene in Enterococcus faecalis isolates susceptible to lincosamides and Streptogramins A.

Antimicrobial agents and chemotherapy 47 (7)
PMID : 12821484  :   DOI  :   10.1128/aac.47.7.2307-2309.2003     PMC  :   PMC161836    
Abstract >>
The lsa gene confers intrinsic resistance to lincosamides and streptogramins A in Enterococcus faecalis, probably by active efflux. The lsa-like genes of two clinical isolates of E. faecalis susceptible to lincosamides and dalfopristin contained mutations that produced premature termination codons. Revertant mutants were obtained by selection on agar plates containing clindamycin.
KeywordMeSH Terms
Codon, Nonsense
Macrolides
4. Lee  EW, Chen  J, Huda  MN, Kuroda  T, Mizushima  T, Tsuchiya  T,     ( 2003 )

Functional cloning and expression of emeA, and characterization of EmeA, a multidrug efflux pump from Enterococcus faecalis.

Biological & pharmaceutical bulletin 26 (2)
PMID : 12576692  :   DOI  :   10.1248/bpb.26.266    
Abstract >>
A fragment of chromosomal DNA from Enterococcus faecalis ATCC 29212 was cloned using Escherichia coli KAM32 host cells lacking major multidrug efflux pumps. E. coli KAM32 cells were sensitive to many antimicrobial agents, and the transformed cells harboring a recombinant plasmid became resistant to several structurally unrelated antimicrobial agents such as tetraphenylphosphonium chloride, 4',6-diamidino-2-phenylindole (DAPI), Hoechst 33342, acriflavine, benzalkonium chloride, norfloxacin and ethidium bromide. This suggests that the cloned DNA fragment carries a gene(s) encoding a multidrug efflux pump. Determination of the nucleotide sequence of the cloned DNA revealed a gene designated as emeA. The transformed E. coli cells showed efflux activity of several antimicrobial agents such as DAPI, Hoechst 33342 and acriflavine. Efflux of DAPI via EmeA was strongly inhibited by reserpine.
KeywordMeSH Terms
Bacterial Proteins
5. Sánchez-Hidalgo  M, Maqueda  M, Gálvez  A, Abriouel  H, Valdivia  E, Martínez-Bueno  M,     ( 2003 )

The genes coding for enterocin EJ97 production by Enterococcus faecalis EJ97 are located on a conjugative plasmid.

Applied and environmental microbiology 69 (3)
PMID : 12620853  :   DOI  :   10.1128/aem.69.3.1633-1641.2003     PMC  :   PMC150074    
Abstract >>
Enterococcus faecalis EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da). The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment. Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain E. faecalis EJ97 to E. faecalis OG1X conferred bacteriocin production and resistance on the recipient. The genetic determinants of enterocin EJ97 were located in an 11.3-kb EcoRI-BglII DNA fragment of pEJ97. This region was cloned and sequenced. It contains the ej97A structural gene plus three open reading frames (ORFs) (ej97B, ej97C, and ej97D) and three putative ORFs transcribed in the opposite direction (orfA, orfB, and orfC). The gene ej97A translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far. The product of ej97B (Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters. The products of ej97C (Ej97C) and ej97D (Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins. There are two additional ORFs (ORF1 and ORF6) flanking the ej97 module, which have been identified as a transposon-like structure (tnp). ORF1 shows similarities to transposase of the Lactococcus lactis element ISS1 and is up to 50% identical to IS1216. This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of ISS1 and IS1216. ORF6 (resEJ97) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from Staphylococcus aureus and E. faecalis. These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon. Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response. These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of E. faecalis, suggesting that they have a common origin.
KeywordMeSH Terms
Conjugation, Genetic
6. Kurenbach  B, Bohn  C, Prabhu  J, Abudukerim  M, Szewzyk  U, Grohmann  E,     ( 2003 )

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region.

Plasmid 50 (1)
PMID : 12826062  :  
Abstract >>
The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMbeta1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G-) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G- Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli.
KeywordMeSH Terms
7. Thompson  JK, Collins  MA,     ( 2003 )

Completed sequence of plasmid pIP501 and origin of spontaneous deletion derivatives.

Plasmid 50 (1)
PMID : 12826055  :  
Abstract >>
The sequence of plasmid pIP501 (30,603 bp) was completed using previously published and newly acquired data. The sites at which two spontaneous deletions had occurred were identified. One was between tracts of repeated heptamers and the other between regions of secondary structure associated with plasmid replication. A high level of identity (>95%) between plasmid pIP501 and part of plasmid pRE25, which had been isolated from Enterococcus faecalis associated with a food source, was confirmed.
KeywordMeSH Terms
Sequence Analysis, DNA
8. Foster  KA, Barnes  MH, Stephenson  RO, Butler  MM, Skow  DJ, LaMarr  WA, Brown  NC,     ( 2003 )

DNA polymerase III of Enterococcus faecalis: expression and characterization of recombinant enzymes encoded by the polC and dnaE genes.

Protein expression and purification 27 (1)
PMID : 12509989  :  
Abstract >>
Enterococcus faecalis (Ef) dnaE and polC, the respective genes encoding the DNA replication-specific DNA polymerase III E and DNA polymerase III C, were cloned and engineered for expression in Escherichia coli as hexahistidine (his6)-tagged recombinant proteins. Each gene expressed a catalytically active DNA polymerase of the expected molecular weight. The recombinant polymerases were purified and each was characterized with respect to catalytic properties, inhibitor sensitivity, and recognition by specific antibody raised against the corresponding DNA polymerase III of the model Gram-positive (Gr(+)) organism, Bacillus subtilis (Bs). In conclusion, the properties of each Enterococcus polymerase enzymes were similar to those of the respective B. subtilis enzymes.
KeywordMeSH Terms
Bacterial Proteins
9. Low  YL, Jakubovics  NS, Flatman  JC, Jenkinson  HF, Smith  AW,     ( 2003 )

Manganese-dependent regulation of the endocarditis-associated virulence factor EfaA of Enterococcus faecalis.

Journal of medical microbiology 52 (Pt 2)
PMID : 12543916  :   DOI  :   10.1099/jmm.0.05039-0    
Abstract >>
There is increasing recognition of the emerging role of manganese regulation and acquisition in some pathogenic bacteria. Expression of the Enterococcus faecalis endocarditis-associated virulence factor EfaA is induced by growth in serum. It is demonstrated here that expression of the efaCBA operon encoding a putative ABC-type transporter is regulated by Mn(2+). Transcription of efaCBA and EfaA production were repressed in Mn(2+)-supplemented medium. A Mn(2+)-responsive transcriptional regulator, EfaR, sharing 27 % identity with the Corynebacterium diphtheriae diphtheria toxin repressor (DtxR), was identified. In the presence of Mn(2+), EfaR protein bound in vitro to the efaC promoter region. Analysis of the E. faecalis V583 genome revealed ten additional putative EfaR-binding sites, suggesting that manganese availability could have a broader regulatory role in infection. The results identify a new Mn(2+)-sensing regulator in enterococci that regulates the expression of a virulence factor implicated in enterococcal endocarditis.
KeywordMeSH Terms
Antigens, Bacterial
10. Barcelona-Andrés  B, Marina  A, Rubio  V,     ( 2002 )

Gene structure, organization, expression, and potential regulatory mechanisms of arginine catabolism in Enterococcus faecalis.

Journal of bacteriology 184 (22)
PMID : 12399499  :   DOI  :   10.1128/jb.184.22.6289-6300.2002     PMC  :   PMC151947    
Abstract >>
Although Enteroccus faecalis is the paradigm for biochemical studies on the arginine deiminase (ADI) pathway of fermentative arginine catabolism, little genetic information exists on this pathway in this organism. We fill this important gap by characterizing, in an 8,228-bp region cloned from a lambdagt11 genomic library of E. faecalis, a five-gene cluster forming a transcriptional unit (revealed by Northern blots and primer extension in E. faecalis) that corresponds to the ADI operon. Four additional genes in the opposite DNA strand and one in the same DNA strand are also identified. Studies on the protein products, including heterologous expression and/or sequence comparisons, allow us to ascertain or propose functions for all but 1 of the 10 genes. The ADI operon genes, arcABCRD, encode, respectively, ADI, ornithine transcarbamylase, carbamate kinase, a putative Crp/Fnr-type regulator (ArcR), and a putative ornithine-arginine antiporter (ArcD). Arginine induces the expression of arcABCRD, most likely by means of two homologous ArgR/AhrC-type regulators encoded by two genes, argR1 and argR2, that precede arcABCRD in each DNA strand and that are transcribed monocistronically, their transcription being influenced differentially by glucose and arginine. Potential ArgR1/ArgR2 (double and single) binding sequences are found in the promoter regions of arcA and of argR1/argR2 themselves. In addition, putative binding sequences for ArcR and for CcpA are found, respectively, in the argR1/argR2 and arcA promoter regions. Of the three other genes identified, two form a transcriptional unit and encode a putative metal-sensitive transcriptional regulator (ArsR) and a cysteine protease.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Multigene Family
11. Francia  MV, Clewell  DB,     ( 2002 )

Amplification of the tetracycline resistance determinant of pAMalpha1 in Enterococcus faecalis requires a site-specific recombination event involving relaxase.

Journal of bacteriology 184 (18)
PMID : 12193637  :   DOI  :   10.1128/jb.184.18.5187-5193.2002     PMC  :   PMC135321    
Abstract >>
The small multicopy plasmid pAMalpha1 (9.75 kb) encoding tetracycline resistance in Enterococcus faecalis is known to generate tandem repeats of a 4.1-kb segment carrying tet(L) when cells are grown extensively in the presence of tetracycline. Here we show that the initial (rate-limiting) step involves a site-specific recombination event involving plasmid-encoded relaxase activity acting at two recombination sequences (RS1 and RS2) that flank the tet determinant. We also present the complete nucleotide sequence of pAMalpha1.
KeywordMeSH Terms
Mutagenesis, Site-Directed
Recombination, Genetic
12. Sutherlin  A, Hedl  M, Sanchez-Neri  B, Burgner  JW, Stauffacher  CV, Rodwell  VW,     ( 2002 )

Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A synthase, an enzyme of isopentenyl diphosphate biosynthesis.

Journal of bacteriology 184 (15)
PMID : 12107122  :   DOI  :   10.1128/jb.184.15.4065-4070.2002     PMC  :   PMC135212    
Abstract >>
Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways. Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni(2+)-agarose to apparent homogeneity and a specific activity of 10 micromol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s(20,w), 5.3). Optimal activity occurred in 2.0 mM MgCl(2) at 37(o)C. The DeltaH(a) was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK(a) of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 +/- 0.2 and that of covalent acetylation was 0.60 +/- 0.02. The K(m) for the hydrolysis of acetyl-CoA was 10 microM. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis.
KeywordMeSH Terms
Hemiterpenes
13. Connil  N, Le Breton  Y, Dousset  X, Auffray  Y, Rincé  A, Prévost  H,     ( 2002 )

Identification of the Enterococcus faecalis tyrosine decarboxylase operon involved in tyramine production.

Applied and environmental microbiology 68 (7)
PMID : 12089039  :   DOI  :   10.1128/aem.68.7.3537-3544.2002     PMC  :   PMC126796    
Abstract >>
Screening of a library of Enterococcus faecalis insertional mutants allowed isolation of a mutant affected in tyramine production. The growth of this mutant was similar to that of the wild-type E. faecalis JH2-2 strain in Maijala broth, whereas high-performance liquid chromatography analyses showed that tyramine production, which reached 1,000 microg ml(-1) for the wild-type strain, was completely abolished. Genetic analysis of the insertion locus revealed a gene encoding a decarboxylase with similarity to eukaryotic tyrosine decarboxylases. Sequence analysis revealed a pyridoxal phosphate binding site, indicating that this enzyme belongs to the family of amino acid decarboxylases using this cofactor. Reverse transcription-PCR analyses demonstrated that the gene (tdc) encoding the putative tyrosine decarboxylase of E. faecalis JH2-2 is cotranscribed with the downstream gene encoding a putative tyrosine-tyramine antiporter and with the upstream tyrosyl-tRNA synthetase gene. This study is the first description of a tyrosine decarboxylase gene in prokaryotes.
KeywordMeSH Terms
Transcription, Genetic
14. Shankar  N, Baghdayan  AS, Gilmore  MS,     ( 2002 )

Modulation of virulence within a pathogenicity island in vancomycin-resistant Enterococcus faecalis.

Nature 417 (6890)
PMID : 12066186  :   DOI  :   10.1038/nature00802    
Abstract >>
Enterococci are members of the healthy human intestinal flora, but are also leading causes of highly antibiotic-resistant, hospital-acquired infection. We examined the genomes of a strain of Enterococcus faecalis that caused an infectious outbreak in a hospital ward in the mid-1980s (ref. 2), and a strain that was identified as the first vancomycin-resistant isolate in the United States, and found that virulence determinants were clustered on a large pathogenicity island, a genetic element previously unknown in this genus. The pathogenicity island, which varies only subtly between strains, is approximately 150 kilobases in size, has a lower G + C content than the rest of the genome, and is flanked by terminal repeats. Here we show that subtle variations within the structure of the pathogenicity island enable strains harbouring the element to modulate virulence, and that these variations occur at high frequency. Moreover, the enterococcal pathogenicity island, in addition to coding for most known auxiliary traits that enhance virulence of the organism, includes a number of additional, previously unstudied genes that are rare in non-infection-derived isolates, identifying a class of new targets associated with disease which are not essential for the commensal behaviour of the organism.
KeywordMeSH Terms
15. Flannagan  SE, Clewell  DB,     ( 2002 )

Identification and characterization of genes encoding sex pheromone cAM373 activity in Enterococcus faecalis and Staphylococcus aureus.

Molecular microbiology 44 (3)
PMID : 11994160  :   DOI  :   10.1046/j.1365-2958.2002.02922.x    
Abstract >>
The sex pheromone cAM373 of Enterococcus faecalis and the related staph-cAM373 of Staphylococcus aureus were found to correspond to heptapeptides located within the C-termini of the signal sequences of putative prelipoproteins. The deduced mature forms of the lipoproteins share no detectable homology and presumably serve unrelated functions in the cells. The chromosomally encoded genetic determinants for production of the pheromones have been identified and designated camE (encoding cAM373) and camS (encoding staph-cAM373). Truncated and full-length clones of camE were generated in Escherichia coli, in which cAM373 activity was expressed. In E. faecalis, insertional inactivation in the middle of camE had no detectable phenotypic effects on the pheromone system. Establishment of an in frame translation stop codon within the signal sequence resulted in reduction of cAM373 activity to 3% of normal levels. The camS determinant has homologues in Staphylococcus epidermidis, Bacillus subtilis and Listeria monocytogenes; however, corresponding heptapeptides present within those sequences do not resemble staph-cAM373 closely. The particular significance of staph-cAM373 as a potential intergeneric inducer of transfer-proficient genetic elements is discussed.
KeywordMeSH Terms
Genes, Bacterial
16. Boyd  DA, Cabral  T, Van Caeseele  P, Wylie  J, Mulvey  MR,     ( 2002 )

Molecular characterization of the vanE gene cluster in vancomycin-resistant Enterococcus faecalis N00-410 isolated in Canada.

Antimicrobial agents and chemotherapy 46 (6)
PMID : 12019119  :   DOI  :   10.1128/aac.46.6.1977-1979.2002     PMC  :   PMC127252    
Abstract >>
The vanE operon was characterized from Enterococcus faecalis N00-410 (MIC of vancomycin = 24 microg/ml). The organization of the vanE operon was identical to that of the vanC1 operon from Enterococcus gallinarum, with protein identities ranging from 46 to 63%. An open reading frame located downstream of the vanE operon showed significant homology to a number of integrase genes, all of which are located downstream of the chromosomal GMP synthase gene guaA.
KeywordMeSH Terms
17. Onodera  Y, Okuda  J, Tanaka  M, Sato  K,     ( 2002 )

Inhibitory activities of quinolones against DNA gyrase and topoisomerase IV of Enterococcus faecalis.

Antimicrobial agents and chemotherapy 46 (6)
PMID : 12019093  :   DOI  :   10.1128/aac.46.6.1800-1804.2002     PMC  :   PMC127212    
Abstract >>
We have cloned the DNA gyrase and topoisomerase IV genes of Enterococcus faecalis to examine the actions of quinolones against E. faecalis genetically and enzymatically. We first generated levofloxacin-resistant mutants of E. faecalis by stepwise selection with increasing drug concentrations and analyzed the quinolone resistance-determining regions of gyrA and parC from the resistant mutants. Isogenic mutants with low-level resistance contained a mutation in gyrA, whereas those with higher levels of resistance had mutations in both gyrA and parC. These results suggested that gyrA is the primary target for levofloxacin in E. faecalis. We then purified the recombinant DNA gyrase and topoisomerase IV enzymes of E. faecalis and measured the in vitro inhibitory activities of quinolones against these enzymes. The 50% inhibitory concentrations (IC(50)s) of levofloxacin, ciprofloxacin, sparfloxacin, tosufloxacin, and gatifloxacin for DNA gyrase were found to be higher than those for topoisomerase IV. In conflict with the genetic data, these results indicated that topoisomerase IV would be the primary target for quinolones in E. faecalis. Among the quinolones tested, the IC(50) of sitafloxacin (DU-6859a), which shows the greatest potency against enterococci, for DNA gyrase was almost equal to that for topoisomerase IV; its IC(50)s were the lowest among those of all the quinolones tested. These results indicated that other factors can modulate the effect of target affinity to determine the bacterial killing pathway, but the highest inhibitory actions against both enzymes correlated with good antienterococcal activities.
KeywordMeSH Terms
Topoisomerase II Inhibitors
18. Haas  W, Shepard  BD, Gilmore  MS,     ( 2002 )

Two-component regulator of Enterococcus faecalis cytolysin responds to quorum-sensing autoinduction.

Nature 415 (6867)
PMID : 11780122  :   DOI  :   10.1038/415084a    
Abstract >>
Bacteria of the genus Enterococcus are the main causes of highly antibiotic-resistant infections that are acquired in hospitals. Many clinical isolates of Enterococcus faecalis produce an exotoxin called cytolysin that contributes to bacterial virulence. In addition to its toxin activity, the cytolysin is bactericidal for nearly all Gram-positive organisms. An understanding of conditions that regulate cytolysin expression has advanced little since its initial description. Here we show that the products of two genes, cylR1 and cylR2, which lack homologues of known function, work together to repress transcription of cytolysin genes. Derepression occurs at a specific cell density when one of the cytolysin subunits reaches an extracellular threshold concentration. These observations form the basis of a model for the autoinduction of the cytolysin by a quorum-sensing mechanism involving a two-component regulatory system.
KeywordMeSH Terms
Feedback, Physiological
Gene Expression Regulation, Bacterial
19. Schwarz  FV, Perreten  V, Teuber  M,     ( 2001 )

Sequence of the 50-kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25.

Plasmid 46 (3)
PMID : 11735367  :   DOI  :   10.1006/plas.2001.1544    
Abstract >>
The complete 50,237-bp DNA sequence of the conjugative and mobilizing multiresistance plasmid pRE25 from Enterococcus faecalis RE25 was determined. The plasmid had 58 putative open reading frames, 5 of which encode resistance to 12 antimicrobials. Chloramphenicol acetyltransferase and the 23S RNA methylase are identical to gene products of the broad-host-range plasmid pIP501 from Streptococcus agalactiae. In addition, a 30.5-kb segment is almost identical to pIP501. Genes encoding an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglycoside phosphotransferase are arranged in tandem on a 7.4-kb fragment as previously reported in Tn5405 from Staphylococcus aureus and in pJH1 from E. faecalis. One interrupted and five complete IS elements as well as three replication genes were also identified. pRE25 was transferred by conjugation to E. faecalis, Listeria innocua, and Lactococcus lactis by means of a transfer region that appears similar to that of pIP501. It is concluded that pRE25 may contribute to the further spread of antibiotic-resistant microorganisms via food into the human community.
KeywordMeSH Terms
20. Franz  CM, Grube  A, Herrmann  A, Abriouel  H, Stärke  J, Lombardi  A, Tauscher  B, Holzapfel  WH,     ( 2002 )

Biochemical and genetic characterization of the two-peptide bacteriocin enterocin 1071 produced by Enterococcus faecalis FAIR-E 309.

Applied and environmental microbiology 68 (5)
PMID : 11976133  :   DOI  :   10.1128/aem.68.5.2550-2554.2002     PMC  :   PMC127528    
Abstract >>
The structural genes for the two-peptide bacteriocin enterocin 1071 (Ent1071) in Enterococcus faecalis FAIR-E 309 were cloned. DNA sequence analysis showed that the enterocin 1071A (Ent1071A) peptide of strain FAIR-E 309 differed by two amino acids from the Ent1071A reported for E. faecalis BFE 1071 (E. Balla, L. M. T. Dicks, M. Du Toit, M. J. van der Merwe, and W. H. Holzapfel, Appl. Environ. Microbiol. 66:1298-1304, 2000), while the Ent1071B gene encoded identical peptides in these strains. However, resequencing of ent1071A from E. faecalis BFE 1071 showed that the Ent1071A peptide sequence reported previously was incorrect in two amino acids. Also, ent1071B in E. faecalis FAIR-E 309 encoded a prepeptide that was three amino acids shorter than that previously reported for E. faecalis BFE 1071 Ent1071B. A presumptive immunity gene (eni1071) was located downstream of the bacteriocin structural genes. This gene was cloned into the heterologous host E. faecalis ATCC 19433 and was shown to confer immunity. A truncated ABC transporter gene was located upstream of the Ent1071 structural genes.
KeywordMeSH Terms
21. An  FY, Clewell  DB,     ( 2002 )

Identification of the cAD1 sex pheromone precursor in Enterococcus faecalis.

Journal of bacteriology 184 (7)
PMID : 11889094  :   DOI  :   10.1128/jb.184.7.1880-1887.2002     PMC  :   PMC134937    
Abstract >>
The Enterococcus faecalis virulence plasmid pAD1 encodes a mating response induced by exposure to an octapeptide sex pheromone, cAD1, secreted by plasmid-free enterococci. The determinant for the pheromone in E. faecalis FA2-2, designated cad, was found to encode a 309-amino-acid lipoprotein precursor with the last 8 residues of its 22-amino acid signal sequence representing the cAD1 moiety. The lipoprotein moiety contained two 77-amino-acid repeats (70% identity) separated by 45 residues. The nonisogenic E. faecalis strain V583 determinant encodes a homologous precursor protein, but it differs at two amino acid positions, both of which are located within the pheromone peptide moiety (positions 2 and 8). Construction of a variant of strain FA2-2 containing the differences present in V583 resulted in cells that did not produce detectable cAD1. The mutant appeared normal under laboratory growth conditions, and while significantly reduced in recipient potential, when carrying pAD1 it exhibited a normal mating response. A mutant of FA2-2 with a truncated lipoprotein moiety appeared normal with respect to recipient potential and, when carrying plasmid DNA, donor potential. A gene encoding a protein designated Eep, believed to be a zinc metalloprotease, had been previously identified as required for pheromone biosynthesis and was believed to be involved in the processing of a pheromone precursor. Our new observation that the pAD1-encoded inhibitor peptide, iAD1, whose precursor is itself a signal sequence, is also dependent on Eep is consistent with the likelihood that such processing occurs at the amino terminus of the cAD1 moiety.
KeywordMeSH Terms
22. Teng  F, Wang  L, Singh  KV, Murray  BE, Weinstock  GM,     ( 2002 )

Involvement of PhoP-PhoS homologs in Enterococcus faecalis virulence.

Infection and immunity 70 (4)
PMID : 11895963  :   DOI  :   10.1128/iai.70.4.1991-1996.2002     PMC  :   PMC127847    
Abstract >>
Eleven PhoP-PhoS homolog pairs were identified by searching the Enterococcus faecalis V583 genome sequence database at The Institute for Genomic Research with the Bacillus subtilis PhoP-PhoS sequences. Each pair appears to be a potential two-component system composed of a response regulator and a sensor kinase. Seven of the homologs were disrupted in E. faecalis strain OG1RF. TX10293, a mutant disrupted in one of these genes (etaR, the first gene of the gene pair designated etaRS), showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. The predicted EtaR protein sequence showed greatest similarity to LisR of Listeria monocytogenes (77%) and CsrR of Streptococcus pyogenes (70%); EtaS is 53% similar to LisK and 54% similar to CsrS. When grown in vitro, the TX10293 mutant was more sensitive to low pH (pH 3.4) and more resistant to high temperature (55 degrees C) than wild-type OG1RF. In conclusion, many potential two-component systems are identified for E. faecalis, one of which, EtaRS, was shown to be involved in stress response and virulence.
KeywordMeSH Terms
Genes, Bacterial
Genes, Regulator
23. Yang  HY, Kim  YW, Chang  HI,     ( 2002 )

Construction of an integration-proficient vector based on the site-specific recombination mechanism of enterococcal temperate phage phiFC1.

Journal of bacteriology 184 (7)
PMID : 11889091  :   DOI  :   10.1128/jb.184.7.1859-1864.2002     PMC  :   PMC134912    
Abstract >>
The genome of temperate phage phiFC1 integrates into the chromosome of Enterococcus faecalis KBL 703 via site-specific recombination. In this study, an integration vector containing the attP site and putative integrase gene mj1 of phage phiFC1 was constructed. A 2,744-bp fragment which included the attP site and mj1 was inserted into a pUC19 derivative containing the cat gene to construct pEMJ1-1. E. faecalis KBL 707, which does not contain the bacteriophage but which has a putative attB site within its genome, could be transformed by pEMJ1-1. Southern hybridization, PCR amplification, and DNA sequencing revealed that pEMJ1-1 was integrated specifically at the putative attB site within the E. faecalis KBL 707 chromosome. This observation suggested that the 2,744-bp fragment carrying mj1 and the attP site of phage phiFC1 was sufficient for site-specific recombination and that pEMJ1-1 could be used as a site-specific integration vector. The transformation efficiency of pEMJ1-1 was as high as 6 x 10(3) transformants/microg of DNA. In addition, a vector (pATTB1) containing the 290-bp attB region was constructed. pATTB1 was transformed into Escherichia coli containing a derivative of the pET14b vector carrying attP and mj1. This resulted in the formation of chimeric plasmids by site-specific recombination between the cloned attB and attP sequences. The results indicate that the integration vector system based on the site-specific recombination mechanism of phage phiFC1 can be used for genetic engineering in E. faecalis and in other hosts.
KeywordMeSH Terms
Recombination, Genetic
24. Francia  MV, Haas  W, Wirth  R, Samberger  E, Muscholl-Silberhorn  A, Gilmore  MS, Ike  Y, Weaver  KE, An  FY, Clewell  DB,     ( 2001 )

Completion of the nucleotide sequence of the Enterococcus faecalis conjugative virulence plasmid pAD1 and identification of a second transfer origin.

Plasmid 46 (2)
PMID : 11591137  :   DOI  :   10.1006/plas.2001.1533    
Abstract >>
pAD1 is a 59.3-kb plasmid in Enterococcus faecalis that has been the subject of intense investigation with regard to its pheromone-inducible conjugation behavior as well as its contribution to virulence. Approximately two-thirds of the pAD1 nucleotide sequence has been previously reported. Here we report on an analysis of the final approximately 22 kb, a significant portion of which is believed to encode structural genes associated with conjugation. The conjugation-related region was also found to contain a new (second) origin of conjugative transfer (oriT). A list of open reading frames covering the entire plasmid is presented.
KeywordMeSH Terms
25. Duez  C, Zorzi  W, Sapunaric  F, Amoroso  A, Thamm  I, Coyette  J,     ( 2001 )

The penicillin resistance of Enterococcus faecalis JH2-2r results from an overproduction of the low-affinity penicillin-binding protein PBP4 and does not involve a psr-like gene.

Microbiology (Reading, England) 147 (Pt 9)
PMID : 11535796  :   DOI  :   10.1099/00221287-147-9-2561    
Abstract >>
A penicillin-resistant mutant, JH2-2r (MIC 75 microg ml(-1)), was isolated from Enterococcus faecalis JH2-2 (MIC 5 microg ml(-1)) by successive passages on plates containing increasing concentrations of benzylpenicillin. A comparison of the penicillin-binding protein (PBP) profiles in the two strains revealed a more intensely labelled PBP4 in JH2-2r. Because the sequences of the JH2-2 and JH2-2r pbp4 genes were strictly identical, even in their promoter regions, this intensive labelling could only be associated with an overproduction of the low-affinity PBP4. No psr gene analogous to that proposed to act as a regulator of PBP5 synthesis in Enterococcus hirae and Enterococcus faecium could be identified in the vicinity of pbp4 in E. faecalis JH2-2 and JH2-2r. However, a psr-like gene distant from pbp4 was identified. The cloning and sequencing of that psr-like gene from both E. faecalis strains indicated that they were identical. It is therefore postulated that the PBP4 overproduction in E. faecalis JH2-2r results from the modification of an as yet unidentified factor.
KeywordMeSH Terms
Bacterial Proteins
Hexosyltransferases
Peptidyl Transferases
26. Cellier  MF, Bergevin  I, Boyer  E, Richer  E,     ( 2001 )

Polyphyletic origins of bacterial Nramp transporters.

Trends in genetics : TIG 17 (7)
PMID : 11418195  :  
Abstract >>
The redox-active metals iron and manganese are required for energy metabolism, protection against oxidative stress and defense against infections. In eukaryotes, both divalent metals are transported by Nramp transporters. The sequence of these transporters was remarkably conserved during evolution. Several bacterial Nramp homologs (MntH) are also proton-dependent manganese transporters. Here, we present phylogenetic evidence for the polyphyletic origins of three groups of MntH proteins and for possible Nramp horizontal gene transfer with eukaryotes. We propose that the evolution of the MntH/Nramp family is related to adaptation to oxidative environments, including those arising during infection of animals and plants.
KeywordMeSH Terms
Bacterial Proteins
Cation Transport Proteins
Evolution, Molecular
Phylogeny
27. Lu  JJ, Perng  CL, Ho  MF, Chiueh  TS, Lee  WH,     ( 2001 )

High prevalence of VanB2 vancomycin-resistant Enterococcus faecium in Taiwan.

Journal of clinical microbiology 39 (6)
PMID : 11376048  :   DOI  :   10.1128/JCM.39.6.2140-2145.2001     PMC  :   PMC88102    
Abstract >>
Thirty-six VanB glycopeptide-resistant Enterococcus faecium isolates were collected from patients in five different hospitals in Taiwan. The vancomycin resistance genes were amplified by the long vanB PCR, which amplifies the 6,373-bp vanB gene cluster including the vanR(B2), vanS(B2), vanY(B2), vanW(B2), vanH(B2), vanB2, and vanX(B2) genes. The deduced amino acid sequences were found to be 95 to 98% homologous to those of the vanB1 gene cluster: VanR(B1), 97%; VanS(B1), 97%; VanY(B1), 96%; VanH(B1), 95%; VanB1, 96%; and VanX(B1), 98%. Restriction enzyme analysis of the long vanB PCR products revealed that all 36 isolates had the same vanB2-specific pattern. DNA sequence analysis of the vanB2 gene, which is a D-Ala-D-Lac ligase gene, revealed that none of the 36 sequences were identical to the previously published vanB2 sequence. Thirty-one isolates had 1 nucleotide different from the published vanB2 sequence. The sequences of the other five isolates differed from the published vanB2 sequence by 2 or 3 nucleotides. Four isolates with a low or moderate resistance to vancomycin (MIC = 4 to 32 microg/ml) were found to have the same leucine-to-methionine change at amino acid position 308 of the vanB2 gene. The genomic DNAs of all 36 isolates were digested with SmaI and then typed by pulsed-field gel electrophoresis (PFGE). Eight different PFGE types (I to VIII) were observed, and type I was found to be prevalent in all hospitals examined in this study. This result suggests that intra- and interhospital dissemination of this E. faecium strain has occurred in Taiwan.
KeywordMeSH Terms
28. Matveyev  AV, Young  KT, Meng  A, Elhai  J,     ( 2001 )

DNA methyltransferases of the cyanobacterium Anabaena PCC 7120.

Nucleic acids research 29 (7)
PMID : 11266551  :   DOI  :   10.1093/nar/29.7.1491     PMC  :   PMC31280    
Abstract >>
From the characterization of enzyme activities and the analysis of genomic sequences, the complement of DNA methyltransferases (MTases) possessed by the cyanobacterium ANABAENA PCC 7120 has been deduced. ANABAENA has nine DNA MTases. Four are associated with Type II restriction enzymes (AVAI, AVAII, AVAIII and the newly recognized inactive AVAIV), and five are not. Of the latter, four may be classified as solitary MTases, those whose function lies outside of a restriction/modification system. The group is defined here based on biochemical and genetic characteristics. The four solitary MTases, DmtA/M.AVAVI, DmtB/M.AVAVII, DmtC/M. AVAVIII and DmtD/M.AVAIX, methylate at GATC, GGCC, CGATCG and rCCGGy, respectively. DmtB methylates cytosines at the N4 position, but its sequence is more similar to N6-adenine MTases than to cytosine-specific enzymes, indicating that it may have evolved from the former. The solitary MTases, appear to be of ancient origin within cyanobacteria, while the restriction MTases appear to have arrived by recent horizontal transfer as did five now inactive Type I restriction systems. One Mtase, M.AVAV, cannot reliably be classified as either a solitary or restriction MTase. It is structurally unusual and along with a few proteins of prokaryotic and eukaryotic origin defines a structural class of MTases distinct from all previously described.
KeywordMeSH Terms
29. Dalet  K, Briand  C, Cenatiempo  Y, Héchard  Y,     ( 2000 )

The rpoN gene of Enterococcus faecalis directs sensitivity to subclass IIa bacteriocins.

Current microbiology 41 (6)
PMID : 11080395  :  
Abstract >>
The final sigma 54 factor has been previously described to be involved in Listeria monocytogenes sensitivity to mesentericin Y105, a subclass IIa bacteriocin. Here, we identified the rpoN gene, encoding final sigma 54, of Enterococcus faecalis JH2-2 and showed that its interruption leads to E. faecalis resistance to different subclass IIa bacteriocins. Moreover, this rpoN mutant remained sensitive to nisin, a class I bacteriocin, suggesting that final sigma 54 is especially involved in sensitivity to subclass IIa bacteriocins.
KeywordMeSH Terms
DNA-Binding Proteins
30. Rince  A, Capiaux  H, Giard  JC,     ( 2000 )

Inactivation of the stress- and starvation-inducible gls24 operon has a pleiotrophic effect on cell morphology, stress sensitivity, and gene expression in Enterococcus faecalis.

Journal of bacteriology 182 (16)
PMID : 10913085  :   DOI  :   10.1128/jb.182.16.4512-4520.2000     PMC  :   PMC94623    
Abstract >>
Enterococcus faecalis induces the synthesis of at least 42 proteins during 24 h of glucose starvation. Because of its induction during carbohydrate and complete starvation (incubation in tap water) and CdCl(2) and bile salts stresses, one of these proteins (Gls24) was qualified as a "general stress protein" and was analyzed at the molecular level. Its corresponding gene, gls24, seems to be the penultimate gene of an operon composed, altogether, of six open reading frames (ORFs). The ORF preceding gls24 (orf4) showed very strong identity with gls24. The deduced polypeptides of these two genes showed similarity with a 20-kDa hypothetical protein from Lactococcus lactis and an alkaline stress protein from Staphylococcus aureus with no previously known biological significance. Data from the operon sequence and Northern analysis led to the conclusions that (i) gls24 possesses its own promoter which is especially induced at the onset of starvation and (ii) the operon promoter is stress inducible in exponential-phase cells. A mutation in the gls24 gene led to a severe reduction of growth rate and reduction of survival against 0.3% bile salts in the 24-h-starved cells compared to the wild-type strain. Moreover, the chain length of the mutant is significantly reduced during growth. These results argue strongly for a role of the protein Gls24 and/or GlsB in morphological changes and in stress tolerance in E. faecalis. Comparison of two-dimensional protein gels from wild-type cells with those from gls24 mutant cells revealed a pleiotropic effect of the mutation on gene expression. At least nine proteins were present in larger amounts in the mutant. For six of them, the corresponding N-terminal microsequence has been obtained. Three of these sequences map in genes coding for L-lactate dehydrogenase, lipoamide dehydrogenase, and pyruvate decarboxylase, all involved in pyruvate metabolism.
KeywordMeSH Terms
Bacterial Proteins
Gene Expression Regulation, Bacterial
Operon
31. Goh  SH, Facklam  RR, Chang  M, Hill  JE, Tyrrell  GJ, Burns  EC, Chan  D, He  C, Rahim  T, Shaw  C, Hemmingsen  SM,     ( 2000 )

Identification of Enterococcus species and phenotypically similar Lactococcus and Vagococcus species by reverse checkerboard hybridization to chaperonin 60 gene sequences.

Journal of clinical microbiology 38 (11)
PMID : 11060051  :   PMC  :   PMC87524    
Abstract >>
Data from four recent studies (S. H. Goh et al., J. Clin. Microbiol. 36:2164-2166, 1998; S. H. Goh et al., J. Clin. Microbiol. 34:818-823, 1996; S. H. Goh et al., J. Clin. Microbiol. 35:3116-3121, 1997; A. Y. C. Kwok et al., Int. J. Syst. Bacteriol. 49:1181-1192, 1999) suggest that an approximately 600-bp region of the chaperonin 60 (Cpn60) gene, amplified by PCR with a single pair of degenerate primers, has utility as a potentially universal target for bacterial identification (ID). This Cpn60 gene ID method correctly identified isolates representative of numerous staphylococcal species and Streptococcus iniae, a human and animal pathogen. We report herein that this method enabled us to distinguish clearly between 17 Enterococcus species (Enterococcus asini, Enterococcus rattus, Enterococcus dispar, Enterococcus gallinarum, Enterococcus hirae, Enterococcus durans, Enterococcus cecorum, Enterococcus faecalis, Enterococcus mundtii, Enterococcus casseliflavus, Enterococcus faecium, Enterococcus malodoratus, Enterococcus raffinosus, Enterococcus avium, Enterococcus pseudoavium, Enterococcus new sp. strain Facklam, and Enterococcus saccharolyticus), and Vagococcus fluvialis, Lactococcus lactis, and Lactococcus garvieae. From 123 blind-tested samples, only two discrepancies were observed between the Facklam and Collins phenotyping method (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27:731-734, 1989) and the Cpn60 ID method. In each case, the discrepancies were resolved in favor of the Cpn60 ID method. The species distributions of the 123 blind-tested isolates were Enterococcus new sp. strain Facklam (ATCC 700913), 3; E. asini, 1; E. rattus, 4; E. dispar, 2; E. gallinarum, 20; E. hirae, 9; E. durans, 9; E. faecalis, 12; E. mundtii, 3; E. casseliflavus, 8; E. faecium, 25; E. malodoratus, 3; E. raffinosus, 8; E. avium, 4; E. pseudoavium, 1; an unknown Enterococcus clinical isolate, sp. strain R871; Vagococcus fluvialis, 4; Lactococcus garvieae, 3; Lactococcus lactis, 3; Leuconostoc sp., 1; and Pediococcus sp., 1. The Cpn60 gene ID method, coupled with reverse checkerboard hybridization, is an effective method for the identification of Enterococcus and related organisms.
KeywordMeSH Terms
32. Ingraham  KA, Iordanescu  S, So  CY, Holmes  DJ, Chalker  AF, Bryant  AP, Brown  JR, Wilding  EI,     ( 2000 )

Identification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram-positive cocci.

Journal of bacteriology 182 (15)
PMID : 10894743  :   DOI  :   10.1128/jb.182.15.4319-4327.2000     PMC  :   PMC101949    
Abstract >>
The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)-pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike Bacillus subtilis and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of Streptococcus pneumoniae under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.
KeywordMeSH Terms
Hemiterpenes
33. McKessar  SJ, Berry  AM, Bell  JM, Turnidge  JD, Paton  JC,     ( 2000 )

Genetic characterization of vanG, a novel vancomycin resistance locus of Enterococcus faecalis.

Antimicrobial agents and chemotherapy 44 (11)
PMID : 11036060  :   DOI  :   10.1128/aac.44.11.3224-3228.2000     PMC  :   PMC101640    
Abstract >>
Enterococcus faecalis strain WCH9 displays a moderate level of resistance to vancomycin (MIC = 16 microgram/ml) and full susceptibility to teicoplanin but is negative by PCR analysis using primers specific for all known enterococcal vancomycin resistance genotypes (vanA, vanB, vanC, vanD, and vanE). We have isolated and sequenced a novel putative vancomycin resistance locus (designated vanG), which contains seven open reading frames, from this strain. These are organized differently from those of all the other enterococcal van loci, and, furthermore, the individual vanG gene products exhibit less than 50% amino acid sequence identity to other van gene products.
KeywordMeSH Terms
Drug Resistance, Microbial
Peptide Synthases
34. Taourit  S, Glaser  P, Garnier  F,     ( 2000 )

Characterization of transposon Tn1549, conferring VanB-type resistance in Enterococcus spp.

Microbiology (Reading, England) 146 (Pt 6) (N/A)
PMID : 10846226  :   DOI  :   10.1099/00221287-146-6-1481    
Abstract >>
Transfer of VanB-type resistance to glycopeptides among enterococci has been reported to be associated with the movement of large chromosomal genetic elements or of plasmids. The authors report the characterization of the 34 kb transposon Tn1549 borne by a plasmid related to pAD1 and conferring vancomycin resistance in clinical isolates of Enterococcus spp. Tn1549 contained 30 ORFs and appeared to be organized like the Tn916 family of conjugative transposons into three functional regions: (i) the right end, implicated in the excision-integration process; (ii) the central part, in which the vanB2 operon replaces the tet(M) gene; and (iii) the left extremity, in which eight of the 18 ORFs could be implicated in the conjugative transfer.
KeywordMeSH Terms
35. Lundblad  EW, Rokenes  TP, Dahl  KH,     ( 2000 )

Genetic linkage of the vanB2 gene cluster to Tn5382 in vancomycin-resistant enterococci and characterization of two novel insertion sequences.

Microbiology (Reading, England) 146 (Pt 6) (N/A)
PMID : 10846225  :   DOI  :   10.1099/00221287-146-6-1469    
Abstract >>
VanB-type vancomycin resistance is encoded by the vanB gene cluster, which disseminates by horizontal gene transfer and clonal spread of vancomycin-resistant enterococci (VRE). Genetic linkage of the vanB gene cluster to transposon Tn5382 and the insertion sequences IS16 and IS256-like has previously been shown. In this study linkage of defined vanB gene cluster subtypes to these elements was examined. All the vanB2 subtype strains studied (n=14) revealed co-hybridization of vanB and Tn5382, whereas the strains of vanB1 (n=8) and vanB3 (n=1) subtypes were Tn5382 negative. Conjugative cotransfer of the vanB2 gene cluster and Tn5382 was demonstrated for two strains. DNA sequencing of the vanX(B)-ORFC region in vanB2 strains confirmed that the vanB2 gene cluster is an integral part of Tn5382. No general pattern of linkage was observed with regard to IS16 and IS256-like. Two novel insertion sequences were identified in specific vanB2 subtype strains. (i) A 1611 bp element (ISEnfa110) was detected in the left flank of Tn5382. Its insertion site, lack of terminal inverted and direct repeats, and two conserved motifs in its putative transposase all conform to the conventions of the IS110 family. (ii) A 787 bp element (ISEnfa200) was detected in the vanS(B)-vanY(B) intergenic region. Its ORF encoded a putative protein with 60-70% identity to transposases of the IS200 family. No further copies of ISEnfa110 were found by colony hybridization of 181 enterococcal isolates, whereas ISEnfa200 was found in four additional vanB2 strains from the USA. The five strains had identical ISEnfa200 element insertion sites, and Tn5382 was located downstream from a pbp5 gene conferring high-level ampicillin resistance. These isolates showed related PFGE patterns, suggesting possible clonal spread of a VRE strain harbouring a Tn5382-vanB2-ISEnfa200 element linked to a pbp5 gene conferring ampicillin resistance.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
36. De Boever  EH, Clewell  DB, Fraser  CM,     ( 2000 )

Enterococcus faecalis conjugative plasmid pAM373: complete nucleotide sequence and genetic analyses of sex pheromone response.

Molecular microbiology 37 (6)
PMID : 10998166  :   DOI  :   10.1046/j.1365-2958.2000.02072.x    
Abstract >>
pAM373 is a 36.7 kb conjugative plasmid in Enterococcus faecalis that encodes a response to a peptide sex pheromone, cAM373, secreted by plasmid-free (recipient) strains of enterococci. It was identified over 15 years ago as one of five plasmids in E. faecalis strain RC73 and was of interest because a related pheromone activity could be detected in culture supernatants of Staphylococcus aureus and Streptococcus gordonii. Because of increased clinical concern relating to the possibility of mobilizing vancomycin resistance determinants from enterococci, where they are becoming common, into pathogens such as S. aureus, efforts were initiated to characterize pAM373 further. The results of a complete nucleotide sequence determination of pAM373, as well as a genetic analysis of key genes related to regulation of the pheromone response, are reported here. With regard to determinants related to conjugation, the plasmid has a structural organization similar to other known pheromone-responsive plasmids such as pAD1, pCF10 and pPD1; however, there are several unique features. Although there are significant homologues relating to a pheromone-binding surface protein (TraC) and a negatively regulating protein (TraA), there is an absence of a determinant equivalent to traB of pAD1 (reduces endogenous pheromone) and a determinant for surface-exclusion protein. The precursor structure of the inhibitor peptide iAM373 was identified, and its determinant (iam373) was found to be about 500 nt upstream of an apparent transcription terminator t1. Tn917-lac insertion analyses provided interesting insights into aspects of control of the pheromone response and showed that, although the traA product is sensitive to pheromone, it appears to act differently from the traA homologue of pAD1.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
37. González  C, Langdon  GM, Bruix  M, Gálvez  A, Valdivia  E, Maqueda  M, Rico  M,     ( 2000 )

Bacteriocin AS-48, a microbial cyclic polypeptide structurally and functionally related to mammalian NK-lysin.

Proceedings of the National Academy of Sciences of the United States of America 97 (21)
PMID : 11005847  :   DOI  :   10.1073/pnas.210301097     PMC  :   PMC17181    
Abstract >>
The solution structure of bacteriocin AS-48, a 70-residue cyclic polypeptide from Enterococcus faecalis, consists of a globular arrangement of five alpha-helices enclosing a compact hydrophobic core. The head-to-tail union lies in the middle of helix 5, a fact that is shown to have a pronounced effect on the stability of the three-dimensional structure. Positive charges in the side chains of residues in helix 4 and in the turn linking helix 4 to helix 5 form a cluster that most probably determine its antibacterial activity by promoting pore formation in cell membranes. A similar five-helix structural motif has been found in the antimicrobial NK-lysin, an effector polypeptide of T and natural killer (NK) cells. Bacteriocin AS-48 lacks the three disulfide bridges characteristic of the saposin fold present in NK-lysin, and has no sequence homology with it. Nevertheless, the similar molecular architecture and high positive charge strongly suggest a common mechanism of antibacterial action.
KeywordMeSH Terms
38. Martínez-Bueno  M, Valdivia  E, Gálvez  A, Maqueda  M,     ( 2000 )

pS86, a new theta-replicating plasmid from Enterococcus faecalis.

Current microbiology 41 (4)
PMID : 10977892  :  
Abstract >>
The complete nucleotide sequence of the small (5149 bp) and cryptic plasmid pS86 from Enterococcus faecalis ssp. faecalis S-86 has been determined. Sequence analysis revealed six putative open reading frames (ORFs) encoding polypeptides of 28.3, 11.5, 8.4, 65.1, 7.3, and 11.96 kDa each. Based on sequence similarity, two cassettes have been identified in pS86: ORF1 codes for the replication initiation protein (Rep); ORF4 codes for a putative mobilization protein that shows similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. No function could be assigned to the other putative ORFs found. According to our results, pS86 plasmid could use a theta-mode of replication, similar to the recently described theta-type replicons from pUCL287 (Tetragenococcus halophila) and pLA1 or pLA105 (Lactobacillus acidophilus) plasmids.
KeywordMeSH Terms
DNA-Binding Proteins
39. Nallapareddy  SR, Singh  KV, Duh  RW, Weinstock  GM, Murray  BE,     ( 2000 )

Diversity of ace, a gene encoding a microbial surface component recognizing adhesive matrix molecules, from different strains of Enterococcus faecalis and evidence for production of ace during human infections.

Infection and immunity 68 (9)
PMID : 10948146  :   DOI  :   10.1128/iai.68.9.5210-5217.2000     PMC  :   PMC101780    
Abstract >>
Our previous work reported that most Enterococcus faecalis strains adhered to the extracellular matrix proteins collagen types I and IV and laminin after growth at 46 degrees C, but not 37 degrees C, and we subsequently identified an E. faecalis sequence, ace, that encodes a bacterial adhesin similar to the collagen binding protein Cna of Staphylococcus aureus. In this study, we examined the diversity of E. faecalis-specific ace gene sequences among different isolates obtained from various geographic regions as well as from various clinical sources. A comparison of nucleotide and deduced amino acid sequences of Ace from nine E. faecalis strains identified a highly conserved N-terminal A domain, followed by a variable B domain which contains two to five repeats of 47 amino acids in tandem array, preceded by a 20-amino-acid partial repeat. Using 17 other strains collected worldwide, the 5' region of ace that encodes the A domain was sequenced, and these sequences showed > or =97.5% identity. Among the previously reported five amino acids critical for collagen binding by Cna of S. aureus, four were found to be identical in Ace from all strains tested. Polyclonal immune rabbit serum prepared against recombinant Ace A derived from E. faecalis strain OG1RF detected Ace in mutanolysin extracts of seven of nine E. faecalis strains after growth at 46 degrees C; Ace was detected in four different molecular sizes that correspond to the variation in the B repeat region. To determine if there was any evidence to indicate that Ace might be produced under physiological conditions, we quantitatively assayed sera collected from patients with enterococcal infections for the presence of anti-Ace A antibodies. Ninety percent of sera (19 of 21) from patients with E. faecalis endocarditis showed reactivity with titers from 1:32 to >1:1,024; the only 2 sera which lacked antibodies to Ace A had considerably lower titers of antibodies to other E. faecalis antigens as well. Human-derived, anti-Ace A immunoglobulins G purified from an E. faecalis endocarditis patient serum inhibited adherence of 46 degrees C-grown E. faecalis OG1RF to collagen types I and IV and laminin. In conclusion, these results show that ace is highly conserved among isolates of E. faecalis, with at least four variants related to the differences in the B domain, is expressed by different strains during infection in humans, and human-derived antibodies can block adherence to these extracellular matrix proteins.
KeywordMeSH Terms
Bacterial Proteins
Genetic Variation
40. Singh  KV, Weinstock  GM, Qin  X,     ( 2000 )

Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence.

Infection and immunity 68 (5)
PMID : 10768947  :   DOI  :   10.1128/iai.68.5.2579-2586.2000     PMC  :   PMC97462    
Abstract >>
Three agr-like genes (fsrA, fsrB, and fsrC, for Enterococcus faecalis regulator) were found upstream of the previously reported gelatinase gene (gelE) and a downstream putative serine protease gene (sprE; accession number Z12296) of Enterococcus faecalis OG1RF. The deduced amino acid sequence of fsrA shows 26% identity and 38% similarity to Staphylococcus aureus AgrA (the response regulator of the accessory gene regulator system in the agr locus), FsrB shows 23% identity and 41% similarity to S. aureus AgrB, and FsrC shows 23% identity and 36% similarity to S. aureus AgrC (the sensor transducer of Agr system). Northern blot analysis suggested that gelE and sprE are cotranscribed and that fsrB and fsrC are also cotranscribed in OG1RF. Northern blot analysis of fsrA, fsrB, fsrC, gelE, and sprE insertion mutants showed that fsrB, fsrC, gelE, and sprE are not expressed in fsrA, fsrB, and fsrC mutants, while insertion in an open reading frame further upstream of fsrA did not effect the expression of these genes, suggesting that agr-like genes may be autoregulated and that they regulate gelE and sprE expression, as further confirmed by complementation of fsr gene mutations with a 6-kb fragment which contains all three fsr genes in the shuttle vector, pAT18. Testing of 95 other isolates of E. faecalis showed that 62% produced gelatinase (Gel(+)), while 91% (including all Gel(+) strains) hybridized to a gelE probe; 71% (including all Gel(+) strains) hybridized to an fsr probe, corroborating the conclusion that both gelE and fsr are necessary for gelatinase production. Testing of fsrA, fsrB, and sprE mutants in a mouse peritonitis model showed that sprE and agr-like gene mutants resulted in highly significantly prolonged survival compared to the parent strain OG1RF, a finding similar to what we had previously shown for a gelE mutant. These results suggest that sprE and agr-like genes contribute to the virulence of E. faecalis OG1RF in this model.
KeywordMeSH Terms
DNA-Binding Proteins
Escherichia coli Proteins
Hemeproteins
NADH, NADPH Oxidoreductases
Trans-Activators
41. Dicks  LM, Du Toit  M, Balla  E,     ( 2000 )

Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071.

Applied and environmental microbiology 66 (4)
PMID : 10742203  :   DOI  :   10.1128/aem.66.4.1298-1304.2000     PMC  :   PMC91984    
Abstract >>
The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in G?ttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100 degrees C, 60 min; 50% of activity remained after 15 min at 121 degrees C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the alpha and beta peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.
KeywordMeSH Terms
Cloning, Molecular
42. Auffray  Y, Leboeuf  C,     ( 2000 )

Cloning, sequencing and characterization of the ccpA gene from Enterococcus faecalis.

International journal of food microbiology 55 (1��3��)
PMID : 10791727  :  
Abstract >>
Enzymes involved in the metabolism of complex carbon and energy sources are unnecessary under conditions of abundant, readily metabolisable nutrients such as glucose or fructose. The repression of these enzymes by glucose has been termed carbon catabolite repression. Mechanisms involved in the carbon catabolite repression in gram-positive bacteria are known to differ from those of gram-negative bacteria such as Escherichia coli. It appears to be mediated by transcriptional repression, requiring trans-acting CcpA, a member of the LacI-GalR family of bacterial regulatory proteins and a cis-acting consensus sequence, designated cre. Here, we report the cloning and characterisation of the chromosomal ccpA gene from Enterococcus faecalis JH2-2. This gene is predicted to encode a 333 amino acids protein with nearly 75% identity to CcpA of Lactobacillus casei.
KeywordMeSH Terms
Bacterial Proteins
43. Quesnes  G, Poyart  C,     ( 2000 )

Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci.

Journal of clinical microbiology 38 (1)
PMID : 10618129  :   PMC  :   PMC88737    
Abstract >>
Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 438-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 19 enterococcal type strains (Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus seriolicida, Enterococcus solitarius, and Enterococcus sulfureus). Sequence analysis of the sodA(int) fragments enabled reliable identification of 18 enterococcal species, including E. casseliflavus-E. flavescens and E. gallinarum. The sodA(int) fragments of E. casseliflavus and E. flavescens were almost identical (99.5% sequence identity), which suggests that they should be associated in a single species. Our results confirm that the sodA gene constitutes a more discriminative target sequence than 16S rRNA gene in differentiating closely related bacterial species.
KeywordMeSH Terms
Genes, Bacterial
44. Ménard  C, Roy  PH, Martineau  F, Picard  FJ, Ke  D,     ( 1999 )

Development of a PCR assay for rapid detection of enterococci.

Journal of clinical microbiology 37 (11)
PMID : 10523541  :   PMC  :   PMC85677    
Abstract >>
Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR-based assay is capable of detecting all clinically important enterococci and has potential for use in clinical microbiology laboratories.
KeywordMeSH Terms
45. Ross  RP, Rothschild  CB, Parsonage  D, Nittayajarn  A, Lee  MH,     ( 1999 )

Characterization of Enterococcus faecalis alkaline phosphatase and use in identifying Streptococcus agalactiae secreted proteins.

Journal of bacteriology 181 (18)
PMID : 10482522  :   PMC  :   PMC94101    
Abstract >>
We have identified and characterized an Enterococcus faecalis alkaline phosphatase (AP, encoded by phoZ). The predicted gene product shows homology with alkaline phosphatases from a variety of species; it has especially high similarity with two alkaline phosphatases from Bacillus subtilis. Expression of phoZ in Escherichia coli, E. faecalis, Streptococcus agalactiae (group B streptococcus [GBS]), or Streptococcus pyogenes (group A streptococcus [GAS]) produces a blue-colony phenotype on plates containing a chromogenic substrate, 5-bromo-4-chloro-3-indolylphosphate (XP or BCIP). Two tests were made to determine if the activity of the enzyme is dependent upon the enzyme's subcellular location. First, elimination of the signal sequence reduced AP activity to 3% of the wild-type activity (or less) in three species of gram-positive bacteria. Restoration of export, using the signal sequence from C5a peptidase, restored AP activity to at least 50% of that of the wild type. Second, we engineered two chimeric proteins in which AP was fused to either a periplasmic domain or a cytoplasmic domain of lactose permease (a membrane protein). In E. coli, the periplasmic fusion had 17-fold-higher AP activity than the cytoplasmic fusion. We concluded that AP activity is export dependent. The signal sequence deletion mutant, phoZDeltass, was used to identify random genomic fragments from GBS that encode exported proteins or integral membrane proteins. Included in this set of fragments were genes that exhibited homology with the Rib protein (a cell wall protein from GBS) or with DppB (an integral membrane protein from GAS). AP acts as a reporter enzyme in GBS, GAS, and E. faecalis and is expected to be useful in a variety of gram-positive bacteria.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
Monosaccharide Transport Proteins
Symporters
46. Claiborne  A, Snoep  JL, Ross  RP, van der Weijden  CC,     ( 1999 )

Catabolism of branched-chain alpha-keto acids in Enterococcus faecalis: the bkd gene cluster, enzymes, and metabolic route.

Journal of bacteriology 181 (17)
PMID : 10464218  :   PMC  :   PMC94053    
Abstract >>
Genes encoding a branched-chain alpha-keto acid dehydrogenase from Enterococcus faecalis 10C1, E1alpha (bkdA), E1beta (bkdB), E2 (bkdC), and E3 (bkdD), were found to reside in the gene cluster ptb-buk-bkdDABC. The predicted products of ptb and buk exhibited significant homology to the phosphotransbutyrylase and butyrate kinase, respectively, from Clostridium acetobutylicum. Activity and redox properties of the purified recombinant enzyme encoded by bkdD indicate that E. faecalis has a lipoamide dehydrogenase that is distinct from the lipoamide dehydrogenase associated with the pyruvate dehydrogenase complex. Specific activity of the ptb gene product expressed in Escherichia coli was highest with the substrates valeryl-coenzyme A (CoA), isovaleryl-CoA, and isobutyryl-CoA. In cultures, a stoichiometric conversion of alpha-ketoisocaproate to isovalerate was observed, with a concomitant increase in biomass. We propose that alpha-ketoisocaproate is converted via the BKDH complex to isovaleryl-CoA and subsequently converted into isovalerate via the combined actions of the ptb and buk gene products with the concomitant phosphorylation of ADP. In contrast, an E. faecalis bkd mutant constructed by disruption of the bkdA gene did not benefit from having alpha-ketoisocaproate in the growth medium, and conversion to isovalerate was less than 2% of the wild-type conversion. It is concluded that the bkd gene cluster encodes the enzymes that constitute a catabolic pathway for branched-chain alpha-keto acids that was previously unidentified in E. faecalis.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
47. Courvalin  P, Sahm  DF, Perichon  B, Reynolds  P,     ( 1999 )

VanE, a new type of acquired glycopeptide resistance in Enterococcus faecalis BM4405.

Antimicrobial agents and chemotherapy 43 (9)
PMID : 10471558  :   PMC  :   PMC89440    
Abstract >>
Enterococcus faecalis BM4405 was resistant to low levels of vancomycin (MIC, 16 microg/ml) and was susceptible to teicoplanin (MIC, 0.5 microg/ml). No PCR product was obtained when the total DNA of this clinical isolate was used as a template with primers specific for glycopeptide resistance genes vanA, vanB, vanC, and vanD. However, a 604-bp PCR fragment was obtained when V1 and V2 degenerate primers were used and total DNA was digested with HindIII as a template. The product was cloned and sequenced. The deduced amino acid sequence had greater identity (55%) with VanC than with VanA (45%), VanB (43%), or VanD (44%). This was consistent with the fact that BM4405 synthesized peptidoglycan precursors that terminated in D-serine residues. After induction with vancomycin, weak D,D-dipeptidase and penicillin-insensitive D,D-carboxypeptidase activities were detected in cytoplasmic extracts of BM4405, whereas a serine racemase activity was found in the membrane preparation. This new type of acquired glycopeptide resistance was named VanE.
KeywordMeSH Terms
48. Owens  RT, LaBrenz  S, Narayana  SV, Kreikemeyer  B, Rich  RL,     ( 1999 )

Ace is a collagen-binding MSCRAMM from Enterococcus faecalis.

The Journal of biological chemistry 274 (38)
PMID : 10480905  :   DOI  :   10.1074/jbc.274.38.26939    
Abstract >>
A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecalis was identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM from Staphylococcus aureus. Ace was predicted to have a molecular mass of 71 kDa and contains features characteristic of cell surface proteins on Gram-positive bacteria, including a LPXTG motif for cross-linking to the cell wall. The N-terminal region of Ace contained a region (residues 174-319) in which 56% of the residues are identical or similar when compared with the minimal ligand-binding region of Cna (Cna 151-318); the remainder of the Ace A domain has 46% similarity with the corresponding region of the Cna A domain. Antibodies raised against recombinant Ace A domain were used to verify the cell surface expression of Ace on E. faecalis. These antibodies also effectively inhibited the adhesion of enterococcal cells to a collagen substrate, suggesting that Ace is a functional collagen-binding MSCRAMM. Structural modeling of the conserved region in Ace (residues 174-319) suggested a structure very similar to that reported for residues 151-318 of the Cna collagen-binding domain in which the ligand-binding site was identified as a trench transversing a beta-sheet face (Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., DeLucas, L. J., H??k, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 10, 833-838). Biochemical analyses of recombinant Ace and Cna A domains supported the modeling data in that the secondary structures were similar as determined by CD spectroscopy and both proteins bound at multiple sites in type I collagen with micromolar affinities, but with different apparent kinetics. We conclude that Ace is a collagen-binding MSCRAMM on enterococci and is structurally and functionally related to the staphylococcal Cna protein.
KeywordMeSH Terms
49. Hancock  LE, Booth  MC, Gilmore  MS,     ( 1999 )

A novel means of self-protection, unrelated to toxin activation, confers immunity to the bactericidal effects of the Enterococcus faecalis cytolysin.

Infection and immunity 67 (7)
PMID : 10377111  :   PMC  :   PMC116516    
Abstract >>
Enterococcus faecalis has become a pervasive clinical problem due to the emergence of resistance to most antibiotics. The cytolysin of E. faecalis is a novel bacterial toxin that contributes to the severity of disease. It consists of two structural subunits, which together possess both hemolytic and bactericidal activity. Both toxin subunits are encoded in a complex operon frequently harbored on pheromone-responsive plasmids. E. faecalis strains lacking such plasmids are susceptible to the bactericidal effects of the cytolysin. A novel cytolysin immunity determinant at the 3' end of the pAD1 cytolysin operon is described in the present study. Deletion analysis and specific mutagenesis isolated the immunity function to a single open reading frame. Specific mutagenesis experiments demonstrate that cytolysin immunity is unrelated to cytolysin activator (CylA) expression as previously proposed. Cytolysin immunity is, however, encoded on the same transcript as and 3' to CylA, and previous associations between immunity and CylA can be ascribed to the polar behavior of Tn917 insertion.
KeywordMeSH Terms
50. Dahl  KH, Sundsfjord  A, Olsvik  O,     ( 1999 )

Heterogeneity in the vanB gene cluster of genomically diverse clinical strains of vancomycin-resistant enterococci.

Antimicrobial agents and chemotherapy 43 (5)
PMID : 10223921  :   PMC  :   PMC89118    
Abstract >>
Molecular analysis of 17 genomically unrelated clinical VanB-type vancomycin-resistant enterococcus isolates from hospital patients in Germany, Norway, Sweden, the United Kingdom and the United States revealed three subtypes of the vanB gene cluster-vanB1, vanB2, and vanB3-which was in accordance with previous subtyping of the ligase gene sequence. There was no correlation between vanB subtype and levels of vancomycin resistance. All strains studied carried a structurally conserved vanB gene cluster as shown by long-range PCR (long PCR) covering 5,959 bp of the published sequence in vanB1 strain V583. Restriction analysis of long PCR amplicons displayed one unique vanB1 pattern and a second vanB2- and vanB3-specific pattern. The vanSB-vanYB intergenic sequences with flanking coding regions were identical within each vanB subtype with one exception. A U.S. vanB2 isolate had a 789-bp enlargement of this region containing a putative open reading frame (ORF) with substantial homology to an ORF in the Clostridium perfringens IS1469 insertion element. The molecular heterogeneity within the vanB gene cluster has implications for the selection of PCR primers, as the primers must ensure detection of all vanB subtypes, and is of importance when considering reservoirs and dissemination of vanB resistance. The molecular identity within the vanB1 and the vanB2 subtype indicates horizontal transmission of both gene clusters between isolates in different geographical areas. Restriction analysis of long PCR vanB amplicons may reveal specific varieties that can be used as epidemiological markers for mobile determinants conferring VanB-type resistance. The finding of three distinct vanB gene clusters should encourage a search for different environmental reservoirs of vanB resistance determinants.
KeywordMeSH Terms
Genome, Bacterial
Multigene Family
51. Steussy  CN, Robison  AD, Tetrick  AM, Knight  JT, Rodwell  VW, Stauffacher  CV, Sutherlin  AL,     ( 2006 )

A structural limitation on enzyme activity: the case of HMG-CoA synthase.

Biochemistry 45 (48)
PMID : 17128980  :   DOI  :   10.1021/bi061505q    
Abstract >>
Recent structural studies of the HMG-CoA synthase members of the thiolase superfamily have shown that the catalytic loop containing the nucleophilic cysteine follows the phi and psi angle pattern of a II' beta turn. However, the i + 1 residue is conserved as an alanine, which is quite unusual in this position as it must adopt a strained positive phi angle to accommodate the geometry of the turn. To assess the effect of the conserved strain in the catalytic loop, alanine 110 of Enterococcus faecalis 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was mutated to a glycine. Subsequent enzymatic studies showed that the overall reaction rate of the enzyme was increased 140-fold. An X-ray crystallographic study of the Ala110Gly mutant enzyme demonstrated unanticipated adjustments in the active site that resulted in additional stabilization of all three steps of the reaction pathway. The rates of acetylation and hydrolysis of the mutant enzyme increased because the amide nitrogen of Ser308 shifts 0.4 A toward the catalytic cysteine residue. This motion positions the nitrogen to better stabilize the intermediate negative charge that develops on the carbonyl oxygen of the acetyl group during both the formation of the acyl-enzyme intermediate and its hydrolysis. In addition, the hydroxyl of Ser308 rotates 120 degrees to a position where it is able to stabilize the carbanion intermediate formed by the methyl group of the acetyl-S-enzyme during its condensation with acetoacetyl-CoA.
KeywordMeSH Terms
52. Kozlowicz  BK, Shi  K, Gu  ZY, Ohlendorf  DH, Earhart  CA, Dunny  GM,     ( 2006 )

Molecular basis for control of conjugation by bacterial pheromone and inhibitor peptides.

Molecular microbiology 62 (4)
PMID : 17038121  :   DOI  :   10.1111/j.1365-2958.2006.05434.x     PMC  :   PMC2655123    
Abstract >>
In many bacteria expression of lateral gene transfer and of virulence factors is controlled by cell-cell signalling systems. Molecular interactions of microbial signal molecules with their cognate receptors are not well understood. For the Enterococcus faecalis conjugative plasmid pCF10, the PrgX protein serves as a molecular switch controlling expression of conjugation and virulence genes encoded by the plasmid. The induction state of a pCF10-carrying donor cell is determined by the ratio of two signalling peptides, cCF10 pheromone and iCF10 inhibitor. Recent analysis of PrgX/cCF10 interactions suggests a mechanism for conversion to the induced state. However, the means by which iCF10 peptide antagonizes cCF10 activity is unclear, and it has been suggested that inhibitor peptides block import of pheromone peptides. We now show that both of these peptides interact with the same binding pocket of PrgX, but they differentially alter the conformation of the protein and its oligomerization state, resulting in opposing biological activities.
KeywordMeSH Terms
Conjugation, Genetic
53. Lim  SK, Tanimoto  K, Tomita  H, Ike  Y,     ( 2006 )

Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

Applied and environmental microbiology 72 (10)
PMID : 17021204  :   DOI  :   10.1128/AEM.00749-06     PMC  :   PMC1610277    
Abstract >>
The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.
KeywordMeSH Terms
Gene Transfer, Horizontal
Vancomycin Resistance
54. Shi  L, Zheng  M, Xiao  Z, Asakura  M, Su  J, Li  L, Yamasaki  S,     ( 2006 )

Unnoticed spread of class 1 integrons in gram-positive clinical strains isolated in Guangzhou, China.

Microbiology and immunology 50 (6)
PMID : 16785718  :   DOI  :   10.1111/j.1348-0421.2006.tb03815.x    
Abstract >>
A total of 46 gram-positive bacteria isolated from clinical specimens collected in China were subjected to PCR analysis with the intI1-specific primers, and the intI1-positive strains were further analyzed for their resistance gene cassette. All isolates possessed the class 1 integron in their genomes and the array of gene cassettes was dhfrXII-orfF-aadA2, which is very similar to other organisms except in one isolate carrying an additional copy of the class 1 integron containing the aadA2 gene cassette. Altogether, the results indicate that the class 1 integron is widespread in gram-positive clinical strains isolated in Guangzhou, China.
KeywordMeSH Terms
Integrons
55. Martín-Platero  AM, Valdivia  E, Ruíz-Rodríguez  M, Soler  JJ, Martín-Vivaldi  M, Maqueda  M, Martínez-Bueno  M,     ( 2006 )

Characterization of antimicrobial substances produced by Enterococcus faecalis MRR 10-3, isolated from the uropygial gland of the hoopoe (Upupa epops).

Applied and environmental microbiology 72 (6)
PMID : 16751538  :   DOI  :   10.1128/AEM.02940-05     PMC  :   PMC1489579    
Abstract >>
The uropygial gland (preen gland) is a holocrine secretory gland situated at the base of the tail in birds which produces a hydrophobic fatty secretion. In certain birds, such as the hoopoe, Upupa epops, the composition of this secretion is influenced by both seasonal and sexual factors, becoming darker and more malodorous in females and in their nestlings during the nesting phase. The secretion is spread throughout the plumage when the bird preens itself, leaving its feathers flexible and waterproof. It is also thought to play a role in defending the bird against predators and parasites. We have isolated from the uropygial secretion of a nestling a bacterium that grows in monospecific culture which we have identified unambiguously by phenotypic and genotypic means as Enterococcus faecalis. The strain in question produces antibacterial substances that are active against all gram-positive bacteria assayed and also against some gram-negative strains. Its peptide nature identifies it as a bacteriocin within the group known as enterocins. Two peptides were purified to homogeneity (MR10A and MR10B), and matrix-assisted laser desorption ionization-time of flight (mass spectrometry) analysis showed masses of 5201.58 and 5207.7 Da, respectively. Amino acid sequencing of both peptides revealed high similarity with enterocin L50A and L50B (L. M. Cintas, P. Casaus, H. Holo, P. E. Hern?ndez, I. F. Nes, and L. S. H?varstein, J. Bacteriol. 180:1988-1994, 1998). PCR amplification of total DNA from strain MRR10-3 with primers for the L50A/B structural genes and sequencing of the amplified fragment revealed almost identical sequences, except for a single conservative change in residue 38 (Glu-->Asp) in MR10A and two changes in residues 9 (Thr-->Ala) and 15 (Leu-->Phe) in MR10B. This is the first time that the production of bacteriocins by a bacterium isolated from the uropygial gland has been described. The production of these broad-spectrum antibacterial substances by an enterococcal strain living in the uropygial gland may be important to the hygiene of the nest and thus to the health of the eggs and chicks.
KeywordMeSH Terms
56. Boyd  DA, Du  T, Hizon  R, Kaplen  B, Murphy  T, Tyler  S, Brown  S, Jamieson  F, Weiss  K, Mulvey  MR,     ( 2006 )

VanG-type vancomycin-resistant Enterococcus faecalis strains isolated in Canada.

Antimicrobial agents and chemotherapy 50 (6)
PMID : 16723588  :   DOI  :   10.1128/AAC.01541-05     PMC  :   PMC1479100    
Abstract >>
Enterococcus faecalis G1-0247 (vancomycin MIC, 16 microg/ml) was found to harbor a vanG operon 99% identical to the vanG operon in E. faecalis BM4518. E. faecalis N03-0233 (vancomycin MIC, 16 microg/ml) was found to harbor a novel vanG operon, vanG2, on an element in a different chromosomal location than the vanG-harboring elements in G1-0247 and BM4518.
KeywordMeSH Terms
57. Swinfield  TJ, Jannière  L, Ehrlich  SD, Minton  NP,     ( 1991 )

Characterization of a region of the Enterococcus faecalis plasmid pAM beta 1 which enhances the segregational stability of pAM beta 1-derived cloning vectors in Bacillus subtilis.

Plasmid 26 (3)
PMID : 1661428  :  
Abstract >>
The nucleotide sequence of a 2.13-kb EcoRI-HindIII, pAM beta 1-derived fragment, isolated from the gram-positive cloning vector pHV1431, has been determined and shown to encode two ORFs. ORF H encodes for a protein of 23,930 Da which exhibits substantial homology to bacterial site-specific recombinases, particularly the resolvases of the gram-positive transposons Tn917 (30.3% identity) and Tn552 (31.6% identity) and the clostridial plasmid pIP404 (27.1% identity). The second ORF (I) is incomplete and encodes a polypeptide which has significant homology with Escherichia coli topoisomerase I (26.0% identity). Insertion of either the entire 2.13-kb EcoRI-HindIII fragment or a 0.73-kb EcoRI-DraI subfragment encoding only the resolvase into the pAM beta 1-based cloning vector pMTL500E causes a significant enhancement of segregational stability (from 6.5 X 10(-2) to 3.0-4.0 X 10(-3) plasmid loss per cell per generation). Improved segregational stability is mirrored by a reduction in plasmid polymerization. The introduction of a stop codon into the resolvase coding region negates its ability to promote segregational stability. It is proposed that the identified determinant stabilizes pAM beta 1-based vectors in Bacillus subtilis by maintaining the plasmid population in the monomeric state, thereby reducing the chances of producing plasmid-free segregants.
KeywordMeSH Terms
58. Huys  G, D'Haene  K, Swings  J,     ( 2006 )

Genetic basis of tetracycline and minocycline resistance in potentially probiotic Lactobacillus plantarum strain CCUG 43738.

Antimicrobial agents and chemotherapy 50 (4)
PMID : 16569881  :   DOI  :   10.1128/AAC.50.4.1550-1551.2006     PMC  :   PMC1426919    
Abstract >>
The potentially probiotic strain Lactobacillus plantarum CCUG 43738, which displayed atypical phenotypic resistance to tetracycline (MIC, 512 microg/ml) and minocycline (MIC, 256 microg/ml), was found to contain a tet(S) gene located on a plasmid of approximately 14 kb. Plasmid curing with novobiocin eliminated this plasmid and restored the tetracycline-susceptible phenotype of the host strain.
KeywordMeSH Terms
Probiotics
Tetracycline Resistance
59. LeBlanc  DJ, Lee  LN, Inamine  JM,     ( 1991 )

Cloning and nucleotide base sequence analysis of a spectinomycin adenyltransferase AAD(9) determinant from Enterococcus faecalis.

Antimicrobial agents and chemotherapy 35 (9)
PMID : 1659306  :   DOI  :   10.1128/aac.35.9.1804     PMC  :   PMC245272    
Abstract >>
Enterococcus faecalis LDR55, a human clinical isolate, is resistant to tetracycline (Tcr), erythromycin (Emr), and high levels (greater than 2,000 micrograms/ml) of spectinomycin (Spr) but not streptomycin. Filter matings between strain LDR55 and E. faecalis OG1-RF produced transconjugants with the following resistance phenotypes: Tcr Emr Spr, Tcr Emr, Tcr Spr, and Tcr only but never Emr or Spr only. The genetic determinant encoding resistance to spectinomycin was cloned in Streptococcus sanguis Challis from pDL55, a 26-kb plasmid harbored by a Tcr Spr transconjugant. Subcloning experiments yielded a 1.1-kb ClaI-NdeI fragment that encoded very high-level Spr in S. sanguis (10 mg/ml) and Escherichia coli (50 mg/ml). Cell extracts of cultures obtained from Spr strains expressed adenylating activity for spectinomycin but not for streptomycin, indicating that Spr was due to an AAD(9) activity. The nucleotide base sequence of the 1.1-kb ClaI-NdeI fragment contained a single 750-base open reading frame. The protein predicted from the open reading frame consisted of 250 amino acids and had a calculated size of approximately 28,000 daltons, similar to the size estimated from maxicell analysis (29,000 daltons). The deduced amino acid sequence of the streptococcal AAD(9) was compared with that of the AAD(9) encoded by staphylococcal transposon Tn554. The two proteins shared approximately 39% amino acid identity, which was expanded to 53% when conservative amino acid changes were included. When the streptococcal protein was compared with an AAD(3")(9) protein of E. coli, the degrees of identity were 27 and 47%, on the basis of actual amino acids and conservative replacements, respectively. The cloning and nucleotide base sequence analyses of the spectinomycin AAD(9) determinant from E. faecalis that results in high-level Spr when transferred to S. sanguis or E. coli are presented.
KeywordMeSH Terms
60. Trieu-Cuot  P, Carlier  C, Poyart-Salmeron  C, Courvalin  P,     ( 1991 )

An integrative vector exploiting the transposition properties of Tn1545 for insertional mutagenesis and cloning of genes from gram-positive bacteria.

Gene 106 (1)
PMID : 1657722  :   DOI  :   10.1016/0378-1119(91)90561-o    
Abstract >>
We have constructed and used an integrative vector, pAT112, that takes advantage of the transposition properties (integration and excision) of transposon Tn1545. This 4.9-kb plasmid is composed of: (i) the replication origin of pACYC184; (ii) the attachment site (att) of Tn1545; (iii) erythromycin-and kanamycin-resistance-encoding genes for selection in Gram- and Gram+ bacteria; and (iv) the transfer origin of IncP plasmid RK2, which allows mobilization of the vector from Escherichia coli to various Gram+ recipients. Integration of pAT112 requires the presence of the transposon-encoded integrase, Int-Tn, in the new host. This vector retains the insertion specificity of the parental element Tn1545 and utilises it to carry out insertional mutagenesis, as evaluated in Enterococcus faecalis. Since pAT112 contains the pACYC184 replicon and lacks most of the restriction sites that are commonly used for molecular cloning, a gene from a Gram+ bacterium disrupted with this vector can be recovered in E. coli by cleavage of genomic DNA, intramolecular ligation and transformation. Regeneration of the gene, by excision of pAT112, can be obtained in an E. coli strain expressing the excisionase and integrase of Tn1545. The functionality of this system was illustrated by characterization of an IS30-like structure in the chromosome of En. faecalis. Derivatives pAT113 and pAT114 contain ten unique cloning sites that allow screening of recombinants having DNA inserts by alpha-complementation in E. coli carrying the delta M15 deletion of lacZ alpha. These vectors are useful to clone and introduce foreign genes into the genomes of Gram+ bacteria.
KeywordMeSH Terms
DNA Transposable Elements
Genetic Vectors
Mutagenesis
Plasmids
61. Agersø  Y, Pedersen  AG, Aarestrup  FM,     ( 2006 )

Identification of Tn5397-like and Tn916-like transposons and diversity of the tetracycline resistance gene tet(M) in enterococci from humans, pigs and poultry.

The Journal of antimicrobial chemotherapy 57 (5)
PMID : 16565159  :   DOI  :   10.1093/jac/dkl069    
Abstract >>
To analyse the sequence diversity of the tetracycline resistance gene tet(M) and its location on mobile elements in Enterococcus faecium and Enterococcus faecalis from humans, pigs and poultry in Denmark. A total of 76 isolates were screened for Tn916/Tn1545-like and Tn5397-like transposons using PCR. tet(M) was sequenced in 15 of the isolates and compared with tet(M) sequences submitted to GenBank (phylogenetic analysis and signs of recombination). Plasmids were extracted, filter-mating experiments were performed and Tn5397-like transposons were further characterized in selected isolates. In 8 of 13 isolates of E. faecium from broilers, tet(M) was present on Tn5397-like transposons, whereas tet(M) was predominantly associated with Tn916/Tn1545-like transposons in E. faecium from pigs and humans, as well as in E. faecalis from humans, pigs and broilers (50 of 63 isolates). The tet(M) genes were divided into three major subgroups according to the phylogenetic analysis. Subgroup I consisted of tet(M) from Clostridium difficile and E. faecium associated with Tn5397-like elements, subgroup II consisted of tet(M) located on Tn916/Tn1545 family transposons and subgroup III consisted of tet(M) associated with composite elements containing several resistance genes. We found evidence of recombination both within and between these groups. Moreover, we identified an E. faecium isolate with both Tn916/Tn1545-like and Tn5397-like elements. This study showed that enterococci contain diverse tet(M) genes present on different mobile elements, which may suggest that enterococci play an important role in the evolution and horizontal spread of mobile elements carrying tet(M). This is the first report of Tn5397-like elements in enterococci.
KeywordMeSH Terms
DNA Transposable Elements
Genetic Variation
62. Giard  JC, Riboulet  E, Verneuil  N, Sanguinetti  M, Auffray  Y, Hartke  A,     ( 2006 )

Characterization of Ers, a PrfA-like regulator of Enterococcus faecalis.

FEMS immunology and medical microbiology 46 (3)
PMID : 16553815  :   DOI  :   10.1111/j.1574-695X.2005.00049.x    
Abstract >>
We have identified a transcriptional regulator, named Ers (for enterococcal regulator of survival), of Enterococcus faecalis, an important opportunistic bacterium commonly recovered from hospitalized patients. Ers is a member of the Crp/Fnr family and is 69% similar to Srv, a PrfA-like regulator of Streptococcus pyogenes implicated in virulence, and is the E. faecalis protein most closely related to PrfA, a positive regulator of virulence genes in Listeria monocytogenes. In an in vivo-in vitro macrophage infection model, the survival of an ers mutant was highly significantly decreased compared with that of the parental strain JH2-2. This mutant was more than 10-fold more sensitive to oxidative challenge by hydrogen peroxide. In order to identify genes whose expression was under Ers control, the RNA levels of 31 likely candidates were measured by real-time quantitative PCR. The results indicate that ers may be autoregulated and that the locus ef0082 appears to be positively regulated by Ers. Nevertheless, mutation of ef0082 did not result in any detectable changes in the survival of the bacterium within murine macrophages.
KeywordMeSH Terms
63. Launay  A, Ballard  SA, Johnson  PD, Grayson  ML, Lambert  T,     ( 2006 )

Transfer of vancomycin resistance transposon Tn1549 from Clostridium symbiosum to Enterococcus spp. in the gut of gnotobiotic mice.

Antimicrobial agents and chemotherapy 50 (3)
PMID : 16495268  :   DOI  :   10.1128/AAC.50.3.1054-1062.2006     PMC  :   PMC1426432    
Abstract >>
The vancomycin resistance vanB2 gene cluster is disseminated worldwide and has been found in phylogenetically remote bacterial genera. The vanB2 operon is part of conjugative transposons Tn1549/Tn5382, but conjugative transposition of these elements has not been demonstrated. We have obtained transfer of a Tn1549-like element (referred to herein as "Tn1549-like") from Clostridium symbiosum MLG101 to Enterococcus faecium 64/3 and Enterococcus faecalis JH2-2 in the digestive tract of gnotobiotic mice and to E. faecium 64/3 in vitro. Retransfer of Tn1549-like from an E. faecium transconjugant also containing Tn916 to E. faecium BM77 was obtained in vitro, albeit at a very low frequency. Transfer efficiency was found to be both donor and recipient dependent. Pulsed-field gel electrophoresis analysis of total SmaI-digested DNA of 48 transconjugants indicated in 27 instances the acquisition of ca. 34 kb of DNA. Two transconjugants harbored two copies of the transposon. Sequencing of the flanking regions of Tn1549-like in 48 transconjugants revealed 29 integration events in 26 loci in the E. faecium genome, and two hot spots for insertion were identified. Integration of the transposon was associated with the acquisition of 5 (n = 18) or 6 (n = 7) bp of donor DNA or with 5-bp duplications of target DNA in the remaining transconjugants. These data demonstrate functionality of the Tn1549-like element and attest that the transfer of the vanB operon between enterococci and human commensal anaerobes occurs in the intestinal environment.
KeywordMeSH Terms
DNA Transposable Elements
Germ-Free Life
64. An  FY, Clewell  DB,     ( 1991 )

Tn917 transposase. Sequence correction reveals a single open reading frame corresponding to the tnpA determinant of Tn3-family elements.

Plasmid 25 (2)
PMID : 1650004  :  
Abstract >>
A nucleotide sequence correction on the Enterococcus faecalis transposon Tn917 indicates that what was formerly thought to be two open reading frames (ORF5 and ORF6) is actually one reading frame (ORF5). The latter exhibits homology with the Tn3-family transposase determinants known as tnpA.
KeywordMeSH Terms
DNA Transposable Elements
65. Galli  D, Friesenegger  A, Wirth  R,     ( 1992 )

Transcriptional control of sex-pheromone-inducible genes on plasmid pAD1 of Enterococcus faecalis and sequence analysis of a third structural gene for (pPD1-encoded) aggregation substance.

Molecular microbiology 6 (10)
PMID : 1640831  :   DOI  :   10.1111/j.1365-2958.1992.tb00851.x    
Abstract >>
The expression of several neighbouring genes on plasmid pAD1 that are necessary for conjugation depend on induction with sex pheromone cAD1. Analyses of transcripts by Northern blot hybridization demonstrated that the genes sea1 (encoding surface exclusion protein) and asa1 (encoding aggregation substance) are transcribed independently. Both genes are organized in different operons together with neighbouring open reading frames of unknown function. Several transcripts could be identified for sea1 and asa1. Their transcriptional start sites were determined by primer extension experiments, confirming the results of the Northern blot experiments. We also could identify sea1- and iad- (encoding an inhibitory peptide counteracting sex pheromone cAD1) specific transcripts which are expressed constitutively, but to a lower extent relative to induced conditions. In addition, we localized the asp1 gene coding for aggregation substance of sex pheromone plasmid pPD1 and determined its DNA sequence, which was found to be highly homologous to asa1 (aggregation substance gene of pAD1) and prgB (aggregation substance gene of pCF10). The structural genes were found to be organized more or less identically on the three sex-pheromone plasmids pAD1, pCF10, and pPD1, and to be highly conserved. Regions supposed to be of crucial importance for regulatory functions, however, were found to differ. We also could identify some conserved DNA motifs which might be potential target sites for transcriptional regulators. In combination these data allowed us to formulate a model for the regulation of sex-pheromone-inducible genes of plasmid pAD1. Its main statement is that only in the presence of cAD1 can the gene traE1 be transcribed. The positive regulatory factor TraE1 then can trigger expression of the structural genes sea1 and asa1.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
Transcription, Genetic
66. Nishimoto  Y, Kobayashi  N, Alam  MM, Ishino  M, Uehara  N, Watanabe  N,     ( 2005 )

Analysis of the prevalence of tetracycline resistance genes in clinical isolates of Enterococcus faecalis and Enterococcus faecium in a Japanese hospital.

Microbial drug resistance (Larchmont, N.Y.) 11 (2)
PMID : 15910229  :   DOI  :   10.1089/mdr.2005.11.146    
Abstract >>
Prevalence of seven tetracycline resistance (TC(R)) genes--tet(L), tet(M), tet(K), tet(O), tet(S), tet(T), and tet(U)--which are known to be distributed to gram-positive cocci was analyzed for 224 Enterococcus faecalis and 46 Enterococcus faecium clinical isolates obtained in a Japanese hospital. Any of the TC(R) genes was detected in 75.9% of all the enterococcal strains. The tet(M) was detected at highest rates in both E. faecalis (75.0%) and E. faecium (69.6%), followed by tet(L), which was harbored in 6.7% of E. faecalis isolates and 30.4% of E. faecium isolates. The tet(O), tet(S), and tet(T) were detected in E. faecalis at low frequencies mostly associated with tet(M), while tet(K) and tet(U) were not detected. Nucleotide sequences of tet(S) from E. faecalis strains were identical to that reported in Listeria monocytogenes. Sequences of tet(O) from two E. faecalis strains were almost identical to each other and also to those from Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, Campylobacter jejuni, and Campylobacter coli, although minor sequence divergence was observed. The tet(T), which had been reported only in Streptococcus pyogenes, was found in five E. faecalis strains. Sequence of the enterococcal tet(T) differed from that of S. pyogenes by only four nucleotides (four amino acids) and showed high sequence identity (99.8%, amino acid level). Enterococcal strains with any one TC(R) gene or those with two TC(R) genes showed generally similar MICs of tetracyclines, and no evident difference in resistance level was observed.
KeywordMeSH Terms
67. Ono  S, Muratani  T, Matsumoto  T,     ( 2005 )

Mechanisms of resistance to imipenem and ampicillin in Enterococcus faecalis.

Antimicrobial agents and chemotherapy 49 (7)
PMID : 15980374  :   DOI  :   10.1128/AAC.49.7.2954-2958.2005     PMC  :   PMC1168717    
Abstract >>
We found ampicillin- and imipenem-resistant isolates of vanA-possessing Enterococcus faecalis with MICs of 8 to 16 microg/ml and 4 to 32 microg/ml, respectively. There have been few reports about penicillin- and imipenem-resistant E. faecalis. Two mechanisms of beta-lactam resistance in E. faecalis, the production of beta-lactamase and the overproduction of penicillin-binding proteins (PBPs), have been reported. The resistant isolates in the current study did not produce any beta-lactamases and analysis of the PBPs showed no overproduction. However, the affinities of PBP4 for beta-lactams in the resistant strains were lower than those of susceptible strains but the affinities of other PBPs for beta-lactams did not change. Accordingly, whole pbp4 fragments from these resistant isolates were sequenced. Two amino acid substitutions at positions 520 and 605 were observed in the highly resistant strains compared to the susceptible ones, Pro520Ser and Tyr605His, and a single Tyr605His amino acid substitution was found in the low-resistance strains. These two point mutations exist in the region between the active-site-defining motifs SDN and KTG of the penicillin-binding domain, the main target of beta-lactams. A strong correlation was seen between these substitutions and decreasing affinities of PBP4 to beta-lactams. In E. faecalis, resistance due to mutations in PBPs has not been reported, though it has in Enterococcus faecium. Our results suggest that development of high-level resistance to penicillins and imipenem depends on point mutations of PBP4 at positions 520 and 605.
KeywordMeSH Terms
68. Su  J, Shi  L, Yang  L, Xiao  Z, Li  X, Yamasaki  S,     ( 2006 )

Analysis of integrons in clinical isolates of Escherichia coli in China during the last six years.

FEMS microbiology letters 254 (1)
PMID : 16451182  :   DOI  :   10.1111/j.1574-6968.2005.00025.x    
Abstract >>
A multiple PCR for the detection of the integrase genes of the three classes of integrons was carried out, and their gene cassettes were characterized in 111 clinical strains of Escherichia coli isolated in Guangzhou City, China during the last 6 years. IntI1 and intI2 genes were detected in 95 isolates (85.6%) and four isolates (3.6%), respectively. No intI3 gene was detected. Six different gene cassettes were found in these strains, and a high prevalence of dfr and aad genes was observed. The E. coli isolates that contained a 1664-bp amplicon of dfrA17-aadA5 in class 1 integron were found to be phylogenetically unrelated to each other by using the enterobacterial repetitive intergenic consensus PCR, as the cassette could be transferred to recipient strains, indicating that the gene cassettes might be disseminated in the clinical strains by a horizontal gene transfer. Therefore, it is important that guidelines for the prudent use of antimicrobial agents are adopted and surveillance programs are established.
KeywordMeSH Terms
Integrons
69. Verneuil  N, Rincé  A, Sanguinetti  M, Posteraro  B, Fadda  G, Auffray  Y, Hartke  A, Giard  JC,     ( 2005 )

Contribution of a PerR-like regulator to the oxidative-stress response and virulence of Enterococcus faecalis.

Microbiology (Reading, England) 151 (Pt 12)
PMID : 16339944  :   DOI  :   10.1099/mic.0.28325-0    
Abstract >>
PerR is one of the most important transcriptional regulators involved in the oxidative-stress response in Bacillus subtilis. Here, the homologous gene in Enterococcus faecalis, ranked among the leading causes of nosocomial infection, was characterized and analysed. Phenotype analysis showed that the perR mutant was significantly more resistant to H2O2 challenge (P < 0.05). Expression of eight genes with potential roles in the oxidative-stress response was determined in the wild-type and perR-mutant strains by real-time quantitative PCR. Surprisingly, low quantitative differences in the transcriptional activity of these genes in the mutant versus wild-type were observed. Likewise, this locus was not involved in survival within murine macrophages, but in the mouse peritonitis model, the perR mutant appeared less lethal than the JH2-2 wild-type strain. The combined results show that PerR affects E. faecalis virulence and that its implication in the transcriptional regulation in this bacterium deviates from the B. subtilis model.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
70. Shi  K, Brown  CK, Gu  ZY, Kozlowicz  BK, Dunny  GM, Ohlendorf  DH, Earhart  CA,     ( 2005 )

Structure of peptide sex pheromone receptor PrgX and PrgX/pheromone complexes and regulation of conjugation in Enterococcus faecalis.

Proceedings of the National Academy of Sciences of the United States of America 102 (51)
PMID : 16339309  :   DOI  :   10.1073/pnas.0506163102     PMC  :   PMC1317922     DOI  :   10.1073/pnas.0506163102     PMC  :   PMC1317922    
Abstract >>
Many bacterial activities, including expression of virulence factors, horizontal genetic transfer, and production of antibiotics, are controlled by intercellular signaling using small molecules. To date, understanding of the molecular mechanisms of peptide-mediated cell-cell signaling has been limited by a dearth of published information about the molecular structures of the signaling components. Here, we present the molecular structure of PrgX, a DNA- and peptide-binding protein that regulates expression of the conjugative transfer genes of the Enterococcus faecalis plasmid pCF10 in response to an intercellular peptide pheromone signal. Comparison of the structures of PrgX and the PrgX/pheromone complex suggests that pheromone binding destabilizes PrgX tetramers, opening a 70-bp pCF10 DNA loop required for conjugation repression.
KeywordMeSH Terms
71. Naser  S, Thompson  FL, Hoste  B, Gevers  D, Vandemeulebroecke  K, Cleenwerck  I, Thompson  CC, Vancanneyt  M, Swings  J,     ( 2005 )

Phylogeny and identification of Enterococci by atpA gene sequence analysis.

Journal of clinical microbiology 43 (5)
PMID : 15872246  :   DOI  :   10.1128/JCM.43.5.2224-2230.2005     PMC  :   PMC1153757    
Abstract >>
The relatedness among 91 Enterococcus strains representing all validly described species was investigated by comparing a 1,102-bp fragment of atpA, the gene encoding the alpha subunit of ATP synthase. The relationships observed were in agreement with the phylogeny inferred from 16S rRNA gene sequence analysis. However, atpA gene sequences were much more discriminatory than 16S rRNA for species differentiation. All species were differentiated on the basis of atpA sequences with, at a maximum, 92% similarity. Six members of the Enterococcus faecium species group (E. faecium, E. hirae, E. durans, E. villorum, E. mundtii, and E. ratti) showed > 99% 16S rRNA gene sequence similarity, but the highest value of atpA gene sequence similarity was only 89.9%. The intraspecies atpA sequence similarities for all species except E. faecium strains varied from 98.6 to 100%; the E. faecium strains had a lower atpA sequence similarity of 96.3%. Our data clearly show that atpA provides an alternative tool for the phylogenetic study and identification of enterococci.
KeywordMeSH Terms
72. Ramirez-Arcos  S, Liao  M, Marthaler  S, Rigden  M, Dillon  JA,     ( 2005 )

Enterococcus faecalis divIVA: an essential gene involved in cell division, cell growth and chromosome segregation.

Microbiology (Reading, England) 151 (Pt 5)
PMID : 15870448  :   DOI  :   10.1099/mic.0.27718-0    
Abstract >>
Enterococcus faecalis divIVA (divIVAEf) is an essential gene implicated in cell division and chromosome segregation. This gene was disrupted by insertional inactivation creating E. faecalis JHSR1, which was viable only when a wild-type copy of divIVAEf was expressed in trans, confirming the essentiality of the gene. The absence of DivIVAEf in E. faecalis JHSR1 inhibited proper cell division, which resulted in abnormal cell clusters possessing enlarged cells of altered shape instead of the characteristic diplococcal morphology of enterococci. The lower viability of the divIVAEf mutant is caused by improper nucleoid segregation and impaired septation within the numerous cells generated in each cluster. Overexpression of DivIVAEf in Escherichia coli KJB24 resulted in enlarged cells with disrupted cell division, suggesting that this round E. coli mutant strain could be used as an indicator for functionality of DivIVAEf. A Bacillus subtilis divIVA mutant was not complemented by DivIVAEf, indicating that this protein does not recognize DivIVA-specific target sites in B. subtilis, or that it does not interact with other proteins of the cell division machinery of this micro-organism. DivIVAEf also failed to complement a Streptococcus pneumoniae divIVA mutant, supporting the phylogenetic distance between Enterococcus and Streptococcus. Our results indicate that DivIVA is a species-specific multifunctional protein implicated in cell division and chromosome segregation in E. faecalis.
KeywordMeSH Terms
Cell Division
Chromosome Segregation
Genes, Essential
73. Steussy  CN, Vartia  AA, Burgner  JW, Sutherlin  A, Rodwell  VW, Stauffacher  CV,     ( 2005 )

X-ray crystal structures of HMG-CoA synthase from Enterococcus faecalis and a complex with its second substrate/inhibitor acetoacetyl-CoA.

Biochemistry 44 (43)
PMID : 16245942  :   DOI  :   10.1021/bi051487x    
Abstract >>
Biosynthesis of the isoprenoid precursor, isopentenyl diphosphate, is a critical function in all independently living organisms. There are two major pathways for this synthesis, the non-mevalonate pathway found in most eubacteria and the mevalonate pathway found in animal cells and a number of pathogenic bacteria. An early step in this pathway is the condensation of acetyl-CoA and acetoacetyl-CoA into HMG-CoA, catalyzed by the enzyme HMG-CoA synthase. To explore the possibility of a small molecule inhibitor of the enzyme functioning as a non-cell wall antibiotic, the structure of HMG-CoA synthase from Enterococcus faecalis (MVAS) was determined by selenomethionine MAD phasing to 2.4 A and the enzyme complexed with its second substrate, acetoacetyl-CoA, to 1.9 A. These structures show that HMG-CoA synthase from Enterococcus is a member of the family of thiolase fold enzymes and, while similar to the recently published HMG-CoA synthase structures from Staphylococcus aureus, exhibit significant differences in the structure of the C-terminal domain. The acetoacetyl-CoA binary structure demonstrates reduced coenzyme A and acetoacetate covalently bound to the active site cysteine through a thioester bond. This is consistent with the kinetics of the reaction that have shown acetoacetyl-CoA to be a potent inhibitor of the overall reaction, and provides a starting point in the search for a small molecule inhibitor.
KeywordMeSH Terms
74. Balla  E, Dicks  LM,     ( 2005 )

Molecular analysis of the gene cluster involved in the production and secretion of enterocins 1071A and 1071B and of the genes responsible for the replication and transfer of plasmid pEF1071.

International journal of food microbiology 99 (1)
PMID : 15718027  :   DOI  :   10.1016/j.ijfoodmicro.2004.08.008    
Abstract >>
Plasmid pEF1071 (9328 bp), bearing the structural genes of enterocins 1071A and 1071B, had been sequenced and nine genes were identified. The genes responsible for the production and transfer of enterocins 1071A and 1071B are arranged in two operons. The first operon (EntABI) contains the genes ent1071A, ent1071B and entI that encodes enterocin 1071A, enterocin 1071B and immunity to these bacteriocins, respectively. The second operon (EntTD) contains two genes, viz. abc (also named entT) and entD, encoding a putative ABC transporter (697 aa) and an accessory protein (697 aa), respectively. Three genes (mobC, mobA and mobX), situated downstream of the second operon, encode proteins of 127, 346 and 224 amino acids, respectively, and are presumably involved in the mobilization of plasmid pEF1071. The ninth gene was identified as a putative repA gene encoding a protein of 327 amino acids. The transcription initiation sites of the genes ent1071A, abc, mobA, mobX and repA were determined by primer extension. By inserting the cat gene into the ent1071B gene, and thereby disrupting the operon structure, we have shown that bacteriocins Ent1071A and Ent1071B act independently against target bacteria.
KeywordMeSH Terms
75. Abbani  M, Iwahara  M, Clubb  RT,     ( 2005 )

The structure of the excisionase (Xis) protein from conjugative transposon Tn916 provides insights into the regulation of heterobivalent tyrosine recombinases.

Journal of molecular biology 347 (1)
PMID : 15733914  :   DOI  :   10.1016/j.jmb.2005.01.019    
Abstract >>
Heterobivalent tyrosine recombinases play a prominent role in numerous bacteriophage and transposon recombination systems. Their enzymatic activities are frequently regulated at a structural level by excisionase factors, which alter the ability of the recombinase to assemble into higher-order recombinogenic nucleoprotein structures. The Tn916 conjugative transposon spreads antibiotic resistance in pathogenic bacteria and is mobilized by a heterobivalent recombinase (Tn916Int), whose activity is regulated by an excisionase factor (Tn916Xis). Unlike the well-characterized (lambda)Xis excisionase from bacteriophage lambda, Tn916Xis stimulates excision in vitro and in Escherichia coli only modestly. To gain insights into this functional difference, we have performed in vitro DNA-binding studies of Tn916Xis and Tn916Int, and we have solved the solution structure of Tn916Xis. We show that the heterobivalent Tn916Int protein is capable of bridging the DR2-type and core-type sites on the left arm of the tranpsoson. Consistent with the notion that Tn916Int is regulated only loosely, we find that Tn916Xis binding does not alter the stability of DR2-Tn916Int-core bridges or the ability of Tn916Int to recognize the arms of the transposon in vitro. Despite a high degree of divergence at the primary sequence level, we show that Tn916Xis and (lambda)Xis adopt related prokaryotic winged-helix structures. However, they differ at their C termini, with Tn916Xis replacing the flexible integrase contacting tail found in (lambda)Xis with a positively charged alpha-helix. This difference provides a structural explanation for why Tn916Xis does not interact cooperatively with its cognate integrase in vitro, and reveals how subtle changes in the winged-helix fold can modulate the functional properties of excisionase factors.
KeywordMeSH Terms
Conjugation, Genetic
DNA Transposable Elements
Protein Structure, Tertiary
76. Tanimoto  K, Nomura  T, Hamatani  H, Xiao  YH, Ike  Y,     ( 2005 )

A vancomycin-dependent VanA-type Enterococcus faecalis strain isolated in Japan from chicken imported from China.

Letters in applied microbiology 41 (2)
PMID : 16033514  :   DOI  :   10.1111/j.1472-765X.2005.01722.x    
Abstract >>
The characterization of KC122.1, which is a vancomycin-dependent VRE (Vancomycin-resistant enterococci) (Enterococcus faecalis) and the first case in Japan of a VRE isolate obtained from chicken meat imported from China. PCR amplification of vanA, vanS and ddl gene and direct sequencing of the PCR products were performed. KC122.1 was a VanA-type VRE showing high-level vancomycin resistance and low-level teicoplanin resistance, and its vanS gene had three point mutations. The ddl gene of KC122.1 was sequenced and two changes were found at the ninth codon (GCC-GAC) and the stop codon (TAA-CAA). The latter change was also found in the laboratory strain E. faecalis FA2-2. Three point mutations in vanS resulted in high-level vancomycin resistance and low-level teicoplanin resistance. The change at the ninth codon resulted in the inactivation of the ddl gene and vancomycin-dependent growth. An eight amino acid extension at the C-terminal did not impair the function of the D-Ala : D-Ala ligase. This is the first example of the isolation of VRE from chicken meat imported from China and the first vancomycin-dependent VRE from a nonhuman source.
KeywordMeSH Terms
77. Hirt  H, Manias  DA, Bryan  EM, Klein  JR, Marklund  JK, Staddon  JH, Paustian  ML, Kapur  V, Dunny  GM,     ( 2005 )

Characterization of the pheromone response of the Enterococcus faecalis conjugative plasmid pCF10: complete sequence and comparative analysis of the transcriptional and phenotypic responses of pCF10-containing cells to pheromone induction.

Journal of bacteriology 187 (3)
PMID : 15659682  :   DOI  :   10.1128/JB.187.3.1044-1054.2005     PMC  :   PMC545727    
Abstract >>
The sex pheromone plasmids in Enterococcus faecalis are one of the most efficient conjugative plasmid transfer systems known in bacteria. Plasmid transfer rates can reach or exceed 10(-1) transconjugants per donor in vivo and under laboratory conditions. We report the completion of the DNA sequence of plasmid pCF10 and the analysis of the transcription profile of plasmid genes, relative to conjugative transfer ability following pheromone induction. These experiments employed a mini-microarray containing all 57 open reading frames of pCF10 and a set of selected chromosomal genes. A clear peak of transcription activity was observed 30 to 60 min after pheromone addition, with transcription subsiding 2 h after pheromone induction. The transcript activity correlated with the ability of donor cells to transfer pCF10 to recipient cells. Remarkably, aggregation substance (Asc10, encoded by the prgB gene) was present on the cell surface for a long period of time after pheromone-induced transcription of prgB and plasmid transfer ability had ceased. This observation could have relevance for the virulence of E. faecalis.
KeywordMeSH Terms
78. Weidlich  G, Wirth  R, Galli  D,     ( 1992 )

Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis.

Molecular & general genetics : MGG 233 (1��2��)
PMID : 1603060  :   DOI  :   10.1007/bf00587575    
Abstract >>
During conjugative transfer of sex pheromone plasmids of Enterococcus faecalis a so-called surface exclusion protein reduces the frequency with which these plasmids are transferred to cells already possessing the same plasmid. We report here the DNA sequence of a 3.8 kb fragment of the sex pheromone plasmid pAD1 containing the structural gene sea1 for surface exclusion protein and a small open reading frame (ORF) upstream of sea1. Surface exclusion protein Sea1 was found to be highly homologous to the surface exclusion protein Sec10 encoded by the sex pheromone plasmid pCF10. Hybridization studies with DNA probes derived from the structural gene sea1 demonstrated that, with the exception of pAM373, all known sex pheromone plasmids carry a homologous gene. These studies also indicated that the genetic organization is similar in these plasmids, with the structural gene for surface exclusion protein being located 5' to that for aggregation substance.
KeywordMeSH Terms
Plasmids
79. Sprincova  A, Stovcik  V, Javorsky  P, Pristas  P,     ( 2005 )

Occurrence of pS86/pEF47-related plasmids in gram-positive cocci.

Current microbiology 51 (3)
PMID : 16091849  :   DOI  :   10.1007/s00284-005-4564-z    
Abstract >>
A small, low-copy-number cryptic 5.5-kb plasmid, designated pEF47, was isolated from a rumen bacterium Enterococcus faecalis 47 and partially characterized. Comparisons by restriction and partial sequence analysis of pEF47 demonstrated high homology to pS86 plasmid, recently isolated from a human clinical strain E. faecalis S-86. PCR followed by sequence analysis and Southern hybridization showed that pS86/pEF47-related replicons are regularly encountered in plasmids from gram-positive cocci.
KeywordMeSH Terms
80. Benachour  A, Muller  C, Dabrowski-Coton  M, Le Breton  Y, Giard  JC, Rincé  A, Auffray  Y, Hartke  A,     ( 2005 )

The Enterococcus faecalis sigV protein is an extracytoplasmic function sigma factor contributing to survival following heat, acid, and ethanol treatments.

Journal of bacteriology 187 (3)
PMID : 15659680  :   DOI  :   10.1128/JB.187.3.1022-1035.2005     PMC  :   PMC545719    
Abstract >>
Analysis of the genome sequence of Enterococcus faecalis allowed the identification of two genes whose protein products showed 33 and 34% identity with those of sigV and yrhM of Bacillus subtilis, respectively. These genes, named sigV and rsiV, are predicted to encode members of the extracytoplasmic function subfamily of eubacterial RNA polymerase sigma and anti-sigma factors, respectively. This group of sigma factors has been shown to regulate gene expression in response to stress conditions. sigV and rsiV were shown to be under the control of the same promoter. The transcriptional start site was determined, and the 1.5-kb mRNA transcript was shown to be overexpressed under glucose and complete starvation, as well as under physicochemical treatments. Three mutants, affected in sigV, rsiV, and both genes, were constructed by double-crossover recombination within the genome of E. faecalis strain JH2-2. Compared with the wild type and the rsiV mutant, the sigV mutants were more susceptible to heat shock, acid, and ethanol treatments and displayed decreased survival during long-term starvation. A nisin-inducible sigV gene construction used in complementation assays restored the wild phenotype of the sigV mutants, confirming the involvement of SigV in the heat shock, ethanol, and acid stress responses. Northern blot analysis carried out with the three mutant strains revealed the inhibition of sigV expression by the related anti-sigma factor gene rsiV. In addition, putative candidates of the sigV regulon determined by computer search for the sigV promoter sequence were analyzed.
KeywordMeSH Terms
81. Singh  KV, Murray  BE,     ( 2005 )

Differences in the Enterococcus faecalis lsa locus that influence susceptibility to quinupristin-dalfopristin and clindamycin.

Antimicrobial agents and chemotherapy 49 (1)
PMID : 15616272  :   DOI  :   10.1128/AAC.49.1.32-39.2005     PMC  :   PMC538898    
Abstract >>
We have previously shown that the Enterococcus faecalis lsa gene, encoding the putative ABC protein Lsa, influences resistance to quinupristin-dalfopristin (Q-D) and clindamycin (CLI). We have now found that, while cloned lsa from E. faecalis strain V583 (lsa(V)) fully restored resistance to Q-D, CLI, and dalfopristin (DAL) lost by the OG1 lsa disruption mutant TX5332 and also caused increased MICs for Lactococcus lactis LM2301, cloned lsa from OG1 (lsa(OG)) did not cause any increase in MICs for either species. Sequencing of ca. 2 kb of these two lsa alleles found differences between lsa(OG) and lsa(V) in the upstream region as well as in the 5' and 3' halves of the lsa gene. To investigate the reason for the phenotypic differences expressed by the two cloned loci, 5' half plus 3' half hybrid constructs were created. When introduced into both TX5332 and L. lactis, cloned lsa(V5)(')(OG3)(') conferred increases in MICs of Q-D, CLI, and DAL similar to those of cloned lsa(V) while cloned lsa(OG5)(')(V3)(') showed a moderate increase in MICs relative to those of lsa(OG), indicating that both halves of the locus can influence resistance expression. After site-directed mutagenesis of the cloned lsa alleles at positions -131 and -133 (relative to the putative Lsa start codon ATG), which converted two A's of lsa(V) to the G and T of lsa(OG) and vice versa, MIC testing showed that mutagenized lsa(OG) (lsa(OG-M)) was strongly influenced by these changes in terms of conferring increased MICs of Q-D, CLI, and DAL relative to lsa(OG) while the phenotype of mutagenized lsa(V) (lsa(V-M)) was less influenced, with moderately decreased MICs, primarily to CLI, relative to lsa(V). In conclusion, this study found that changes in different regions of the E. faecalis lsa locus influence the ability of cloned lsa to confer resistance to Q-D, CLI, and DAL.
KeywordMeSH Terms
Mutation
82. Jauhangeer  BR, Wren  MW, MacDonald  RA, Perry  D, Greenwell  R,     ( 2004 )

Use of bioinformatics and PCR in the search for ABC transporter homology among various bacteria.

British journal of biomedical science 61 (4)
PMID : 15649009  :  
Abstract >>
Bioinformatics databases and search tools are utilised to produce polymerase chain reaction (PCR) primers for the amplification of an ABC transporter gene from the clinically important anaerobe Finegoldia magna. On sequencing, a 450 base pair amplicon showed homology with the amino acid transporter of Enterococcus faecalis. Little sequence data is available for F. magna and the newly isolated DNA could be a useful tool in the identification of this organism in clinical specimens.
KeywordMeSH Terms
83. Tsai  JC, Hsueh  PR, Lin  HM, Chang  HJ, Ho  SW, Teng  LJ,     ( 2005 )

Identification of clinically relevant enterococcus species by direct sequencing of groES and spacer region.

Journal of clinical microbiology 43 (1)
PMID : 15634977  :   DOI  :   10.1128/JCM.43.1.235-241.2005     PMC  :   PMC540105    
Abstract >>
We determined the groESL sequences (groES, groEL, and the intergenic spacer) of 10 clinically relevant Enterococcus species and evaluated the feasibility of identifying Enterococcus species on the basis of these sequences. Seven common clinical Enterococcus species, E. faecalis, E. faecium, E. casseliflavus, E. gallinarum, E. avium, E. raffinosus, and E. hirae, and three less common Enterococcus species, E. cecorum, E. durans, and E. mundtii, were examined in this study. We found that the groES genes of these enterococcal species are identical in length (285 nucleotides) and contain an unusual putative start codon, GTG. The lengths and sequences of the intergenic regions (spacers between the groES and groEL genes) are quite variable (17 to 57 bp in length) among Enterococcus species but are conserved in strains within each species, with only a few exceptions. Considerable variation of groES or groEL sequences was also observed. The evolutionary trees of groES or groEL sequences revealed similarities among Enterococcus species. However, the overall intraspecies variation of groES was less than that of groEL. The high interspecies variation and low intraspecies variation indicate that the groES and spacer sequences are more useful than groEL for identification of clinically relevant Enterococcus species. The sequences of these two genetic traits, groES and spacer, can be determined by a single PCR and direct sequencing and may provide important information for the differentiation of closely related species of Enterococcus.
KeywordMeSH Terms
Bacterial Typing Techniques
Sequence Analysis, DNA
84. Jiang  Y, Cronan  JE,     ( 2005 )

Expression cloning and demonstration of Enterococcus faecalis lipoamidase (pyruvate dehydrogenase inactivase) as a Ser-Ser-Lys triad amidohydrolase.

The Journal of biological chemistry 280 (3)
PMID : 15528186  :   DOI  :   10.1074/jbc.M408612200    
Abstract >>
Enterococcus faecalis lipoamidase was discovered almost 50 years ago (Reed, L. J., Koike, M., Levitch, M. E., and Leach, F. R. (1958) J. Biol. Chem. 232, 143-158) as an enzyme activity that cleaved lipoic acid from small lipoylated molecules and from pyruvate dehydrogenase thereby inactivating the enzyme. Although the partially purified enzyme was a key reagent in proving the crucial role of protein-bound lipoic acid in the reaction mechanism of the 2-oxoacid dehydrogenases, the identity of the lipoamidase protein and the encoding gene remained unknown. We report isolation of the lipoamidase gene by screening an expression library made in an unusual cosmid vector in which the copy number of the vector is readily varied from 1-2 to 40-80 in an appropriate Escherichia coli host. Although designed for manipulation of large genome segments, the vector was also ideally suited to isolation of the gene encoding the extremely toxic lipoamidase. The gene encoding lipoamidase was isolated by screening for expression in E. coli and proved to encode an unexpectedly large protein (80 kDa) that contained the sequence signature of the Ser-Ser-Lys triad amidohydrolase family. The hexa-histidine-tagged protein was expressed in E. coli and purified to near-homogeneity. The purified enzyme was found to cleave both small molecule lipoylated and biotinylated substrates as well as lipoic acid from two 2-oxoacid dehydrogenases and an isolated lipoylated lipoyl domain derived from the pyruvate dehydrogenase E2 subunit. Lipoamidase-mediated inactivation of the 2-oxoacid dehydrogenases was observed both in vivo and in vitro. Mutagenesis studies showed that the residues of the Ser-Ser-Lys triad were required for activity on both small molecule and protein substrates and confirmed that lipoamidase is a member of the Ser-Ser-Lys triad amidohydrolase family.
KeywordMeSH Terms
85. Rumpel  S, Razeto  A, Pillar  CM, Vijayan  V, Taylor  A, Giller  K, Gilmore  MS, Becker  S, Zweckstetter  M,     ( 2004 )

Structure and DNA-binding properties of the cytolysin regulator CylR2 from Enterococcus faecalis.

The EMBO journal 23 (18)
PMID : 15359276  :   DOI  :   10.1038/sj.emboj.7600367     PMC  :   PMC517608    
Abstract >>
Enterococcus faecalis is one of the major causes for hospital-acquired antibiotic-resistant infections. It produces an exotoxin, called cytolysin, which is lethal for a wide range of Gram-positive bacteria and is toxic to higher organisms. Recently, the regulation of the cytolysin operon was connected to autoinduction by a quorum-sensing mechanism involving the CylR1/CylR2 two-component regulatory system. We report here the crystal structure of CylR2 and its properties in solution as determined by heteronuclear NMR spectroscopy. The structure reveals a rigid dimer containing a helix-turn-helix DNA-binding motif as part of a five-helix bundle that is extended by an antiparallel beta-sheet. We show that CylR2 is a DNA-binding protein that binds specifically to a 22 bp fragment of the cytolysin promoter region. NMR chemical shift perturbation experiments identify surfaces involved in DNA binding and are in agreement with a model for the CylR2/DNA complex that attributes binding specificity to a complex network of CylR2/DNA interactions. Our results propose a mechanism where repression is achieved by CylR2 obstruction of the promoter preventing biosynthesis of the cytolysin operon transcript.
KeywordMeSH Terms
86. Abadía-Patiño  L, Christiansen  K, Bell  J, Courvalin  P, Périchon  B,     ( 2004 )

VanE-type vancomycin-resistant Enterococcus faecalis clinical isolates from Australia.

Antimicrobial agents and chemotherapy 48 (12)
PMID : 15561872  :   DOI  :   10.1128/AAC.48.12.4882-4885.2004     PMC  :   PMC529234    
Abstract >>
Three distinct Enterococcus faecalis VanE-type isolates-BM4574, BM4575, and BM4576-obtained in Australia were studied. Expression of the resistance genes was constitutive in BM4575, probably due to a 2-bp deletion into the vanSE gene, and inducible in BM4574 and BM4576. Transcription analysis of the vanE operons suggested that the five genes were cotranscribed from an initiation site located 25 bp upstream from the ATG start codon of vanE.
KeywordMeSH Terms
87. Manson  JM, Keis  S, Smith  JM, Cook  GM,     ( 2004 )

Acquired bacitracin resistance in Enterococcus faecalis is mediated by an ABC transporter and a novel regulatory protein, BcrR.

Antimicrobial agents and chemotherapy 48 (10)
PMID : 15388429  :   DOI  :   10.1128/AAC.48.10.3743-3748.2004     PMC  :   PMC521867    
Abstract >>
Bacitracin resistance (bacitracin MIC, >/=256 microg ml(-1)) has been reported in Enterococcus faecalis, and in the present study we report on the genetic basis for this resistance. Mutagenesis was carried out with transposon Tn917 to select for E. faecalis mutants with decreased resistance to bacitracin. Two bacitracin-sensitive mutants (MICs, 32 microg ml(-1)) were obtained and Tn917 insertions were mapped to genes designated bcrA and bcrB. The amino acid sequences of BcrA (ATP-binding domain) and BrcB (membrane-spanning domain) are predicted to constitute a homodimeric ATP-binding cassette (ABC) transporter, the function of which is essential for bacitracin resistance in E. faecalis. The bcrA and bcrB genes were organized in an operon with a third gene, bcrD, that had homology to undecaprenol kinases. Northern analysis demonstrated that bcrA, bcrB, and bcrD were transcribed as a polycistronic message that was induced by increasing concentrations of bacitracin but not by other cell wall-active antimicrobials (e.g., vancomycin). Upstream of the bcrABD operon was a putative regulatory gene, bcrR. The bcrR gene was expressed constitutively, and deletion of bcrR resulted in a bacitracin-sensitive phenotype. No bcrABD expression was observed in a bcrR mutant, suggesting that BcrR is an activator of genes essential for bacitracin resistance (i.e., bcrABD). The bacitracin resistance genes were found to be located on a plasmid that transferred at a high frequency to E. faecalis strain JH2-2. This report represents the first description of genes that are essential for acquired bacitracin resistance in E. faecalis.
KeywordMeSH Terms
88. Hill  JE, Penny  SL, Crowell  KG, Goh  SH, Hemmingsen  SM,     ( 2004 )

cpnDB: a chaperonin sequence database.

Genome research 14 (8)
PMID : 15289485  :   DOI  :   10.1101/gr.2649204     PMC  :   PMC509277    
Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
KeywordMeSH Terms
89. Novais  C, Coque  TM, Sousa  JC, Baquero  F, Peixe  L, N/A  N/A,     ( 2004 )

Local genetic patterns within a vancomycin-resistant Enterococcus faecalis clone isolated in three hospitals in Portugal.

Antimicrobial agents and chemotherapy 48 (9)
PMID : 15328141  :   DOI  :   10.1128/AAC.48.9.3613-3617.2004     PMC  :   PMC514734    
Abstract >>
Eight pulsed-field gel electrophoresis subtypes and six Tn1546 variants were identified among Enterococcus faecalis isolates of a single clone recovered in three geographically separate Portuguese hospitals. Some clonal subtypes were found in particular hospitals, and Tn1546 variants were either widespread or confined to some of them. We also report on the first Tn1546 transposon containing an ISEf1 insertion.
KeywordMeSH Terms
90. Mohamed  JA, Huang  W, Nallapareddy  SR, Teng  F, Murray  BE,     ( 2004 )

Influence of origin of isolates, especially endocarditis isolates, and various genes on biofilm formation by Enterococcus faecalis.

Infection and immunity 72 (6)
PMID : 15155680  :   DOI  :   10.1128/IAI.72.6.3658-3663.2004     PMC  :   PMC415661    
Abstract >>
Endocarditis isolates of Enterococcus faecalis produced biofilm significantly more often than nonendocarditis isolates, and 39% of 79 versus 6% of 84 isolates produced strong biofilm (P < 0.0001). esp was not required, but its presence was associated with higher amounts of biofilm (P < 0.001). Mutants disrupted in dltA, efaA, ace, lsa, and six two-component regulatory systems were largely unaltered, while disruptions in epa (encoding enterococcal polysaccharide antigen), atn (encoding an autolysin), gelE (encoding gelatinase), and fsr (encoding the E. faecalis regulator) [corrected] resulted in fewer attached bacteria, as determined using phase-contrast microscopy, and less biofilm (P < 0.0001).
KeywordMeSH Terms
Biofilms
Gene Expression Regulation, Bacterial
91. Verneuil  N, Sanguinetti  M, Le Breton  Y, Posteraro  B, Fadda  G, Auffray  Y, Hartke  A, Giard  JC,     ( 2004 )

Effects of the Enterococcus faecalis hypR gene encoding a new transcriptional regulator on oxidative stress response and intracellular survival within macrophages.

Infection and immunity 72 (8)
PMID : 15271899  :   DOI  :   10.1128/IAI.72.8.4424-4431.2004     PMC  :   PMC470598    
Abstract >>
In order to identify regulators of the oxidative stress response in Enterococcus faecalis, an important human pathogen, several genes annotated as coding for transcriptional regulators were inactivated by insertional mutagenesis. One mutant, affected in the ef2958 locus (designated hypR [hydrogen peroxide regulator]), appeared to be highly sensitive to oxidative challenge caused by hydrogen peroxide. Moreover, testing of the hypR mutant by using an in vivo-in vitro macrophage infection model resulted in a highly significant reduction in survival compared to the survival of parent strain JH2-2. Northern blot analyses were carried out with probes specific for genes encoding known antioxidant enzymes, and they showed that the ahpCF (alkyl hydroperoxide reductase) transcript was expressed less in mutant cells. Mobility shift protein-DNA binding assays revealed that HypR regulated directly the expression of hypR itself and the ahpCF operon. Our combined results showed that HypR appeared to be directly involved in the expression of ahpCF genes under oxidative stress conditions and suggested that this regulator could contribute to the virulence of E. faecalis.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Heat-Shock Response
Oxidative Stress
Transcription, Genetic
92. Staddon  JH, Bryan  EM, Manias  DA, Dunny  GM,     ( 2004 )

Conserved target for group II intron insertion in relaxase genes of conjugative elements of gram-positive bacteria.

Journal of bacteriology 186 (8)
PMID : 15060042  :   DOI  :   10.1128/jb.186.8.2393-2401.2004     PMC  :   PMC412114    
Abstract >>
The lactococcal group II intron Ll.ltrB interrupts the ltrB relaxase gene within a region that encodes a conserved functional domain. Nucleotides essential for the homing of Ll.ltrB into an intronless version of ltrB are found exclusively at positions required to encode amino acids broadly conserved in a family of relaxase proteins of gram-positive bacteria. Two of these relaxase genes, pcfG from the enterococcal plasmid pCF10 and the ORF4 gene in the streptococcal conjugative transposon Tn5252, were shown to support Ll.ltrB insertion into the conserved motif at precisely the site predicted by sequence homology with ltrB. Insertion occurred through a mechanism indistinguishable from retrohoming. Splicing and retention of conjugative function was demonstrated for pCF10 derivatives containing intron insertions. Ll.ltrB targeting of a conserved motif of a conjugative element suggests a mechanism for group II intron dispersal among bacteria. Additional support for this mechanism comes from sequence analysis of the insertion sites of the E.c.I4 family of bacterial group II introns.
KeywordMeSH Terms
Conjugation, Genetic
Genes, Bacterial
93. Johansson  A, Greko  C, Engström  BE, Karlsson  M,     ( 2004 )

Antimicrobial susceptibility of Swedish, Norwegian and Danish isolates of Clostridium perfringens from poultry, and distribution of tetracycline resistance genes.

Veterinary microbiology 99 (3��4��)
PMID : 15066727  :   DOI  :   10.1016/j.vetmic.2004.01.009    
Abstract >>
This study was undertaken to determine the in vitro susceptibility of Clostridium perfringens, isolated from poultry to antimicrobials used in poultry production. The minimal inhibitory concentration (MIC) of eight antimicrobials, including the ionophoric coccidiostat narasin, was determined for 102 C. perfringens isolates, 58 from Sweden, 24 from Norway and 20 from Denmark. Susceptibility to each antimicrobial compound was determined by broth microdilution. The isolates were obtained from broilers (89), laying hens (9) and turkeys (4), affected by necrotic enteritis (NE) or by C. perfringens associated hepatitis (CPH), and from healthy broilers. All strains, regardless of origin, proved inherently susceptible to ampicillin, narasin, avilamycin, erythromycin and vancomycin. A low frequency of resistance to virginiamycin and bacitracin was also found. Resistance to tetracycline was found in strains isolated in all three countries; Sweden (76%), Denmark (10%) and Norway (29%). In 80% of the tetracycline-resistant isolates, the two resistance genes tetA(P) and tetB(P) were amplified by PCR whereas in 20% only the tetA(P) gene was detected. No tetM gene amplicon was obtained from any of the tetracycline-resistant isolates. The uniform susceptibility to narasin revealed in this study shows that the substance can still be used to control clostridiosis. In this study, C. perfringens also showed a low degree of resistance to most other antimicrobials tested. Despite the small amounts of tetracycline used in poultry, a considerable degree of resistance to tetracycline was found in C. perfringens isolates from Swedish broilers.
KeywordMeSH Terms
Chickens
Turkeys
94. Collin  M, Fischetti  VA,     ( 2004 )

A novel secreted endoglycosidase from Enterococcus faecalis with activity on human immunoglobulin G and ribonuclease B.

The Journal of biological chemistry 279 (21)
PMID : 15028731  :   DOI  :   10.1074/jbc.M402156200    
Abstract >>
The human pathogen Enterococcus faecalis can degrade the N-linked glycans of human RNase B to acquire nutrients, but no gene or protein has been associated with this activity. We identified an 88-kDa secreted protein, endoglycosidase (Endo) E, which is most likely responsible for this activity. EndoE, encoded by ndoE, consists of an alpha-domain with a family 18 glycosyl hydrolase motif and a beta-domain similar to family 20 glycosyl hydrolases. Phylogenetic analysis of EndoE indicates that the alpha-domain is related to human chitobiases, and the beta-domain is related to bacterial and human hexosaminidases. Recombinant expression of full-length EndoE or EndoEalpha, site-directed mutagenesis of the catalytic residues, mass spectroscopy, and homology modeling shows that EndoEalpha hydrolyzes the glycan on human RNase B, whereas EndoEbeta hydrolyzes the conserved glycan on IgG. Denaturation experiments indicate that the chitinase activity on RNase B is not dependent on the tertiary structure, although it is on IgG. The ndoE gene and secreted EndoE are present in most E. faecalis but not in Enterococcus faecium isolates. Correspondingly, E. faecalis, but not E. faecium, degrades the glycan on RNase B during growth. Thus, we have identified a secreted enzyme from E. faecalis, EndoE, which by two distinct activities hydrolyzes the glycans on RNase B and IgG. Both activities could be important for the molecular pathogenesis and persistence of E. faecalis during human infections.
KeywordMeSH Terms
95. Duez  C, Hallut  S, Rhazi  N, Hubert  S, Amoroso  A, Bouillenne  F, Piette  A, Coyette  J,     ( 2004 )

The ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class A penicillin-binding protein.

Journal of bacteriology 186 (13)
PMID : 15205448  :   DOI  :   10.1128/JB.186.13.4412-4416.2004     PMC  :   PMC421628    
Abstract >>
A soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli 14C-labeled lipid II precursor. As a DD- peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k2/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
Penicillin-Binding Proteins
96. Drancourt  M, Roux  V, Fournier  PE, Raoult  D,     ( 2004 )

rpoB gene sequence-based identification of aerobic Gram-positive cocci of the genera Streptococcus, Enterococcus, Gemella, Abiotrophia, and Granulicatella.

Journal of clinical microbiology 42 (2)
PMID : 14766807  :   DOI  :   10.1128/jcm.42.2.497-504.2004     PMC  :   PMC344509    
Abstract >>
We developed a new molecular tool based on rpoB gene (encoding the beta subunit of RNA polymerase) sequencing to identify streptococci. We first sequenced the complete rpoB gene for Streptococcus anginosus, S. equinus, and Abiotrophia defectiva. Sequences were aligned with these of S. pyogenes, S. agalactiae, and S. pneumoniae available in GenBank. Using an in-house analysis program (SVARAP), we identified a 740-bp variable region surrounded by conserved, 20-bp zones and, by using these conserved zones as PCR primer targets, we amplified and sequenced this variable region in an additional 30 Streptococcus, Enterococcus, Gemella, Granulicatella, and Abiotrophia species. This region exhibited 71.2 to 99.3% interspecies homology. We therefore applied our identification system by PCR amplification and sequencing to a collection of 102 streptococci and 60 bacterial isolates belonging to other genera. Amplicons were obtained in streptococci and Bacillus cereus, and sequencing allowed us to make a correct identification of streptococci. Molecular signatures were determined for the discrimination of closely related species within the S. pneumoniae-S. oralis-S. mitis group and the S. agalactiae-S. difficile group. These signatures allowed us to design a S. pneumoniae-specific PCR and sequencing primer pair.
KeywordMeSH Terms
97. Huys  G, D'Haene  K, Collard  JM, Swings  J,     ( 2004 )

Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food.

Applied and environmental microbiology 70 (3)
PMID : 15006778  :   DOI  :   10.1128/aem.70.3.1555-1562.2004     PMC  :   PMC368340    
Abstract >>
In the present study, a collection of 187 Enterococcus food isolates mainly originating from European cheeses were studied for the phenotypic and genotypic assessment of tetracycline (TC) resistance. A total of 45 isolates (24%) encompassing the species Enterococcus faecalis (n = 33), E. durans (n = 7), E. faecium (n = 3), E. casseliflavus (n = 1), and E. gallinarum (n = 1) displayed phenotypic resistance to TC with MIC ranges of 16 to 256 microg/ml. Eight of these strains exhibited multiresistance to TC, erythromycin, and chloramphenicol. By PCR detection, TC resistance could be linked to the presence of the tet(M) (n = 43), tet(L) (n = 16), and tet(S) (n = 1) genes. In 15 isolates, including all of those for which the MIC was 256 micro g/ml, both tet(M) and tet(L) were found. Furthermore, all tet(M)-containing enterococci also harbored a member of the Tn916-Tn1545 conjugative transposon family, of which 12 erythromycin-resistant isolates also contained the erm(B) gene. Filter mating experiments revealed that 10 E. faecalis isolates, 3 E. durans isolates, and 1 E. faecium isolate could transfer either tet(M), tet(L), or both of these genes to E. faecalis recipient strain JH2-2. In most cases in which only tet(M) was transferred, no detectable plasmids were acquired by JH2-2 but instead all transconjugants contained a member of the Tn916-Tn1545 family. Sequencing analysis of PCR amplicons and evolutionary modeling showed that a subset of the transferable tet(M) genes belonged to four sequence homology groups (SHGs) showing an internal homology of > or = 99.6%. Two of these SHGs contained tet(M) mosaic structures previously found in Tn916 elements and on Lactobacillus and Neisseria plasmids, respectively, whereas the other two SHGs probably represent new phylogenetic lineages of this gene.
KeywordMeSH Terms
Food Microbiology
Tetracycline Resistance
98. Gueneau de Novoa  P, Williams  KP,     ( 2004 )

The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts.

Nucleic acids research 32 (Database issue)
PMID : 14681369  :   DOI  :   10.1093/nar/gkh102     PMC  :   PMC308836    
Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
KeywordMeSH Terms
Databases, Nucleic Acid
Evolution, Molecular
Internet
99. Richer  E, Courville  P, Bergevin  I, Cellier  MF,     ( 2003 )

Horizontal gene transfer of "prototype" Nramp in bacteria.

Journal of molecular evolution 57 (4)
PMID : 14708570  :   DOI  :   10.1007/s00239-003-2472-z    
Abstract >>
Eukaryotic Nramp genes encode divalent metal ion permeases important for nutrition and resistance to microbial infection. Bacterial homologs encode proton-dependent transporters of manganese (MntH), and other divalent metal ions. Bacterial MntH were classified in three homology groups (A, B, C) and MntH C further subdivided in Calpha, Cbeta, Cgamma. The proteins from C. tepidum (MntH B) and E. faecalis (MntH Cbeta1, 2), divergent in sequence and hydropathy profile, conferred increased metal sensitivity when expressed in E. coli, suggesting conservation of divalent metal transport function in MntH B and C. Several genomic evidence suggest horizontal gene transfer (HGT) of mntH C genes: (i) The enterobacteria Wigglesworthia mntH Cbeta gene is linked to an Asn t-RNA, and its sequence most conserved with Gram positive bacteria homologs; (ii) all the Cbeta genes identified in oral streptococcaceae are associated with different potentially mobile DNA elements; (iii) Lactococcus lactis and Burkholderia mallei genomes contain an mntH gene prematurely terminated and a novel full-length mntH C gene; (iv) remarkable sequence relatedness between the unicellular alga C. reinhardtii "prototype" Nramp and some MntH Calpha (e.g., Nostoc spp., Listeria spp.) suggests HGT between Eukarya and Bacteria. Other "prototype" Nramp genes (intronless, encoding proteins strongly conserved with MntH A and B proteins) identified in invertebrates represent a possible source for transfer of Nramp genes toward opportunistic bacteria. This study demonstrates complex evolution of MntH in Bacteria. It is proposed that "prototype" Nramp are ancestors of bacterial MntH C proteins, which could facilitate bacterial infection.
KeywordMeSH Terms
Bacterial Proteins
Escherichia coli Proteins
100. Cobbe  N, Heck  MM,     ( 2004 )

The evolution of SMC proteins: phylogenetic analysis and structural implications.

Molecular biology and evolution 21 (2)
PMID : 14660695  :   DOI  :   10.1093/molbev/msh023    
Abstract >>
The SMC proteins are found in nearly all living organisms examined, where they play crucial roles in mitotic chromosome dynamics, regulation of gene expression, and DNA repair. We have explored the phylogenetic relationships of SMC proteins from prokaryotes and eukaryotes, as well as their relationship to similar ABC ATPases, using maximum-likelihood analyses. We have also investigated the coevolution of different domains of eukaryotic SMC proteins and attempted to account for the evolutionary patterns we have observed in terms of available structural data. Based on our analyses, we propose that each of the six eukaryotic SMC subfamilies originated through a series of ancient gene duplication events, with the condensins evolving more rapidly than the cohesins. In addition, we show that the SMC5 and SMC6 subfamily members have evolved comparatively rapidly and suggest that these proteins may perform redundant functions in higher eukaryotes. Finally, we propose a possible structure for the SMC5/SMC6 heterodimer based on patterns of coevolution.
KeywordMeSH Terms
Evolution, Molecular
Phylogeny
101. Sánchez-Barrena  MJ, Martínez-Ripoll  M, Gálvez  A, Valdivia  E, Maqueda  M, Cruz  V, Albert  A,     ( 2003 )

Structure of bacteriocin AS-48: from soluble state to membrane bound state.

Journal of molecular biology 334 (3)
PMID : 14623193  :   DOI  :   10.1016/j.jmb.2003.09.060    
Abstract >>
The bacteriocin AS-48 is a membrane-interacting peptide, which displays a broad anti-microbial spectrum against Gram-positive and Gram-negative bacteria. The NMR structure of AS-48 at pH 3 has been solved. The analysis of this structure suggests that the mechanism of AS-48 anti-bacterial activity involves the accumulation of positively charged molecules at the membrane surface leading to a disruption of the membrane potential. Here, we report the high-resolution crystal structure of AS-48 and sedimentation equilibrium experiments showing that this bacteriocin is able to adopt different oligomeric structures according to the physicochemical environment. The analysis of these structures suggests a mechanism for molecular function of AS-48 involving a transition from a water-soluble form to a membrane-bound state upon membrane binding.
KeywordMeSH Terms
Bacterial Proteins
Protein Conformation
102. Depardieu  F, Bonora  MG, Reynolds  PE, Courvalin  P,     ( 2003 )

The vanG glycopeptide resistance operon from Enterococcus faecalis revisited.

Molecular microbiology 50 (3)
PMID : 14617152  :   DOI  :   10.1046/j.1365-2958.2003.03737.x    
Abstract >>
Acquired VanG-type resistance to vancomycin (MIC = 16 micro g ml(-1)) but susceptibility to teicoplanin in Enterococcus faecalis BM4518 and WCH9 is due to the inducible synthesis of peptidoglycan precursors ending in d-alanine-d-serine. The vanG cluster, assigned to a chromosomal location, was composed of genes recruited from various van operons. The 3' end encoded VanG, a d-Ala:d-Ser ligase, VanXY(G), a putative bifunctional d,d-peptidase and VanT(G), a serine racemase: VanG and VanT(G) were implicated in the synthesis of d-Ala:d-Ser as in VanC- and VanE-type strains. Upstream from the structural genes for these proteins were vanW(G) with unknown function and vanY(G) containing a frameshift mutation which resulted in premature termination of the encoded protein and accounted for the lack of UDP-MurNAc-tetrapeptide in the cytoplasm. Without the frameshift mutation, VanY(G) had homology with Zn2+ dependent d,d-carboxypeptidases. The 5' end of the gene cluster contained three genes vanU(G), vanR(G) and vanS(G) encoding a putative regulatory system, which were co-transcribed constitutively from the PY(G) promoter, whereas transcription of vanY(G),W(G),G,XY(G),T(G) was inducible and initiated from the P(YG) promoter. Transfer of VanG-type glycopeptide resistance to E. faecalis JH2-2 was associated with the movement, from chromosome to chromosome, of genetic elements of c. 240 kb carrying also ermB-encoded erythromycin resistance. Sequence determination of the flanking regions of the vanG cluster in donor and transconjugants revealed the same 4 bp direct repeats and 22 bp imperfect inverted repeats that delineated the large element.
KeywordMeSH Terms
Operon
Serine-Type D-Ala-D-Ala Carboxypeptidase
103. Burdett  V,     ( 1990 )

Nucleotide sequence of the tet(M) gene of Tn916.

Nucleic acids research 18 (20)
PMID : 2172929  :   DOI  :   10.1093/nar/18.20.6137     PMC  :   PMC332426    
Abstract >>
N/A
KeywordMeSH Terms
DNA Transposable Elements
Genes, Bacterial
104. Gilmore  MS, Segarra  RA, Booth  MC,     ( 1990 )

An HlyB-type function is required for expression of the Enterococcus faecalis hemolysin/bacteriocin.

Infection and immunity 58 (12)
PMID : 2123826  :   PMC  :   PMC313755    
Abstract >>
The nucleotide sequence of a 3,422-bp internal restriction fragment from the Enterococcus faecalis pAD1 hemolysin/bacteriocin-encoding region was determined. This fragment was associated with expression of hemolysin/bacteriocin component L and contained a 2,142-bp open reading frame. The inferred amino acid sequence revealed a protein which shared extensive similarity with HlyB of the Escherichia coli alpha-hemolysin operon. The inferred protein, CylB, was observed to be independently expressed in E. coli and capable of complementing an insertion mutation in the cloned hemolysin/bacteriocin operon in trans. Despite the extensive similarity to HlyB, CylB was incapable of complementing an insertion mutation in hlyB. Cytolysin determinants possessing an HlyB-type transport function are widely dispersed throughout gram-negative genera. We believe this to be the first example of an HlyB-type protein encoded within a cytolysin determinant from a gram-positive bacterium.
KeywordMeSH Terms
105. Nakayama  J, Nagasawa  H, Isogai  A, Clewell  DB, Suzuki  A,     ( 1990 )

Amino acid sequence of pheromone-inducible surface protein in Enterococcus faecalis, that is encoded on the conjugative plasmid pPD1.

FEBS letters 267 (1)
PMID : 2114324  :   DOI  :   10.1016/0014-5793(90)80293-r    
Abstract >>
The major pheromone-inducible protein, PD78, believed to contribute to bacterial conjugation, was purified from Enterococcus (formerly Streptococcus) faecalis cells containing the plasmid pPD1. A cloned EcoRI-BglII 3.6-kbp fragment of the plasmid pAM351 (pPD1::Tn916) contained an open reading frame corresponding to 467 amino acid residues representing PD78. In a central region of the deduced protein, there is a repeated sequence of X-X-Pro that is repeated 15 times. This is analogous to the Gln-Gln-Pro repeat in the C-terminal region of TraD product encoded on the R100 plasmid in Escherichia coli.
KeywordMeSH Terms
Conjugation, Genetic
106. Gruene  T, Cho  MK, Karyagina  I, Kim  HY, Grosse  C, Giller  K, Zweckstetter  M, Becker  S,     ( 2011 )

Integrated analysis of the conformation of a protein-linked spin label by crystallography, EPR and NMR spectroscopy.

Journal of biomolecular NMR 49 (2)
PMID : 21271275  :   DOI  :   10.1007/s10858-011-9471-y     PMC  :   PMC3042103    
Abstract >>
Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein-protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.
KeywordMeSH Terms
Spin Labels
107. Swinfield  TJ, Oultram  JD, Thompson  DE, Brehm  JK, Minton  NP,     ( 1990 )

Physical characterisation of the replication region of the Streptococcus faecalis plasmid pAM beta 1.

Gene 87 (1)
PMID : 2110101  :  
Abstract >>
The complete nucleotide (nt) sequence of a 5.1-kb EcoRI DNA restriction fragment carrying the replication region of the Streptococcus faecalis plasmid pAM beta 1 has been determined. Of the seven major open reading frames (ORF A-G) identified within this fragment, two (C and E) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that synthesis of the polypeptide (Mr 57,380) encoded by the largest ORF (E) was essential for replication. Deletion analysis indicated that the minimum unit of DNA required for replication resided on a 2.59-kb AccI-HpaI subfragment. ORF C resided outside of this fragment and encompassed an extensive region of directly repeated nt sequence. The encoded polypeptide (Mr 30,471) was therefore composed of large tracts of reiterated amino acid sequence (11 x VDP and 35 x TEP tripeptides) which probably caused the observed anomalous electrophoretic mobility of the synthesised protein (equivalent to 61 kDa). Deletion of a 416-bp segment of DNA between unique KpnI and StyI sites caused an increase in copy number, which correlated with the in vitro production of higher levels of ORF E polypeptide. Although homology was detected between the sequenced DNA, and the replicon of a closely related streptococcal plasmid (pSM19035), none was evident to any other characterised Gram+ plasmid.
KeywordMeSH Terms
DNA Replication
Plasmids
108. Brantl  S, Behnke  D, Alonso  JC,     ( 1990 )

Molecular analysis of the replication region of the conjugative Streptococcus agalactiae plasmid pIP501 in Bacillus subtilis. Comparison with plasmids pAM beta 1 and pSM19035.

Nucleic acids research 18 (16)
PMID : 2118624  :   DOI  :   10.1093/nar/18.16.4783     PMC  :   PMC331945    
Abstract >>
The large conjugative plasmid pIP501 was originally isolated from Streptococcus agalactiae. To study the molecular basis of pIP501 replication we determined the nucleotide sequence of a 2.2 kb DNA segment which is essential and sufficient for autonomous replication of pIP501 derived plasmids, in Bacillus subtilis cells. This region can be divided into two functionally discrete segments: a 496 bp region (oriR) that acts as an origin of replication, and a 1488 bp segment coding for an essential replication protein (RepR). The RepR protein, which has a molecular mass of 57.4 kDa, could complement in trans a thermosensitive replicon bearing the pIP501 origin. Chimeric Rep proteins and replicons were obtained by domain swapping between rep genes of closely related streptococcal plasmids belonging to the inc18 group (pIP501, pAM beta 1 and pSM19035). The chimeras were functional in B. subtilis.
KeywordMeSH Terms
DNA Replication
DNA-Binding Proteins
Plasmids
109. Zhu  W, Murray  PR, Huskins  WC, Jernigan  JA, McDonald  LC, Clark  NC, Anderson  KF, McDougal  LK, Hageman  JC, Olsen-Rasmussen  M, Frace  M, Alangaden  GJ, Chenoweth  C, Zervos  MJ, Robinson-Dunn  B, Schreckenberger  PC, Reller  LB, Rudrik  JT, Patel  JB,     ( 2010 )

Dissemination of an Enterococcus Inc18-Like vanA plasmid associated with vancomycin-resistant Staphylococcus aureus.

Antimicrobial agents and chemotherapy 54 (10)
PMID : 20660665  :   DOI  :   10.1128/AAC.00185-10     PMC  :   PMC2944587    
Abstract >>
Of the 9 vancomycin-resistant Staphylococcus aureus (VRSA) cases reported to date in the literature, 7 occurred in Michigan. In 5 of the 7 Michigan VRSA cases, an Inc18-like vanA plasmid was identified in the VRSA isolate and/or an associated vancomycin-resistant Enterococcus (VRE) isolate from the same patient. This plasmid may play a critical role in the emergence of VRSA. We studied the geographical distribution of the plasmid by testing 1,641 VRE isolates from three separate collections by PCR for plasmid-specific genes traA, repR, and vanA. Isolates from one collection (phase 2) were recovered from surveillance cultures collected in 17 hospitals in 13 states. All VRE isolates from 2 Michigan institutions (n = 386) and between 60 and 70 VRE isolates (n = 883) from the other hospitals were tested. Fifteen VRE isolates (3.9%) from Michigan were positive for an Inc18-like vanA plasmid (9 E. faecalis [12.5%], 3 E. faecium [1.0%], 2 E. avium, and 1 E. raffinosus). Six VRE isolates (0.6%) from outside Michigan were positive (3 E. faecalis [2.7%] and 3 E. faecium [0.4%]). Of all E. faecalis isolates tested, 6.0% were positive for the plasmid, compared to 0.6% for E. faecium and 3.0% for other spp. Fourteen of the 15 plasmid-positive isolates from Michigan had the same Tn1546 insertion site location as the VRSA-associated Inc18-like plasmid, whereas 5 of 6 plasmid-positive isolates from outside Michigan differed in this characteristic. Most plasmid-positive E. faecalis isolates demonstrated diverse patterns by PFGE, with the exception of three pairs with indistinguishable patterns, suggesting that the plasmid is mobile in nature. Although VRE isolates with the VRSA-associated Inc18-like vanA plasmid were more common in Michigan, they remain rare. Periodic surveillance of VRE isolates for the plasmid may be useful in predicting the occurrence of VRSA.
KeywordMeSH Terms
110. Göbl  C, Kosol  S, Stockner  T, Rückert  HM, Zangger  K,     ( 2010 )

Solution structure and membrane binding of the toxin fst of the par addiction module.

Biochemistry 49 (31)
PMID : 20677831  :   DOI  :   10.1021/bi1005128     PMC  :   PMC2914490    
Abstract >>
The par toxin-antitoxin system is required for the stable inheritance of the plasmid pAD1 in its native host Enterococcus faecalis. It codes for the toxin Fst and a small antisense RNA which inhibits translation of toxin mRNA, and it is the only known antisense regulated toxin-antitoxin system in Gram-positive bacteria. This study presents the structure of the par toxin Fst, the first atomic resolution structure of a component of an antisense regulated toxin-antitoxin system. The mode of membrane binding was determined by relaxation enhancements in a paramagnetic environment and molecular dynamics simulation. Fst forms a membrane-binding alpha-helix in the N-terminal part and contains an intrinsically disordered region near the C-terminus. It binds in a transmembrane orientation with the C-terminus likely pointing toward the cytosol. Membrane-bound, alpha-helical peptides are frequently found in higher organisms as components of the innate immune system. Despite similarities to these antimicrobial peptides, Fst shows neither hemolytic nor antimicrobial activity when applied externally to a series of bacteria, fungal cells, and erythrocytes. Moreover, its charge distribution, orientation in the membrane, and structure distinguish it from antimicrobial peptides.
KeywordMeSH Terms
111. Glazunova  OO, Raoult  D, Roux  V,     ( 2010 )

Partial recN gene sequencing: a new tool for identification and phylogeny within the genus Streptococcus.

International journal of systematic and evolutionary microbiology 60 (Pt 9)
PMID : 19880633  :   DOI  :   10.1099/ijs.0.018176-0    
Abstract >>
Partial sequences of the recN gene (1249 bp), which encodes a recombination and repair protein, were analysed to determine the phylogenetic relationship and identification of streptococci. The partial sequences presented interspecies nucleotide similarity of 56.4-98.2 % and intersubspecies similarity of 89.8-98 %. The mean DNA sequence similarity of recN gene sequences (66.6 %) was found to be lower than those of the 16S rRNA gene (94.1 %), rpoB (84.6 %), sodA (74.8 %), groEL (78.1 %) and gyrB (73.2 %). Phylogenetically derived trees revealed six statistically supported groups: Streptococcus salivarius, S. equinus, S. hyovaginalis/S. pluranimalium/S. thoraltensis, S. pyogenes, S. mutans and S. suis. The 'mitis' group was not supported by a significant bootstrap value, but three statistically supported subgroups were noted: Streptococcus sanguinis/S. cristatus/S. sinensis, S. anginosus/S. intermedius/S. constellatus (the 'anginosus' subgroup) and S. mitis/S. infantis/S. peroris/S. oralis/S. oligofermentans/S. pneumoniae/S. pseudopneumoniae. The partial recN gene sequence comparison highlighted a high percentage of divergence between Streptococcus dysgalactiae subsp. dysgalactiae and S. dysgalactiae subsp. equisimilis. This observation is confirmed by other gene sequence comparisons (groEL, gyrB, rpoB and sodA). A high percentage of similarity was found between S. intermedius and S. constellatus after sequence comparison of the recN gene. To study the genetic diversity among the 'anginosus' subgroup, recN, groEL, sodA, gyrB and rpoB sequences were determined for 36 clinical isolates. The results that were obtained confirmed the high genetic diversity within this group of streptococci.
KeywordMeSH Terms
Phylogeny
112. Burdett  V,     ( 1991 )

Purification and characterization of Tet(M), a protein that renders ribosomes resistant to tetracycline.

The Journal of biological chemistry 266 (5)
PMID : 1993661  :  
Abstract >>
The tet(M) tetracycline resistance gene has been found in a wide variety of clinically important bacteria. It has been shown previously (Burdett, V. (1986) J. Bacteriol. 165, 564-569) that the tet(M) gene product mediates resistance at the level of protein synthesis as judged by in vitro assay. Using this assay, large amounts of protein were purified from an Escherichia coli overproducer expressing the gene under control of a T7 promoter. The purified activity consists of a single polypeptide of molecular weight 68,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was confirmed to be the tet(M) gene product by amino-terminal sequence analysis. Purified Tet(M) has an associated ribosome-dependent GTPase with the specific activity being similar to that of the corresponding activity associated with elongation factor G. Since Tet(M) also displays substantial homology to elongation factor G throughout its length, Tet(M) may function as an analog of this elongation factor.
KeywordMeSH Terms
113. Zscheck  KK, Murray  BE,     ( 1991 )

Nucleotide sequence of the beta-lactamase gene from Enterococcus faecalis HH22 and its similarity to staphylococcal beta-lactamase genes.

Antimicrobial agents and chemotherapy 35 (9)
PMID : 1952840  :   DOI  :   10.1128/aac.35.9.1736     PMC  :   PMC245260    
Abstract >>
The nucleotide sequence of the constitutively produced beta-lactamase (Bla) gene from Enterococcus faecalis HH22 was shown to be identical to the published sequences of three of four staphylococcal type A beta-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred forty nucleotides upstream of the beta-lactamase start codon were determined for an inducible staphylococcal beta-lactamase and were identical to those of the constitutively expressed enterococcal gene, indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The genes for the enterococcal Bla and an inducible staphylococcal Bla were each cloned into a shuttle vector and transformed into enterococcal and staphylococcal recipients. The major difference between the backgrounds of the two hosts was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene. The location of the enzyme was found to be host dependent, since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell-bound enzyme in the enterococcus. On the basis of the identities of the enterococcal Bla and several staphylococcal Bla sequences, these data suggest the recent spread of beta-lactamase to enterococci and also suggest the loss of a functional repressor.
KeywordMeSH Terms
114. McMahon  SA, Roberts  GA, Johnson  KA, Cooper  LP, Liu  H, White  JH, Carter  LG, Sanghvi  B, Oke  M, Walkinshaw  MD, Blakely  GW, Naismith  JH, Dryden  DT,     ( 2009 )

Extensive DNA mimicry by the ArdA anti-restriction protein and its role in the spread of antibiotic resistance.

Nucleic acids research 37 (15)
PMID : 19506028  :   DOI  :   10.1093/nar/gkp478     PMC  :   PMC2731889    
Abstract >>
The ardA gene, found in many prokaryotes including important pathogenic species, allows associated mobile genetic elements to evade the ubiquitous Type I DNA restriction systems and thereby assist the spread of resistance genes in bacterial populations. As such, ardA contributes to a major healthcare problem. We have solved the structure of the ArdA protein from the conjugative transposon Tn916 and find that it has a novel extremely elongated curved cylindrical structure with defined helical grooves. The high density of aspartate and glutamate residues on the surface follow a helical pattern and the whole protein mimics a 42-base pair stretch of B-form DNA making ArdA by far the largest DNA mimic known. Each monomer of this dimeric structure comprises three alpha-beta domains, each with a different fold. These domains have the same fold as previously determined proteins possessing entirely different functions. This DNA mimicry explains how ArdA can bind and inhibit the Type I restriction enzymes and we demonstrate that 6 different ardA from pathogenic bacteria can function in Escherichia coli hosting a range of different Type I restriction systems.
KeywordMeSH Terms
Molecular Mimicry
115. Vermette  CJ, Russell  AH, Desai  AR, Hill  JE,     ( 2010 )

Resolution of phenotypically distinct strains of Enterococcus spp. in a complex microbial community using cpn60 universal target sequencing.

Microbial ecology 59 (1)
PMID : 19844647  :   DOI  :   10.1007/s00248-009-9601-1    
Abstract >>
Characterization of complex microbial communities is frequently based on the examination of polymerase chain reaction amplified sequences from a single phylogenetic marker, usually the 16S rRNA gene. However, this commonly used target often does not offer robust resolution of species or sub-species and is thus not a sufficiently informative target for understanding microbial population dynamics occurring at the strain level. We have used the cpn60 universal target sequence to characterize Enterococcus isolates from feces of growing pigs and have shown that sub-species groups, not detected using 16S rRNA sequences, can be resolved. Furthermore, groups resolved by cpn60-based phylogenetic analysis have distinct phenotypes. We report changes in the structure and function of Enterococcus communities in pig feces sampled from individual animals at three times, from suckling through to maturity. Enterococcus faecalis was largely replaced by Enterococcus hirae between suckling and 9 weeks of age, and a shift from one sub-species group of E. hirae to another was observed in all animals between 9 and 15 weeks. Conversely, E. faecalis strains remained consistent throughout the study period. Our results demonstrate that cpn60 sequences can be used to detect strain level changes in Enterococcus populations during succession in the fecal microbiota of growing pigs.
KeywordMeSH Terms
116. Maldonado-Barragán  A, Caballero-Guerrero  B, Jiménez  E, Jiménez-Díaz  R, Ruiz-Barba  JL, Rodríguez  JM,     ( 2009 )

Enterocin C, a class IIb bacteriocin produced by E. faecalis C901, a strain isolated from human colostrum.

International journal of food microbiology 133 (1��2��)
PMID : 19501421  :   DOI  :   10.1016/j.ijfoodmicro.2009.05.008    
Abstract >>
Enterocin C (EntC), a class IIb bacteriocin was purified from culture supernatants of Enterococcus faecalis C901, a strain isolated from human colostrum. Enterocin C consists of two distinct peptides, named EntC1 and EntC2, whose complementary action is required for full antimicrobial activity. The structural genes entC1 and entC2 encoding enterocins EntC1 and EntC2, respectively, and that encoding the putative immunity protein (EntCI) are located in the 9-kb plasmid pEntC, harboured by E. faecalis C901. The N-terminal sequence of both antimicrobial peptides revealed that EntC1 (4284 Da) is identical to Ent1071A, one of the two peptides that form enterocin 1071 (Ent1071), a bacteriocin produced by E. faecalis BFE 1071. In contrast, EntC2 (3867 Da) presents the non-polar alanine residue at position 17 (Ala(17)) instead of the polar threonine residue (Thr(17)) in Ent1071B, the second peptide constituting Ent1071. In spite of peptide similarities, EntC differs from Ent1071 in major aspects, including the complementary activity among its constitutive peptides and its wider inhibitory spectrum of activity. Different amphiphilic alpha-helical conformations between EntC2 and Ent1071B could explain both, acquired complementary activity and increased antimicrobial spectrum.
KeywordMeSH Terms
Genes, Bacterial
117. Izquierdo  E, Wagner  C, Marchioni  E, Aoude-Werner  D, Ennahar  S,     ( 2009 )

Enterocin 96, a novel class II bacteriocin produced by Enterococcus faecalis WHE 96, isolated from Munster cheese.

Applied and environmental microbiology 75 (13)
PMID : 19411428  :   DOI  :   10.1128/AEM.02772-08     PMC  :   PMC2704840    
Abstract >>
Enterococcus faecalis WHE 96, a strain isolated from soft cheese based on its anti-Listeria activity, produced a 5,494-Da bacteriocin that was purified to homogeneity by ultrafiltration and cation-exchange and reversed-phase chromatographies. The amino acid sequence of this bacteriocin, named enterocin 96, was determined by Edman degradation, and its structural gene was sequenced, revealing a double-glycine leader peptide. After a comparison with other bacteriocins, it was shown that enterocin 96 was a new class II bacteriocin that showed very little similarity with known structures. Enterocin 96 was indeed a new bacteriocin belonging to class II bacteriocins. The activity spectrum of enterocin 96 covered a wide range of bacteria, with strong activity against most gram-positive strains but very little or no activity against gram-negative strains.
KeywordMeSH Terms
118. Zheng  B, Tomita  H, Inoue  T, Ike  Y,     ( 2009 )

Isolation of VanB-type Enterococcus faecalis strains from nosocomial infections: first report of the isolation and identification of the pheromone-responsive plasmids pMG2200, Encoding VanB-type vancomycin resistance and a Bac41-type bacteriocin, and pMG2201, encoding erythromycin resistance and cytolysin (Hly/Bac).

Antimicrobial agents and chemotherapy 53 (2)
PMID : 19029325  :   DOI  :   10.1128/AAC.00754-08     PMC  :   PMC2630599    
Abstract >>
Eighteen identical VanB-type Enterococcus faecalis isolates that were obtained from different hospitalized patients were examined for their drug resistance and plasmid DNAs. Of the 18 strains, 12 strains exhibited resistance to erythromycin (Em), gentamicin (Gm), kanamycin (Km), tetracycline (Tc), and vancomycin (Van) and produced cytolysin (Hly/Bac) and a bacteriocin (Bac) active against E. faecalis strains. Another six of the strains exhibited resistance to Gm, Km, Tc, and Van and produced a bacteriocin. Em and Van resistance was transferred individually to E. faecalis FA2-2 strains at a frequency of about 10(-4) per donor cell by broth mating. The Em-resistant transconjugants and the Van-resistant transconjugants harbored a 65.7-kbp plasmid and a 106-kbp plasmid, respectively. The 106-kbp and 65.7-kbp plasmids isolated from the representative E. faecalis NKH15 strains were designated pMG2200 and pMG2201, respectively. pMG2200 conferred vancomycin resistance and bacteriocin activity on the host strain and responded to the synthetic pheromone cCF10 for pCF10, while pMG2201 conferred erythromycin resistance and cytolysin activity on its host strain and responded to the synthetic pheromone cAD1 for pAD1. The complete DNA sequence of pMG2200 (106,527 bp) showed that the plasmid carried a Tn1549-like element encoding vanB2-type resistance and the Bac41-like bacteriocin genes of pheromone-responsive plasmid pYI14. The plasmid contained the regulatory region found in pheromone-responsive plasmids and encoded the genes prgX and prgQ, which are the key negative regulatory elements for plasmid pCF10. pMG2200 also encoded TraE1, a key positive regulator of plasmid pAD1, indicating that pMG2200 is a naturally occurring chimeric plasmid that has a resulting prgX-prgQ-traE1 genetic organization in the regulatory region of the pheromone response. The functional oriT region and the putative relaxase gene of pMG2200 were identified and found to differ from those of pCF10 and pAD1. The putative relaxase of pMG2200 was classified as a member of the MOB(MG) family, which is found in pheromone-independent plasmid pHTbeta of the pMG1-like plasmids. This is the first report of the isolation and characterization of a pheromone-responsive highly conjugative plasmid encoding vanB resistance.
KeywordMeSH Terms
119. Folli  C, Mangiarotti  L, Folloni  S, Alfieri  B, Gobbo  M, Berni  R, Rivetti  C,     ( 2008 )

Specificity of the TraA-DNA interaction in the regulation of the pPD1-encoded sex pheromone response in Enterococcus faecalis.

Journal of molecular biology 380 (5)
PMID : 18579153  :   DOI  :   10.1016/j.jmb.2008.05.058    
Abstract >>
The Enterococcus faecalis conjugative plasmid pPD1 encodes proteins responsible for the mating response to the sex pheromone cPD1 secreted by a recipient cell. This response involves the respectively negative and positive determinants traA and traE, the pheromone-inhibitor determinant ipd and structural genes participating in the conjugation process. TraA is capable of binding to key sites within the regulatory gene cluster. The binding of TraA to cognate sites is modulated by the pheromone (cPD1) and the pheromone-inhibitor (iPD1) peptides. Using atomic force microscopy and classic biochemical techniques, we mapped and characterized the TraA-DNA interactions within the pPD1 regulatory gene cluster and the role of TraA in the transcription regulation of the sex pheromone response. A previous report showed that TraA binds to three adjacent sites (tab1, tab2 and tab3) located upstream of the ipd promoter region. Here, we provide direct evidence for such interactions and show that TraA alone or in the presence of iPD1 inhibits ipd transcription by preferentially binding to tab1, whereas in the presence of saturating cPD1, the overall binding to the tab sites decreases, TraA preferentially binds to tab3 and the ipd repression is relieved. Moreover, TraA alone or in the presence of iPD1 binds to two non-adjacent sites within the ipd terminators T1 and T2, an interaction that is also relieved in the presence of cPD1. The binding of TraA to the termination region of ipd may play an important role in controlling traE and traF expression via a transcriptional read-through mechanism already postulated for the pAD1 plasmid. TraA may also regulate its own expression by binding to a site in the proximity of the traA promoter, which has been relocated 200 bp downstream of the ipd gene. A model for the TraA-mediated regulation of the pPD1-encoded sex pheromone response is presented.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
120. Kassem  II, Esseili  MA, Sigler  V,     ( 2008 )

Occurrence of mecA in nonstaphylococcal pathogens in surface waters.

Journal of clinical microbiology 46 (11)
PMID : 18845826  :   DOI  :   10.1128/JCM.01035-08     PMC  :   PMC2576628    
Abstract >>
N/A
KeywordMeSH Terms
Methicillin Resistance
Water Microbiology
121. Boyd  DA, Willey  BM, Fawcett  D, Gillani  N, Mulvey  MR,     ( 2008 )

Molecular characterization of Enterococcus faecalis N06-0364 with low-level vancomycin resistance harboring a novel D-Ala-D-Ser gene cluster, vanL.

Antimicrobial agents and chemotherapy 52 (7)
PMID : 18458129  :   DOI  :   10.1128/AAC.01516-07     PMC  :   PMC2443935    
Abstract >>
Enterococcus faecalis N06-0364, exhibiting a vancomycin MIC of 8 microg/ml, was found to harbor a novel D-Ala-D-Ser gene cluster, designated vanL. The vanL gene cluster was similar in organization to the vanC operon, but the VanT serine racemase was encoded by two separate genes, vanTm(L) (membrane binding) and vanTr(L) (racemase).
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
122. Flannagan  SE, Clewell  DB, Sedgley  CM,     ( 2008 )

A "retrocidal" plasmid in Enterococcus faecalis: passage and protection.

Plasmid 59 (3)
PMID : 18295881  :   DOI  :   10.1016/j.plasmid.2008.01.002    
Abstract >>
Enterococcus faecalis MC4 harbors a 130 kb conjugative, pheromone (cCF10)-responding plasmid, pAMS1, conferring chloramphenicol, streptomycin and tetracycline resistances. A plasmid-borne class IIa bacteriocin (MC4-1) determinant and cognate immunity gene were present, but not expressed in MC4. However, pAMS1 transfer to E. faecalis JH2-2 (but not to the non-isogenic OG1SS) generated the surprising ability to express bacteriocin activity against the plasmid donor, MC4. The bacteriocin target spectrum includes E. faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus hirae, and Listeria monocytogenes. Those donors unable to express bacteriocin or immunity could protect themselves from the "retrocidal" behavior of transconjugants by a switch to bacteriocin resistance at a frequency of approximately 10(-3). Reversion to sensitivity occurred at a relatively high frequency, suggestive of involvement of a phase variation event. These observations concerning a conjugative plasmid with novel "retrocidal" properties, coupled with a defense mechanism independent of plasmid-borne immunity functions, may relate to phenomena exploiting regulatory features with broader ecological and evolutionary implications.
KeywordMeSH Terms
123. Gauntlett  JC, Gebhard  S, Keis  S, Manson  JM, Pos  KM, Cook  GM,     ( 2008 )

Molecular analysis of BcrR, a membrane-bound bacitracin sensor and DNA-binding protein from Enterococcus faecalis.

The Journal of biological chemistry 283 (13)
PMID : 18227063  :   DOI  :   10.1074/jbc.M709503200    
Abstract >>
BcrR has been identified as a novel regulatory protein of high level bacitracin resistance encoded by the bcrABD operon in Enterococcus faecalis. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNA-binding proteins, and topological modeling predicts that the C-terminal domain contains four transmembrane alpha-helices. These data have led to the hypothesis that BcrR functions as both a membrane-bound sensor and transducer of bacitracin availability to regulate bcrABD expression. To characterize the bcrABD promoter and identify the promoter elements to which BcrR binds, a series of bcrA-lacZ fusions were constructed. A 69-bp region was identified that was essential for bacitracin-dependent bcrA-lacZ expression. Mutations that targeted this region were used to identify two inverted repeat sequences, each with the sequence 5'-GACA(N)(7)TGTC-3', on the bcrABD promoter that were required for bcrA-lacZ expression. To study BcrR binding to this region, we over-produced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-beta-d-maltoside, and subsequently purified it via Ni(2+)-nitrilotriacetic acid and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes, and BcrR binding to bcrABD promoter DNA was analyzed using electrophoretic mobility shift assays. Both inverted repeat sequences were required for BcrR binding, both in the presence and absence of bacitracin. These data demonstrate that membrane-bound BcrR binds specifically to the bcrABD promoter, irrespective of bacitracin concentration. We therefore propose that bacitracin-dependent induction of bcrABD expression by BcrR occurs after DNA binding.
KeywordMeSH Terms
124. Chan  YY, Abd Nasir  MH, Yahaya  MA, Salleh  NM, Md Dan  AD, Musa  AM, Ravichandran  M,     ( 2008 )

Low prevalence of vancomycin- and bifunctional aminoglycoside-resistant enterococci isolated from poultry farms in Malaysia.

International journal of food microbiology 122 (1��2��)
PMID : 18187222  :   DOI  :   10.1016/j.ijfoodmicro.2007.11.063    
Abstract >>
A total of 225 samples from poultry farms and the surrounding environment were screened for vancomycin-resistant enterococci (VRE) and bifunctional aminoglycoside-resistant enterococci using conventional microbiological tests and a nanoplex polymerase chain reaction (PCR) assay. Three (1.3%) of the samples were found to contain vancomycin-resistant isolates (MIC>256 microg/mL) that had a vanA genotype. The three vanA positive VRE isolates were identified as different species. Only one isolate (Enterococcus faecium F 4/13_54) was sensitive to teicoplanin (MIC<0. 12-0.35 microg/mL); the other two VRE (E. faecalis A 21_35 and E. gallinarum F 5/10_1) were resistant to teicoplanin (MIC 3.6-->16 microg/mL). The vanC genotype was observed in nine (4%) of the samples collected. High-level gentamicin-resistant (HLGR) enterococci (with MIC ranging between 100 and 500 microg/mL) were detected in 44 samples. However, only 40 of these were found to possess the aac(6')-aph(2'') gene. The overall prevalence of VRE among the samples from the poultry farms and environment was 5.3%, but the prevalence of the clinically significant vanA VRE was 1.3%, and the prevalence of bifunctional aminoglycoside-resistant enterococci was slightly higher, at 19.5%.
KeywordMeSH Terms
Vancomycin Resistance
125. Tomita  H, Kamei  E, Ike  Y,     ( 2008 )

Cloning and genetic analyses of the bacteriocin 41 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI14: a novel bacteriocin complemented by two extracellular components (lysin and activator).

Journal of bacteriology 190 (6)
PMID : 18203826  :   DOI  :   10.1128/JB.01056-07     PMC  :   PMC2258863    
Abstract >>
The conjugative plasmid pYI14 (61 kbp) was isolated from Enterococcus faecalis YI714, a clinical isolate. pYI14 conferred a pheromone response on its host and encoded bacteriocin 41 (bac41). Bacteriocin 41 (Bac41) only showed activity against E. faecalis. Physical mapping of pYI14 showed that it consisted of EcoRI fragments A to P. The clone pHT1100, containing EcoRI fragments A (12.6 kbp) and H (3.5 kbp), conferred the bacteriocin activity on E. faecalis strains. Genetic analysis showed that the determinant was located in a 6.6-kbp region within the EcoRI AH fragments. Six open reading frames (ORFs) were identified in this region and designated ORF7 (bacL1) ORF8 (bacL2), ORF9, ORF10, ORF11 (bacA), and ORF12 (bacI). They were aligned in this order and oriented in the same direction. ORFs bacL1, bacL2, bacA, and bacI were essential for expression of the bacteriocin in E. faecalis. Extracellular complementation of bacteriocin expression was possible for bacL1 and -L2 and bacA mutants. bacL1 and -L2 and bacA encoded bacteriocin component L and activator component A, respectively. The products of these genes are secreted into the culture medium and extracellularly complement bacteriocin expression. bacI encoded immunity, providing the host with resistance to its own bacteriocin activity. The bacL1-encoded protein had significant homology with lytic enzymes that attack the gram-positive bacterial cell wall. Sequence data for the deduced bacL1-encoded protein suggested that it has a domain structure consisting of an N-terminal signal peptide, a second domain with the enzymatic activity, and a third domain with a three-repeat structure directing the proenzyme to its cell surface receptor.
KeywordMeSH Terms
126. Rumpel  S, Becker  S, Zweckstetter  M,     ( 2008 )

High-resolution structure determination of the CylR2 homodimer using paramagnetic relaxation enhancement and structure-based prediction of molecular alignment.

Journal of biomolecular NMR 40 (1)
PMID : 18026911  :   DOI  :   10.1007/s10858-007-9204-4     PMC  :   PMC2758389    
Abstract >>
Structure determination of homooligomeric proteins by NMR spectroscopy is difficult due to the lack of chemical shift perturbation data, which is very effective in restricting the binding interface in heterooligomeric systems, and the difficulty of obtaining a sufficient number of intermonomer distance restraints. Here we solved the high-resolution solution structure of the 15.4 kDa homodimer CylR2, the regulator of cytolysin production from Enterococcus faecalis, which deviates by 1.1 angstroms from the previously determined X-ray structure. We studied the influence of different experimental information such as long-range distances derived from paramagnetic relaxation enhancement, residual dipolar couplings, symmetry restraints and intermonomer Nuclear Overhauser Effect restraints on the accuracy of the derived structure. In addition, we show that it is useful to combine experimental information with methods of ab initio docking when the available experimental data are not sufficient to obtain convergence to the correct homodimeric structure. In particular, intermonomer distances may not be required when residual dipolar couplings are compared to values predicted on the basis of the charge distribution and the shape of ab initio docking solutions.
KeywordMeSH Terms
127. Dufour  M, Manson  JM, Bremer  PJ, Dufour  JP, Cook  GM, Simmonds  RS,     ( 2007 )

Characterization of monolaurin resistance in Enterococcus faecalis.

Applied and environmental microbiology 73 (17)
PMID : 17630314  :   DOI  :   10.1128/AEM.01013-07     PMC  :   PMC2042098    
Abstract >>
There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 microg/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.
KeywordMeSH Terms
Drug Resistance, Bacterial
128. Li  S, Zhang  Z, Mi  ZH,     ( 2007 )

Vancomycin-resistant enterococci in a Chinese hospital.

Current microbiology 55 (2)
PMID : 17619103  :   DOI  :   10.1007/s00284-005-0484-1    
Abstract >>
In investigating the prevalence of vancomycin-resistant enterococci (VREs) in a hospital in China, 338 enterococci were detected by using multiplex polymerase chain reaction. Eleven VREs were found, including one VanA E. faecalis, one VanB E. faecium, three VanB E. faecalis, five VanC(1) E. gallinarum, and one VanC(2) E. flavescens. VITEK 2, microbroth dilution, and E-test were used to determine the susceptibilities of the VREs to certain antimicrobial agents; multiple-drug resistance of the 11 VREs was distinct. Compared with phenotypic methods, multiplex PCR rapidly detected 11 VREs and provided van genotype information. This is the first study that sequenced van genes of VRE isolates in China to date and found type VanB in China. Because of the relatively low isolating rate, early detection of VRE is vital.
KeywordMeSH Terms
129. Clewell  DB, Flannagan  SE, Ike  Y, Jones  JM, Gawron-Burke  C,     ( 1988 )

Sequence analysis of termini of conjugative transposon Tn916.

Journal of bacteriology 170 (7)
PMID : 2838457  :   DOI  :   10.1128/jb.170.7.3046-3052.1988     PMC  :   PMC211247    
Abstract >>
Transposon Tn916 is a 16.4-kilobase, broad-host-range, conjugative transposon originally identified on the chromosome of Enterococcus (Streptococcus) faecalis DS16. Its termini have been sequenced along with the junction regions for two different insertions. The ends were found to contain imperfect inverted repeat sequences with identity at 20 of 26 nucleotides. Further in from the ends, imperfect directly repeated sequences were present, with 24 of 27 nucleotides matching. The transposon junction regions contained homologous segments but of a nature not consistent with a direct duplication of the target sequence. Within the right terminus was a potential outwardly reading promoter. Tn916 is believed to transpose via an excision-insertion mechanism; based on the analyses of the termini, as well as two target sequences (before insertion and after excision), a possible model is suggested.
KeywordMeSH Terms
DNA Transposable Elements
130. Ponnuraj  K, Narayana  SV,     ( 2007 )

Crystal structure of ACE19, the collagen binding subdomain of Enterococus faecalis surface protein ACE.

Proteins 69 (1)
PMID : 17557326  :   DOI  :   10.1002/prot.21464    
Abstract >>
N/A
KeywordMeSH Terms
131. Zhu  XQ, Wang  XM, Li  H, Shang  YH, Pan  YS, Wu  CM, Wang  Y, Du  XD, Shen  JZ,     ( 2017 )

Novel lnu(G) gene conferring resistance to lincomycin by nucleotidylation, located on Tn6260 from Enterococcus faecalis E531.

The Journal of antimicrobial chemotherapy 72 (4)
PMID : 28039271  :   DOI  :   10.1093/jac/dkw549    
Abstract >>
To identify a novel putative lincosamide resistance gene determinant in a swine Enterococcus faecalis E531 exhibiting a lincosamide resistance/macrolide susceptibility (L R M S) phenotype and to determine its location and genetic environment. The whole genomic DNA of E. faecalis E531, which tested negative for the known lincosamide nucleotidyltransferase genes, was sequenced. A putative lincosamide resistance gene determinant was cloned into an Escherichia coli - E. faecalis shuttle vector (pAM401) and transformed into E. faecalis JH2-2. The MICs were determined by the microbroth dilution method. Inactivity of lincomycin was examined by UPLC-MS/MS. Inverse PCR and primer walking were used to explore the genetic environment based on the assembled sequence. A novel resistance gene, designated lnu (G), which encodes a putative lincosamide nucleotidyltransferase, was found in E. faecalis E531. The deduced Lnu(G) amino acid sequence displayed 76.0% identity to Lnu(B) in Enterococcus faecium . Both E. faecalis E531 and E. faecalis JH2-2 harbouring pAM401- lnu (G) showed a 4-fold increase in the MICs of lincomycin, compared with E. faecalis JH2-2 or E. faecalis JH2-2 harbouring empty vector pAM401 only. UPLC-MS/MS demonstrated that the Lnu(G) enzyme catalysed adenylylation of lincomycin. The genetic environment analysis revealed that the lnu (G) gene was embedded into a novel putative transposon, designated Tn 6260 , which was active. A novel lincosamide nucleotidyltransferase gene lnu (G) was identified in E. faecalis . The location of the lnu (G) gene on a mobile element Tn 6260 makes it easy to disseminate.
KeywordMeSH Terms
DNA Transposable Elements
Drug Resistance, Bacterial
132. Gawryszewska  I, Żabicka  D, Hryniewicz  W, Sadowy  E,     ( 2017 )

Linezolid-resistant enterococci in Polish hospitals: species, clonality and determinants of linezolid resistance.

European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology 36 (7)
PMID : 28197728  :   DOI  :   10.1007/s10096-017-2934-7     PMC  :   PMC5495842    
Abstract >>
The significant increase of the linezolid-resistant enterococci (LRE) has been observed in Polish hospitals since 2012 and our study aimed at elucidating the possible reasons for this phenomenon. Polish LRE isolates were analysed by multilocus-sequence typing (MLST) and multiple locus variable-number tandem repeat (VNTR) analysis (MLVA), polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) to establish clonal relatedness and mechanism of linezolid resistance, respectively. Fifty analysed LRE (2008-2015) included mostly Enterococcus faecium (82%) and Enterococcus faecalis (16%). Enterococcus faecium belonged to the hospital-adapted lineages 17/18 and 78, while E. faecalis isolates represented ST6, a hospital-associated type, and ST116, found in both humans and food-production animals. The G2576T 23S rRNA mutation was the most frequent (94%) mechanism of linezolid/tedizolid resistance of LRE. None of the isolates carried the plasmid-associated gene of Cfr methyltransferase, whereas optrA, encoding the ABC-type drug transporter, was identified in two E. faecalis isolates. In these isolates, optrA was located on a plasmid, transferable to both E. faecium and E. faecalis, whose partial (36.3 kb) sequence was 100% identical to the pE394 plasmid, identified previously in China in both clinical and farm animal isolates. The optrA-E. faecium transconjugant displayed a significant growth deficiency, in contrast to the optrA-E. faecalis. Our study indicates the role of mutation acquisition by hospital-adapted clones of enterococci as a major driver of increasing resistance to linezolid and tedizolid. Transferability and apparent lack of a biological cost of resistance suggest that E. faecalis may be a natural reservoir of optrA, an emerging mechanism of oxazolidinone resistance.
KeywordMeSH Terms
Drug Resistance, Bacterial
133. Molale  LG, Bezuidenhout  CC,     ( 2016 )

Virulence determinants and production of extracellular enzymes in Enterococcus spp. from surface water sources.

Water science and technology : a journal of the International Association on Water Pollution Research 73 (8)
PMID : 27120635  :   DOI  :   10.2166/wst.2016.015    
Abstract >>
Virulence factors in Enterococcus may be indicative of potential pathogenicity. The aim of this study was to determine the relationship between the presence of clinically relevant virulence genes, in Enterococcus spp. from environmental water, and their in vitro expression. One hundred and twenty-four Enterococcus isolates (seven species), from five surface water systems in the North West Province, South Africa, were screened for the presence of asa1, cylA, esp, gelE and hyl using polymerase chain reaction. The expression of cylA, hyl and gelE was determined by phenotypic assessments. Sixty-five percent of the isolates were positive for one virulence gene and 13% for two or more. Most frequently detected genes were gelE (32%) and cylA (28%). Enterococcal surface protein was absent in all isolates screened. The presence of virulence genes was correlated with their extracellular enzyme production. The results show that a large percentage of these environmental Enterococcus spp. possess virulence factors that could be expressed in vitro. This is a cause for concern and could have implications for individuals using this water for recreational and cultural purposes. Further investigation is required into the sources of these potential pathogenic Enterococcus isolates and measures to minimize their presence in water sources.
KeywordMeSH Terms
Water Microbiology
134. Seiple  IB, Zhang  Z, Jakubec  P, Langlois-Mercier  A, Wright  PM, Hog  DT, Yabu  K, Allu  SR, Fukuzaki  T, Carlsen  PN, Kitamura  Y, Zhou  X, Condakes  ML, Szczypi?ski  FT, Green  WD, Myers  AG,     ( 2016 )

A platform for the discovery of new macrolide antibiotics.

Nature 533 (7603)
PMID : 27193679  :   DOI  :   10.1038/nature17967     PMC  :   PMC6526944    
Abstract >>
The chemical modification of structurally complex fermentation products, a process known as semisynthesis, has been an important tool in the discovery and manufacture of antibiotics for the treatment of various infectious diseases. However, many of the therapeutics obtained in this way are no longer effective, because bacterial resistance to these compounds has developed. Here we present a practical, fully synthetic route to macrolide antibiotics by the convergent assembly of simple chemical building blocks, enabling the synthesis of diverse structures not accessible by traditional semisynthetic approaches. More than 300 new macrolide antibiotic candidates, as well as the clinical candidate solithromycin, have been synthesized using our convergent approach. Evaluation of these compounds against a panel of pathogenic bacteria revealed that the majority of these structures had antibiotic activity, some efficacious against strains resistant to macrolides in current use. The chemistry we describe here provides a platform for the discovery of new macrolide antibiotics and may also serve as the basis for their manufacture.
KeywordMeSH Terms
135. He  T, Shen  Y, Schwarz  S, Cai  J, Lv  Y, Li  J, Fe?ler  AT, Zhang  R, Wu  C, Shen  J, Wang  Y,     ( 2016 )

Genetic environment of the transferable oxazolidinone/phenicol resistance gene optrA in Enterococcus faecalis isolates of human and animal origin.

The Journal of antimicrobial chemotherapy 71 (6)
PMID : 26903276  :   DOI  :   10.1093/jac/dkw016    
Abstract >>
Aim of this study was to analyse 17 non-related Enterococcus faecalis isolates of human and animal origin for the genetic environment of the novel oxazolidinone/phenicol resistance gene optrA. WGS and de novo assembly were conducted to analyse the flanking sequences of the optrA gene in the 17 E. faecalis isolates. When optrA was located on a plasmid, conjugation assays were performed to check whether the plasmids are conjugative and to confirm the resistance phenotype associated with these plasmids. All nine optrA-carrying plasmids were conjugated into E. faecalis JH2-2 and the transconjugants exhibited the optrA-associated phenotype. In these plasmids, an IS1216E element was detected either upstream and/or downstream of the optrA gene. In eight plasmids, the phenicol exporter gene fexA was found upstream of optrA and in six plasmids, a novel erm(A)-related gene for macrolide-lincosamide-streptogramin B resistance was detected downstream of optrA. When located in the chromosomal DNA, the optrA gene was found downstream of the transcriptional regulator gene araC in four isolates, or downstream of the fexA gene in another four isolates. Integration of the optrA region into a Tn558-Tn554 hybrid, located in the chromosomal radC gene, was seen in two isolates. The findings of the present study extend the current knowledge about the genetic environment of optrA and suggest that IS1216E elements play an important role in the dissemination of optrA among different types of enterococcal plasmids. The mechanism underlying the integration of optrA into the chromosomal DNA requires further investigation.
KeywordMeSH Terms
136. Kommineni  S, Bretl  DJ, Lam  V, Chakraborty  R, Hayward  M, Simpson  P, Cao  Y, Bousounis  P, Kristich  CJ, Salzman  NH,     ( 2015 )

Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract.

Nature 526 (7575)
PMID : 26479034  :   DOI  :   10.1038/nature15524     PMC  :   PMC4978352    
Abstract >>
Enterococcus faecalis is both a common commensal of the human gastrointestinal tract and a leading cause of hospital-acquired infections. Systemic infections with multidrug-resistant enterococci occur subsequent to gastrointestinal colonization. Preventing colonization by multidrug-resistant E. faecalis could therefore be a valuable approach towards limiting infection. However, little is known about the mechanisms E. faecalis uses to colonize and compete for stable gastrointestinal niches. Pheromone-responsive conjugative plasmids encoding bacteriocins are common among enterococcal strains and could modulate niche competition among enterococci or between enterococci and the intestinal microbiota. We developed a model of colonization of the mouse gut with E. faecalis, without disrupting the microbiota, to evaluate the role of the conjugative plasmid pPD1 expressing bacteriocin 21 (ref. 4) in enterococcal colonization. Here we show that E. faecalis harbouring pPD1 replaces indigenous enterococci and outcompetes E. faecalis lacking pPD1. Furthermore, in the intestine, pPD1 is transferred to other E. faecalis strains by conjugation, enhancing their survival. Colonization with an E. faecalis strain carrying a conjugation-defective pPD1 mutant subsequently resulted in clearance of vancomycin-resistant enterococci, without plasmid transfer. Therefore, bacteriocin expression by commensal bacteria can influence niche competition in the gastrointestinal tract, and bacteriocins, delivered by commensals that occupy a precise intestinal bacterial niche, may be an effective therapeutic approach to specifically eliminate intestinal colonization by multidrug-resistant bacteria, without profound disruption of the indigenous microbiota.
KeywordMeSH Terms
137. Murugan  K, Selvanayaki  K, Al-Sohaibani  S,     ( 2016 )

Urinary catheter indwelling clinical pathogen biofilm formation, exopolysaccharide characterization and their growth influencing parameters.

Saudi journal of biological sciences 23 (1)
PMID : 26858552  :   DOI  :   10.1016/j.sjbs.2015.04.016     PMC  :   PMC4705282    
Abstract >>
Self-reproducing microbial biofilm community mainly involved in the contamination of indwelling medical devices including catheters play a vital role in nosocomial infections. The catheter-associated urinary tract infection (CA-UTI) causative Staphylococcus aureus, Enterobacter faecalis, and Pseudomonas aeruginosa were selectively isolated, their phenotypic as well as genotypic biofilm formation, production and monomeric sugar composition of EPS as well as sugar, salt, pH and temperature influence on their in vitro biofilm formation were determined. From 50 culture positive urinary catheters S. aureus (24%), P. aeruginosa (18%), E. faecalis (14%) and others (44%) were isolated. The performed assays revealed their varying biofilm forming ability. The isolated S. aureus ica, E. faecalis esp, and P. aeruginosa cup A gene sequencing and phylogenetic analysis showed their close branching and genetic relationship. The analyzed sugar, salt, pH, and temperature showed that the degree of CA-UTI isolates biofilm formation is an environmentally sensitive process. EPS monosaccharide HPLC analysis showed the presence of neutral sugars (ng/�gl) as follows: glucose (P. aeruginosa: 44.275; E. faecalis: 4.23), lactose (P. aeruginosa: 7.29), mannitol (P. aeruginosa: 2.53; S. aureus: 2.62; E. faecalis: 2.054) and maltose (E. faecalis: 7.0042) revealing species-specific presence and variation. This study may have potential clinical relevance for the easy diagnosis and management of CA-UTI.
KeywordMeSH Terms
Biofilm
CA-UTI
Environmental factors
Exopolysaccharide
HPLC
SEM
138. Wang  Y, Lv  Y, Cai  J, Schwarz  S, Cui  L, Hu  Z, Zhang  R, Li  J, Zhao  Q, He  T, Wang  D, Wang  Z, Shen  Y, Li  Y, Fe?ler  AT, Wu  C, Yu  H, Deng  X, Xia  X, Shen  J,     ( 2015 )

A novel gene, optrA, that confers transferable resistance to oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin.

The Journal of antimicrobial chemotherapy 70 (8)
PMID : 25977397  :   DOI  :   10.1093/jac/dkv116    
Abstract >>
The oxazolidinone-resistant Enterococcus faecalis E349 from a human patient tested negative for the cfr gene and 23S rRNA mutations. Here we report the identification of a novel oxazolidinone resistance gene, optrA, and a first investigation of the extent to which this gene was present in E. faecalis and Enterococcus faecium from humans and food-producing animals. The resistance gene optrA was identified by whole-plasmid sequencing and subsequent cloning and expression in a susceptible Enterococcus host. Transformation and conjugation assays served to investigate the transferability of optrA. All optrA-positive E. faecalis and E. faecium isolates of human and animal origin were analysed for their MICs and their genotype, as well as the location of optrA. The novel plasmid-borne ABC transporter gene optrA from E. faecalis E349 conferred combined resistance or elevated MICs (when no clinical breakpoints were available) to oxazolidinones (linezolid and tedizolid) and phenicols (chloramphenicol and florfenicol). The corresponding conjugative plasmid pE349, on which optrA was located, had a size of 36 331 bp and also carried the phenicol exporter gene fexA. The optrA gene was functionally expressed in E. faecalis, E. faecium and Staphylococcus aureus. It was detected more frequently in E. faecalis and E. faecium from food-producing animals (20.3% and 5.7%, respectively) than from humans (4.2% and 0.6%, respectively). Enterococci with elevated MICs of linezolid and tedizolid should be tested not only for 23S rRNA mutations and the gene cfr, but also for the novel resistance gene optrA.
KeywordMeSH Terms
enterococci
florfenicol
interspecies transfer
linezolid
plasmids
tedizolid
Drug Resistance, Bacterial
Genes, Bacterial
139. Meziane-Cherif  D, Stogios  PJ, Evdokimova  E, Egorova  O, Savchenko  A, Courvalin  P,     ( 2015 )

Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases.

mBio 6 (4)
PMID : 26265719  :   DOI  :   10.1128/mBio.00806-15     PMC  :   PMC4542195    
Abstract >>
Vancomycin resistance in Gram-positive bacteria results from the replacement of the D-alanyl-D-alanine target of peptidoglycan precursors with D-alanyl-D-lactate or D-alanyl-D-serine (D-Ala-D-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of D-Ala-D-Ser-terminating precursors by converting L-Ser to D-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in L-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5'-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the L-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against L-Ser versus L-Ala implied that this enzyme relies on its membrane-bound domain for L-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria. Vancomycin is one of the drugs of last resort against Gram-positive antibiotic-resistant pathogens. However, bacteria have evolved a sophisticated mechanism which remodels the drug target, the D-alanine ending precursors in cell wall synthesis, into precursors terminating with D-lactate or D-serine, to which vancomycin has less affinity. D-Ser is synthesized by VanT serine racemase, which has two unusual characteristics: (i) it is one of the few serine racemases identified in bacteria and (ii) it contains a membrane-bound domain involved in L-Ser uptake. The structure of the catalytic domain of VanTG showed high similarity to alanine racemases, and we identified three specific active site substitutions responsible for L-Ser specificity. The data provide the molecular basis for VanT evolution to a bifunctional enzyme coordinating both transport and racemization. Our findings also illustrate the evolution of the essential alanine racemase into a vancomycin resistance enzyme in response to antibiotic pressure.
KeywordMeSH Terms
140. Cebrián  R, Martínez-Bueno  M, Valdivia  E, Albert  A, Maqueda  M, Sánchez-Barrena  MJ,     ( 2015 )

The bacteriocin AS-48 requires dimer dissociation followed by hydrophobic interactions with the membrane for antibacterial activity.

Journal of structural biology 190 (2)
PMID : 25816760  :   DOI  :   10.1016/j.jsb.2015.03.006    
Abstract >>
The molecular mechanism underlining the antibacterial activity of the bacteriocin AS-48 is not known, and two different and opposite alternatives have been proposed. Available data suggested that the interaction of positively charged amino acids of AS-48 with the membrane would produce membrane destabilization and disruption. Alternatively, it has been proposed that AS-48 activity could rely on the effective insertion of the bacteriocin into the membrane. The biological and structural properties of the AS-48G13K/L40K double mutant were investigated to shed light on this subject. Compared with the wild type, the mutant protein suffered an important reduction in the antibacterial activity. Biochemical and structural studies of AS-48G13K/L40K mutant suggest the basis of its decreased antimicrobial activity. Lipid cosedimentation assays showed that the membrane affinity of AS-48G13K/L40K is 12-fold lower than that observed for the wild type. L40K mutation is responsible for this reduced membrane affinity and thus, hydrophobic interactions are involved in membrane association. Furthermore, the high-resolution crystal structure of AS-48G13K/L40K, together with the study of its dimeric character in solution showed that G13K stabilizes the inactive water-soluble dimer, which displays a reduced dipole moment. Our data suggest that the cumulative effect of these three affected properties reduces AS-48 activity, and point out that the bactericidal effect is achieved by the electrostatically driven approach of the inactive water-soluble dimer towards the membrane, followed by the dissociation and insertion of the protein into the lipid bilayer.
KeywordMeSH Terms
Antimicrobial peptide
Bacteriocin
Lipid binding protein
Membrane bilayer
X-ray crystallography
Models, Molecular
141. Dong  SH, Tang  W, Lukk  T, Yu  Y, Nair  SK, van der Donk  WA,     ( 2015 )

The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold.

eLife 4 (N/A)
PMID : 26226635  :   DOI  :   10.7554/eLife.07607     PMC  :   PMC4550811    
Abstract >>
The enterococcal cytolysin is a virulence factor consisting of two post-translationally modified peptides that synergistically kill human immune cells. Both peptides are made by CylM, a member of the LanM lanthipeptide synthetases. CylM catalyzes seven dehydrations of Ser and Thr residues and three cyclization reactions during the biosynthesis of the cytolysin large subunit. We present here the 2.2 ? resolution structure of CylM, the first structural information on a LanM. Unexpectedly, the structure reveals that the dehydratase domain of CylM resembles the catalytic core of eukaryotic lipid kinases, despite the absence of clear sequence homology. The kinase and phosphate elimination active sites that affect net dehydration are immediately adjacent to each other. Characterization of mutants provided insights into the mechanism of the dehydration process. The structure is also of interest because of the interactions of human homologs of lanthipeptide cyclases with kinases such as mammalian target of rapamycin.
KeywordMeSH Terms
Enterococcus faecalis
biochemistry
biophysics
dehydration
kinase
lanthipeptides
structural biology
142. Goessweiner-Mohr  N, Fercher  C, Arends  K, Birner-Gruenberger  R, Laverde-Gomez  D, Huebner  J, Grohmann  E, Keller  W,     ( 2014 )

The type IV secretion protein TraK from the Enterococcus conjugative plasmid pIP501 exhibits a novel fold.

Acta crystallographica. Section D, Biological crystallography 70 (Pt 4)
PMID : 24699656  :   DOI  :   10.1107/S1399004714001606    
Abstract >>
Conjugative plasmid transfer presents a serious threat to human health as the most important means of spreading antibiotic resistance and virulence genes among bacteria. The required direct cell-cell contact is established by a multi-protein complex, the conjugative type IV secretion system (T4SS). The conjugative core complex spans the cellular envelope and serves as a channel for macromolecular secretion. T4SSs of Gram-negative (G-) origin have been studied in great detail. In contrast, T4SSs of Gram-positive (G+) bacteria have only received little attention thus far, despite the medical relevance of numerous G+ pathogens (e.g. enterococci, staphylococci and streptococci). This study provides structural information on the type IV secretion (T4S) protein TraK of the G+ broad host range Enterococcus conjugative plasmid pIP501. The crystal structure of the N-terminally truncated construct TraK�G was determined to 3.0 ? resolution and exhibits a novel fold. Immunolocalization demonstrated that the protein localizes to the cell wall facing towards the cell exterior, but does not exhibit surface accessibility. Circular dichroism, dynamic light scattering and size-exclusion chromatography confirmed the protein to be a monomer. With the exception of proteins from closely related T4SSs, no significant sequence or structural relatives were found. This observation marks the protein as a very exclusive, specialized member of the pIP501 T4SS.
KeywordMeSH Terms
Enterococcus
antibiotic resistance
conjugative plasmid
pIP501
type IV secretion
143. Maky  MA, Ishibashi  N, Zendo  T, Perez  RH, Doud  JR, Karmi  M, Sonomoto  K,     ( 2015 )

Enterocin F4-9, a Novel O-Linked Glycosylated Bacteriocin.

Applied and environmental microbiology 81 (14)
PMID : 25956765  :   DOI  :   10.1128/AEM.00940-15     PMC  :   PMC4551185    
Abstract >>
Enterococcus faecalis F4-9 isolated from Egyptian salted-fermented fish produces a novel bacteriocin, termed enterocin F4-9. Enterocin F4-9 was purified from the culture supernatant by three steps, and its molecular mass was determined to be 5,516.6 Da by mass spectrometry. Amino acid and DNA sequencing showed that the propeptide consists of 67 amino acid residues, with a leader peptide containing a double glycine cleavage site to produce a 47-amino-acid mature peptide. Enterocin F4-9 is modified by two molecules of N-acetylglucosamine �]-O-linked to Ser37 and Thr46. The O-linked N-acetylglucosamine moieties are essential for the antimicrobial activity of enterocin F4-9. Further analysis of the enterocin F4-9 gene cluster identified enfC, which has high sequence similarity to a glycosyltransferase. The antimicrobial activity of enterocin F4-9 covered a limited range of bacteria, including, interestingly, a Gram-negative strain, Escherichia coli JM109. Enterocin F4-9 is sensitive to protease, active at a wide pH range, and moderately resistant to heat.
KeywordMeSH Terms
144. Meziane-Cherif  D, Stogios  PJ, Evdokimova  E, Savchenko  A, Courvalin  P,     ( 2014 )

Structural basis for the evolution of vancomycin resistance D,D-peptidases.

Proceedings of the National Academy of Sciences of the United States of America 111 (16)
PMID : 24711382  :   DOI  :   10.1073/pnas.1402259111     PMC  :   PMC4000784    
Abstract >>
Vancomycin resistance in Gram-positive bacteria is due to production of cell-wall precursors ending in D-Ala-D-Lac or D-Ala-D-Ser, to which vancomycin exhibits low binding affinities, and to the elimination of the high-affinity precursors ending in D-Ala-D-Ala. Depletion of the susceptible high-affinity precursors is catalyzed by the zinc-dependent D,D-peptidases VanX and VanY acting on dipeptide (D-Ala-D-Ala) or pentapeptide (UDP-MurNac-L-Ala-D-Glu-L-Lys-D-Ala-D-Ala), respectively. Some of the vancomycin resistance operons encode VanXY D,D-carboxypeptidase, which hydrolyzes both di- and pentapeptide. The molecular basis for the diverse specificity of Van D,D-peptidases remains unknown. We present the crystal structures of VanXYC and VanXYG in apo and transition state analog-bound forms and of VanXYC in complex with the D-Ala-D-Ala substrate and D-Ala product. Structural and biochemical analysis identified the molecular determinants of VanXY dual specificity. VanXY residues 110-115 form a mobile cap over the catalytic site, whose flexibility is involved in the switch between di- and pentapeptide hydrolysis. Structure-based alignment of the Van D,D-peptidases showed that VanY enzymes lack this element, which promotes binding of the penta- rather than that of the dipeptide. The structures also highlight the molecular basis for selection of D-Ala-ending precursors over the modified resistance targets. These results illustrate the remarkable adaptability of the D,D-peptidase fold in response to antibiotic pressure via evolution of specific structural elements that confer hydrolytic activity against vancomycin-susceptible peptidoglycan precursors.
KeywordMeSH Terms
antibiotic resistance
enzyme evolution
glycopeptides
metallopeptidases
subfamily M15B
antibiotic resistance
enzyme evolution
glycopeptides
metallopeptidases
subfamily M15B
antibiotic resistance
enzyme evolution
glycopeptides
metallopeptidases
subfamily M15B
Evolution, Molecular
Vancomycin Resistance
145. Murphree  CA, Heist  EP, Moe  LA,     ( 2014 )

Antibiotic resistance among cultured bacterial isolates from bioethanol fermentation facilities across the United States.

Current microbiology 69 (3)
PMID : 24748439  :   DOI  :   10.1007/s00284-014-0583-y    
Abstract >>
Bacterial contamination of fuel ethanol fermentations by lactic acid bacteria (LAB) can have crippling effects on bioethanol production. Producers have had success controlling bacterial growth through prophylactic addition of antibiotics to fermentors, yet concerns have arisen about antibiotic resistance among the LAB. Here, we report on mechanisms used by 32 LAB isolates from eight different US bioethanol facilities to persist under conditions of antibiotic stress. Minimum inhibitory concentration assays with penicillin, erythromycin, and virginiamycin revealed broad resistance to each of the antibiotics as well as high levels of resistance to individual antibiotics. Phenotypic assays revealed that antibiotic inactivation mechanisms contributed to the high levels of individual resistances among the isolates, especially to erythromycin and virginiamycin, yet none of the isolates appeared to use a �]-lactamase. Biofilm formation was noted among the majority of the isolates and may contribute to persistence under low levels of antibiotics. Nearly all of the isolates carried at least one canonical antibiotic resistance gene and many carried more than one. The erythromycin ribosomal methyltransferase (erm) gene class was found in 19 of 32 isolates, yet a number of these isolates exhibit little to no resistance to erythromycin. The erm genes were present in 15 isolates that encoded more than one antibiotic resistance mechanism, suggestive of potential genetic linkages.
KeywordMeSH Terms
Drug Resistance, Bacterial
Industrial Microbiology
146. Goessweiner-Mohr  N, Eder  M, Hofer  G, Fercher  C, Arends  K, Birner-Gruenberger  R, Grohmann  E, Keller  W,     ( 2014 )

Structure of the double-stranded DNA-binding type IV secretion protein TraN from Enterococcus.

Acta crystallographica. Section D, Biological crystallography 70 (Pt 9)
PMID : 25195751  :   DOI  :   10.1107/S1399004714014187    
Abstract >>
Conjugative transfer through type IV secretion multiprotein complexes is the most important means of spreading antimicrobial resistance. Plasmid pIP501, frequently found in clinical Enterococcus faecalis and Enterococcus faecium isolates, is the first Gram-positive (G+) conjugative plasmid for which self-transfer to Gram-negative (G-) bacteria has been demonstrated. The pIP501-encoded type IV secretion system (T4SS) protein TraN localizes to the cytoplasm and shows specific DNA binding. The specific DNA-binding site upstream of the pIP501 origin of transfer (oriT) was identified by a novel footprinting technique based on exonuclease digestion and sequencing, suggesting TraN to be an accessory protein of the pIP501 relaxase TraA. The structure of TraN was determined to 1.35 ? resolution. It revealed an internal dimer fold with antiparallel �]-sheets in the centre and a helix-turn-helix (HTH) motif at both ends. Surprisingly, structurally related proteins (excisionases from T4SSs of G+ conjugative transposons and transcriptional regulators of the MerR family) resembling only one half of TraN were found. Thus, TraN may be involved in the early steps of pIP501 transfer, possibly triggering pIP501 TraA relaxase activity by recruiting the relaxosome to the assembled mating pore.
KeywordMeSH Terms
DNA binding
Enterococcus
Gram positive
internal dimer
type IV secretion
147. Cebrián  R, Rodríguez-Ruano  S, Martínez-Bueno  M, Valdivia  E, Maqueda  M, Montalbán-López  M,     ( 2014 )

Analysis of the promoters involved in enterocin AS-48 expression.

PloS one 9 (3)
PMID : 24594763  :   DOI  :   10.1371/journal.pone.0090603     PMC  :   PMC3942455    
Abstract >>
The enterocin AS-48 is the best characterized antibacterial circular protein in prokaryotes. It is a hydrophobic and cationic bacteriocin, which is ribosomally synthesized by enterococcal cells and post-translationally cyclized by a head-to-tail peptide bond. The production of and immunity towards AS-48 depend upon the coordinated expression of ten genes organized in two operons, as-48ABC (where genes encoding enzymes with processing, secretion, and immunity functions are adjacent to the structural as-48A gene) and as-48C1DD1EFGH. The current study describes the identification of the promoters involved in AS-48 expression. Seven putative promoters have been here amplified, and separately inserted into the promoter-probe vector pTLR1, to create transcriptional fusions with the mCherry gene used as a reporter. The activity of these promoter regions was assessed measuring the expression of the fluorescent mCherry protein using the constitutive pneumococcal promoter PX as a reference. Our results revealed that only three promoters PA, P2(2) and PD1 were recognized in Enterococcus faecalis, Lactococcus lactis and Escherichia coli, in the conditions tested. The maximal fluorescence was obtained with PX in all the strains, followed by the P2(2) promoter, which level of fluorescence was 2-fold compared to PA and 4-fold compared to PD1. Analysis of putative factors influencing the promoter activity in single and double transformants in E. faecalis JH2-2 demonstrated that, in general, a better expression was achieved in presence of pAM401-81. In addition, the P2(2) promoter could be regulated in a negative fashion by genes existing in the native pMB-2 plasmid other than those of the as-48 cluster, while the pH seems to affect differently the as-48 promoter expression.
KeywordMeSH Terms
Promoter Regions, Genetic
148. Tricot  C, De Coen  JL, Momin  P, Falmagne  P, Stalon  V,     ( 1989 )

Evolutionary relationships among bacterial carbamoyltransferases.

Journal of general microbiology 135 (9)
PMID : 2516870  :   DOI  :   10.1099/00221287-135-9-2453    
Abstract >>
An immunological approach was used for the study of ornithine carbamoyltransferase (OTCase) evolution in bacteria. Antisera were prepared against the anabolic and catabolic OTCases of Pseudomonas aeruginosa and Aeromonas formicans as well as against OTCase and putrescine carbamoyltransferases from Streptococcus faecalis; these antisera were then tested against the unpurified OTCases, either anabolic or catabolic, of 34 bacterial strains. Extensive cross-reactions were observed between the antisera to catabolic OTCases from P. aeruginosa, A. formicans and S. faecalis and the catabolic enzymes from other species or genera. These antisera cross-reacted also with the anabolic OTCases of strains of the Enterobacteriaceae but not with the anabolic OTCases of the same species or of other species or genera. The cross-reaction measured between the antisera against P. aeruginosa anabolic OTCase and the anabolic OTCases of other Pseudomonas were largely in agreement with the phylogenic subdivision of Pseudomonas proposed by N. J. Palleroni. The correlation was also significantly higher with the anabolic enzyme of an archaeobacterium, Methanobacterium thermoaceticum, than with the catabolic or anabolic OTCases from other genera in the eubacterial line. The antiserum raised against A. formicans anabolic OTCase was quite specific for its antigen and appeared to be raised against the heaviest of the various oligomeric structures of the enzyme.
KeywordMeSH Terms
Biological Evolution
149. Woegerbauer  M, Zeinzinger  J, Springer  B, Hufnagl  P, Indra  A, Korschineck  I, Hofrichter  J, Kopacka  I, Fuchs  R, Steinwider  J, Fuchs  K, Nielsen  KM, Allerberger  F,     ( 2014 )

Prevalence of the aminoglycoside phosphotransferase genes aph(3')-IIIa and aph(3')-IIa in Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica and Staphylococcus aureus isolates in Austria.

Journal of medical microbiology 63 (Pt 2)
PMID : 24194558  :   DOI  :   10.1099/jmm.0.065789-0    
Abstract >>
The aminoglycoside phosphotransferase aph(3')-IIa primarily inactivates kanamycin and neomycin, whilst aph(3')-IIIa also inactivates amikacin. The aim of this study was to determine the frequency of both resistance genes in major human pathogens to obtain their baseline prevalence in the gene pool of these bacterial populations in Austria. In total, 10 541 Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica and Staphylococcus aureus isolates were collected representatively without selection bias between 2008 and 2011. Isolates were analysed by aph(3')-IIIa/nptIII- and aph(3')-IIa/nptII-specific TaqMan real-time PCR. For positive strains, MICs using Etests were performed and resistance gene sequences were determined. The overall prevalence of aph(3')-IIIa/nptIII was 1.62 % (95 % confidence interval: 1.38-1.88 %). In Escherichia coli, enterococci, Staphylococcus aureus, P. aeruginosa and Salmonella spp., the aph(3')-IIIa/nptIII prevalence was 0.47 % (0-1.47 %), 37.53 % (32.84-42.40 %), 2.90 % (1.51-5.02 %), 0 % (0-0.32 %) and 0 % (0-0.037 %), respectively. Eleven of a total of 169 carriers showed single-nucleotide polymorphisms in the resistance allele. The overall prevalence of aph(3')-IIa/nptII was 0.0096 % (0-0.046 %). Escherichia coli (0-0.70 %), enterococci (0-0.75 %), Staphylococcus aureus (0-0.73 %) and P. aeruginosa (0-0.32 %) did not carry aph(3')-IIa. A single Salmonella isolate was positive, resulting in an aph(3')-IIa prevalence of 0.013 % (0-0.058 %). aph(3')-IIIa/nptIII carriers were moderately prevalent in the strains tested except for in enterococci, which appeared to be an important reservoir for aph(3')-IIIa. aph(3')-IIa/nptII genes were detected at clinically irrelevant frequencies and played no significant role in the aminoglycoside resistance gene pool during the observation period.
KeywordMeSH Terms
Drug Resistance, Bacterial
150. Liu  Y, Wang  Y, Schwarz  S, Wang  S, Chen  L, Wu  C, Shen  J,     ( 2014 )

Investigation of a multiresistance gene cfr that fails to mediate resistance to phenicols and oxazolidinones in Enterococcus faecalis.

The Journal of antimicrobial chemotherapy 69 (4)
PMID : 24272266  :   DOI  :   10.1093/jac/dkt459    
Abstract >>
To investigate the basis of susceptibility to phenicols and oxazolidinones of the porcine Enterococcus faecalis CPPF5 despite the presence of the multiresistance gene cfr. Southern blotting, conjugation and transformation analyses were conducted to confirm the plasmid location and transferability of cfr in CPPF5. The genetic environment of cfr was determined by sequence analysis. Transcription and translation of cfr were examined by RT-PCR and western blotting, respectively, and modifications at A2503 within the 23S rRNA sequence were identified by primer extension. Electrotransformation and Southern blotting indicated that CPPF5 and its transformant 5B2-3 contained two cfr-carrying plasmids ? 50 and ? 12 kb in size. The complete 12,270 bp sequence of the smaller plasmid, pCPPF5, was determined and shared 99.9% (12,269/12,270 bp) identity with the corresponding region of the cfr-carrying plasmid pEF-01 in E. faecalis of cattle origin. Moreover, the genetic environment of cfr in the ? 50 kb plasmid was the same as that in pCPPF5 according to sequencing results. Although cfr mRNA, Cfr protein and a modification at the A2503 site were detected, the cfr-carrying transformant 5B2-3 did not have elevated MICs of chloramphenicol, florfenicol and linezolid, indicating that cfr fails to mediate resistance to the respective antibiotics in E. faecalis. This is the first report of the cfr gene failing to elevate MICs of the corresponding antibiotics. Although the genetic basis for the apparent 'no resistance' phenotype remains to be determined, this finding may have implications for surveillance studies that target the cfr gene.
KeywordMeSH Terms
RNA methylation
linezolid
pigs
transcription
translation
Drug Resistance, Multiple, Bacterial
151. Kanno  M, Katayama  T, Tamaki  H, Mitani  Y, Meng  XY, Hori  T, Narihiro  T, Morita  N, Hoshino  T, Yumoto  I, Kimura  N, Hanada  S, Kamagata  Y,     ( 2013 )

Isolation of butanol- and isobutanol-tolerant bacteria and physiological characterization of their butanol tolerance.

Applied and environmental microbiology 79 (22)
PMID : 24014527  :   DOI  :   10.1128/AEM.02900-13     PMC  :   PMC3811554    
Abstract >>
Despite their importance as a biofuel production platform, only a very limited number of butanol-tolerant bacteria have been identified thus far. Here, we extensively explored butanol- and isobutanol-tolerant bacteria from various environmental samples. A total of 16 aerobic and anaerobic bacteria that could tolerate greater than 2.0% (vol/vol) butanol and isobutanol were isolated. A 16S rRNA gene sequencing analysis revealed that the isolates were phylogenetically distributed over at least nine genera: Bacillus, Lysinibacillus, Rummeliibacillus, Brevibacillus, Coprothermobacter, Caloribacterium, Enterococcus, Hydrogenoanaerobacterium, and Cellulosimicrobium, within the phyla Firmicutes and Actinobacteria. Ten of the isolates were phylogenetically distinct from previously identified butanol-tolerant bacteria. Two relatively highly butanol-tolerant strains CM4A (aerobe) and GK12 (obligate anaerobe) were characterized further. Both strains changed their membrane fatty acid composition in response to butanol exposure, i.e., CM4A and GK12 exhibited increased saturated and cyclopropane fatty acids (CFAs) and long-chain fatty acids, respectively, which may serve to maintain membrane fluidity. The gene (cfa) encoding CFA synthase was cloned from strain CM4A and expressed in Escherichia coli. The recombinant E. coli showed relatively higher butanol and isobutanol tolerance than E. coli without the cfa gene, suggesting that cfa can confer solvent tolerance. The exposure of strain GK12 to butanol by consecutive passages even enhanced the growth rate, indicating that yet-unknown mechanisms may also contribute to solvent tolerance. Taken together, the results demonstrate that a wide variety of butanol- and isobutanol-tolerant bacteria that can grow in 2.0% butanol exist in the environment and have various strategies to maintain structural integrity against detrimental solvents.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
152. Moura  TM, Cassenego  AP, Campos  FS, Ribeiro  AM, Franco  AC, d'Azevedo  PA, Frazzon  J, Frazzon  AP,     ( 2013 )

Detection of vanC1 gene transcription in vancomycin-susceptible Enterococcus faecalis.

Memorias do Instituto Oswaldo Cruz 108 (4)
PMID : 23828012  :   DOI  :   10.1590/S0074-0276108042013009     PMC  :   PMC3970631    
Abstract >>
Here we report the presence and expression levels of the vanC1 and vanC(2/3) genes in vancomycin-susceptible strains of Enterococcus faecalis. The vanC1 and vanC(2/3) genes were located in the plasmid DNA and on the chromosome, respectively. Specific mRNA of the vanC1 gene was detected in one of these strains. Additionally, analysis of the vanC gene sequences showed that these genes are related to the vanC genes of Enterococcus gallinarum and Enterococcus casseliflavus. The presence of vanC genes is useful for the identification of E. gallinarum and E. casseliflavus. Moreover, this is the first report of vanC mRNA in E. faecalis.
KeywordMeSH Terms
153. Lohans  CT, Towle  KM, Miskolzie  M, McKay  RT, van Belkum  MJ, McMullen  LM, Vederas  JC,     ( 2013 )

Solution structures of the linear leaderless bacteriocins enterocin 7A and 7B resemble carnocyclin A, a circular antimicrobial peptide.

Biochemistry 52 (23)
PMID : 23725536  :   DOI  :   10.1021/bi400359z    
Abstract >>
Leaderless bacteriocins are a class of ribosomally synthesized antimicrobial peptides that are produced by certain Gram-positive bacteria without an N-terminal leader section. These bacteriocins are of great interest due to their potent inhibition of many Gram-positive organisms, including food-borne pathogens such as Listeria and Clostridium spp. We now report the NMR solution structures of enterocins 7A and 7B, leaderless bacteriocins recently isolated from Enterococcus faecalis 710C. These are the first three-dimensional structures to be reported for bacteriocins of this class. Unlike most other linear Gram-positive bacteriocins, enterocins 7A and 7B are highly structured in aqueous conditions. Both peptides are primarily �\-helical, adopting a similar overall fold. The structures can be divided into three separate �\-helical regions: the N- and C-termini are both �\-helical, separated by a central kinked �\-helix. The overall structures bear an unexpected resemblance to carnocyclin A, a 60-residue peptide that is cyclized via an amide bond between the C- and N-termini and has a saposin fold. Because of synergism observed for other two-peptide leaderless bacteriocins, it was of interest to probe possible binding interactions between enterocins 7A and 7B. However, despite synergistic activity observed between these peptides, no significant binding interaction was observed based on NMR and isothermal calorimetry.
KeywordMeSH Terms
Enterococcus faecalis
154. Buhnik-Rosenblau  K, Matsko-Efimov  V, Danin-Poleg  Y, Franz  CM, Klein  G, Kashi  Y,     ( 2013 )

Biodiversity of Enterococcus faecalis based on genomic typing.

International journal of food microbiology 165 (1)
PMID : 23685727  :   DOI  :   10.1016/j.ijfoodmicro.2013.04.009    
Abstract >>
Enterococcus faecalis is a common inhabitant of the gastrointestinal tracts of different animals and is also found in other environments, such as plants, soil, food and water. The diverse nature of E. faecalis, which includes pathogenic, commensal and probiotic strains, calls for the development of tools for accurate discrimination and characterization at the strain level. Here we studied the genetic relationships among 106 E. faecalis strains isolated from diverse origins and possessing different degrees of virulence. Strain typing was conducted using a set of selected simple-sequence repeat (SSR) loci combined with multilocus sequence typing (MLST) analysis, which discriminated among the strains and separated them into three main clusters. While pathogenic and commensal isolates were dispersed along the dendrogram, probiotic and cheese-originated strains were highly associated with one specific cluster (cluster 1). The strain panel was further characterized by testing the occurrence of two virulence determinants (esp gene and �]-hemolysis). The two determinants showed low abundance among probiotic and cheese-originated strains within cluster 1 when compared to non-cluster 1 cheese-originated strains, indicating a possible association of cluster 1 with non-virulent strains. Our results further emphasize the importance and challenge of precise characterization of E. faecalis strains from various sources.
KeywordMeSH Terms
Food Microbiology
Genetic Variation
Genomics
155. Wardal  E, Gawryszewska  I, Hryniewicz  W, Sadowy  E,     ( 2013 )

Abundance and diversity of plasmid-associated genes among clinical isolates of Enterococcus faecalis.

Plasmid 70 (3)
PMID : 23906674  :   DOI  :   10.1016/j.plasmid.2013.07.003    
Abstract >>
Enterococcus faecalis, a normal compound of the human intestinal microbiome, plays an important role in hospital-acquired infections. Plasmids make a significant contribution to the acquisition of the novel traits such as antimicrobial resistance and virulence by this pathogen. The study investigated the plasmid content and the diversity of plasmid-associated genes in a group of 152 hospital isolates of E. faecalis. The majority of plasmids visualized by pulsed-field gel electrophoresis of S1 nuclease-digested DNA fell into the range of 50-100 kb. PCR-based screening allowed detection of genes of the rep1(pIP501), rep2(pRE25), rep4(pMBB1), rep6(pS86), rep7(pT181), rep8(pAM373), rep9(pAD1/pTEF2/pCF10), rep10(pIM13) and rep13(pC194) families in 29 different combinations. The par and �s-�`-�a plasmid stabilization systems were ubiquitous (45 isolates, 29.6% and 88 isolates, 57.9%, respectively), while the axe-txe system was not found. The asa1 gene homologues encoding aggregation substance characteristic for the pAD1 and related group of pheromone-responsive plasmids were present in 106 isolates. A variety of sequence variants, including novel ones, of genes associated with pheromone-responsive plasmids, such as rep8(pAM373), rep9(pAD1/pTEF2/pCF10), par, and asa1 were observed. In conclusion, there is a big and only partially characterized pool of diverse plasmids in clinical E. faecalis.
KeywordMeSH Terms
HiRECC
asa
cyl
par
rep
Gene Expression Regulation, Bacterial
Genes, Bacterial
Phylogeny
156. Huang  E, Zhang  L, Chung  YK, Zheng  Z, Yousef  AE,     ( 2013 )

Characterization and application of enterocin RM6, a bacteriocin from Enterococcus faecalis.

BioMed research international 2013 (N/A)
PMID : 23844357  :   DOI  :   10.1155/2013/206917     PMC  :   PMC3697273    
Abstract >>
Use of bacteriocins in food preservation has received great attention in recent years. The goal of this study is to characterize enterocin RM6 from Enterococcus faecalis OSY-RM6 and investigate its efficacy against Listeria monocytogenes in cottage cheese. Enterocin RM6 was purified from E. faecalis culture supernatant using ion exchange column, multiple C18-silica cartridges, followed by reverse-phase high-performance liquid chromatography. The molecular weight of enterocin RM6 is 7145.0823 as determined by mass spectrometry (MS). Tandem mass spectrometry (MS/MS) analysis revealed that enterocin RM6 is a 70-residue cyclic peptide with a head-to-tail linkage between methionine and tryptophan residues. The peptide sequence of enterocin RM6 was further confirmed by sequencing the structural gene of the peptide. Enterocin RM6 is active against Gram-positive bacteria, including L. monocytogenes, Bacillus cereus, and methicillin-resistant Staphylococcus aureus (MRSA). Enterocin RM6 (final concentration in cottage cheese, 80 AU/mL) caused a 4-log reduction in population of L. monocytogenes inoculated in cottage cheese within 30 min of treatment. Therefore, enterocin RM6 has potential applications as a potent antimicrobial peptide against foodborne pathogens in food.
KeywordMeSH Terms
157. VanNice  JC, Skaff  DA, Wyckoff  GJ, Miziorko  HM,     ( 2013 )

Expression in Haloferax volcanii of 3-hydroxy-3-methylglutaryl coenzyme A synthase facilitates isolation and characterization of the active form of a key enzyme required for polyisoprenoid cell membrane biosynthesis in halophilic archaea.

Journal of bacteriology 195 (17)
PMID : 23794621  :   DOI  :   10.1128/JB.00485-13     PMC  :   PMC3754609    
Abstract >>
Enzymes of the isoprenoid biosynthetic pathway in halophilic archaea remain poorly characterized, and parts of the pathway remain cryptic. This situation may be explained, in part, by the difficulty of expressing active, functional recombinant forms of these enzymes. The use of newly available expression plasmids and hosts has allowed the expression and isolation of catalytically active Haloferax volcanii 3-hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (EC 2.3.310). This accomplishment has permitted studies that represent, to the best of our knowledge, the first characterization of an archaeal hydroxymethylglutaryl CoA synthase. Kinetic characterization indicates that, under optimal assay conditions, which include 4 M KCl, the enzyme exhibits catalytic efficiency and substrate saturation at metabolite levels comparable to those reported for the enzyme from nonhalophilic organisms. This enzyme is unique in that it is the first hydroxymethylglutaryl CoA synthase that is insensitive to feedback substrate inhibition by acetoacetyl-CoA. The enzyme supports reaction catalysis in the presence of various organic solvents. Haloferax 3-hydroxy-3-methylglutaryl CoA synthase is sensitive to inactivation by hymeglusin, a specific inhibitor known to affect prokaryotic and eukaryotic forms of the enzyme, with experimentally determined Ki and kinact values of 570 �� 120 nM and 17 �� 3 min(-1), respectively. In in vivo experiments, hymeglusin blocks the propagation of H. volcanii cells, indicating the critical role that the mevalonate pathway plays in isoprenoid biosynthesis by these archaea.
KeywordMeSH Terms
158. Frolkova  P, Ghosh  A, Svec  P, Zurek  L, Literak  I,     ( 2012 )

Use of the manganese-dependent superoxide dismutase gene sodA for rapid identification of recently described enterococcal species.

Folia microbiologica 57 (5)
PMID : 22570141  :   DOI  :   10.1007/s12223-012-0115-8    
Abstract >>
N/A
KeywordMeSH Terms
159. Skaff  DA, Ramyar  KX, McWhorter  WJ, Barta  ML, Geisbrecht  BV, Miziorko  HM,     ( 2012 )

Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin.

Biochemistry 51 (23)
PMID : 22510038  :   DOI  :   10.1021/bi300037k     PMC  :   PMC3431454    
Abstract >>
Hymeglusin (1233A, F244, L-659-699) is established as a specific �]-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 ?) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.
KeywordMeSH Terms
160. Posteraro  B, Giard  JC, Sanguinetti  M, Martini  C, Colone  M, De Carolis  E, Stringaro  A, Florio  AR, Paroni Sterbini  F, Bugli  F,     ( 2012 )

The PavA-like fibronectin-binding protein of Enterococcus faecalis, EfbA, is important for virulence in a mouse model of ascending urinary tract infection.

The Journal of infectious diseases 206 (6)
PMID : 22782954  :   DOI  :   10.1093/infdis/jis440    
Abstract >>
Enterococcus faecalis is an established nosocomial pathogen, yet the pathogenesis of enterococcal infections, particularly of urinary tract infections (UTIs), remains to be fully elucidated. Fibronectin-binding proteins have been identified as potent adhesins in pathogenic Gram-positive cocci. Here, we characterized EfbA, which is encoded by the enterococcal orthologue of Streptococcus pneumoniae pavA. Similar to PavA, the anchorless EfbA protein was localized to the enterococcal cell outer surface and bound to immobilized human fibronectin. In addition to abrogated EfbA expression, deletion of the efbA gene eliminated EfbA from the cell surface and drastically reduced the enterococcal cell binding to immobilized fibronectin. The �GefbA deletion mutant was highly attenuated vs wild-type in a murine ascending UTI model, consistent with an increased tropism for the kidney relative to the bladder. These results provide the first evidence that EfbA of E. faecalis plays a role in UTIs, probably contributing to the pathogenesis in this site.
KeywordMeSH Terms
161. Silva Lopes  Mde F, Serror  P, Barreto Crespo  MT, Gonzalez-Zorn  B, Matos  R, Teixeira  N,     ( 2012 )

Incongruence between the cps type 2 genotype and host-related phenotypes of an Enterococcus faecalis food isolate.

International journal of food microbiology 158 (2)
PMID : 22831818  :   DOI  :   10.1016/j.ijfoodmicro.2012.07.006    
Abstract >>
Enterococcus faecalis is a nosocomial opportunistic pathogen, but is also found in fermented food products where it plays a fundamental role in the fermentation process. Previously, we have described the non-starter E. faecalis cheese isolate QA29b as harboring virulence genes and proven to be virulent in Galleria mellonella virulence model. In this study, we further characterized this food strain concerning traits relevant for the host-pathogen relationship. QA29b was found to belong to sequence type (ST) 72, a common ST among food isolates, and thus we consider it as a good representative of food E. faecalis strains. It demonstrated high ability to form biofilms, to adhere to epithelial cells and was readily eliminated by J774.A1 macrophage cells. Despite carrying the cps locus associated with the capsular polysaccharide CPS 2 type, cps genes were not expressed, likely due to an IS6770 inserted in the cpsC-cpsK promoter region. This work constitutes the first study of traits important for interaction, colonization and infection in the host performed on a good representative of E. faecalis food isolates. Reported results stress the need for a reliable serotyping assay of E. faecalis, as cps genotyping may not be reliable. Overall, QA29b characterization shows that despite its virulence potential in an insect model, this food strain is readily eliminated by mammalian macrophages. Thus, fine tuned approaches combining cellular and mammalian models are needed to address and elucidate the multifactorial aspect of virulence potential associated with food isolates.
KeywordMeSH Terms
Genotype
162. Diaz  L, Kiratisin  P, Mendes  RE, Panesso  D, Singh  KV, Arias  CA,     ( 2012 )

Transferable plasmid-mediated resistance to linezolid due to cfr in a human clinical isolate of Enterococcus faecalis.

Antimicrobial agents and chemotherapy 56 (7)
PMID : 22491691  :   DOI  :   10.1128/AAC.00419-12     PMC  :   PMC3393385    
Abstract >>
Nonmutational resistance to linezolid is due to the presence of cfr, which encodes a methyltransferase responsible for methylation of A2503 in the 23S rRNA. The cfr gene was first described in animal isolates of staphylococci, and more recently, it has been identified in Staphylococcus aureus from human clinical infections, including in an outbreak of methicillin-resistant S. aureus. In enterococci, cfr has been described in an animal isolate of Enterococcus faecalis from China. Here, we report an isolate of linezolid-resistant E. faecalis (603-50427X) recovered from a patient in Thailand who received prolonged therapy with the antibiotic for the treatment of atypical mycobacterial disease. The isolate lacked mutations in the genes coding for 23S rRNA and L3 and L4 ribosomal proteins and belonged to the multilocus sequence type (MLST) 16 (ST16), which is commonly found in enterococcal isolates from animal sources. Resistance to linezolid was associated with the presence of cfr on an ~97-kb transferable plasmid. The cfr gene environment exhibited DNA sequences similar to those of other cfr-carrying plasmids previously identified in staphylococci (nucleotide identity, 99 to 100%). The cfr-carrying plasmid was transferable by conjugation to a laboratory strain of E. faecalis (OG1RF) but not to Enterococcus faecium or S. aureus. The cfr gene was flanked by IS256-like sequences both upstream and downstream. This is the first characterization of the potential horizontal transferability of the cfr gene from a human linezolid-resistant isolate of E. faecalis.
KeywordMeSH Terms
163. Teixeira  N, Santos  S, Marujo  P, Yokohata  R, Iyer  VS, Nakayama  J, Hancock  LE, Serror  P, Silva Lopes  Mde F,     ( 2012 )

The incongruent gelatinase genotype and phenotype in Enterococcus faecalis are due to shutting off the ability to respond to the gelatinase biosynthesis-activating pheromone (GBAP) quorum-sensing signal.

Microbiology (Reading, England) 158 (Pt 2)
PMID : 22117005  :   DOI  :   10.1099/mic.0.055574-0     PMC  :   PMC4083509    
Abstract >>
The concomitant presence of a complete fsr quorum-sensing system and gelE-sprE operons in Enterococcus faecalis is known to be essential for the detection of gelatinase activity. However, there are reports of the absence of gelatinase activity despite the presence of complete fsr and gelE loci. In order to understand this incongruence between genotype and phenotype we sequenced fsr and gelE loci of the E. faecalis LN68 strain, which was previously found to carry both operons but to lack gelatinase activity. Of the 59 nucleotide differences detected compared with the gelatinase-positive V583 strain, we found a nonsense mutation (a premature STOP codon) predicted to truncate the ATPase sensor domain of the FsrC protein, responsible for sensing and transducing the signal from the quorum-sensing molecule. Strain LN68 was highly affected in the expression of the gelE and sprE genes, further supporting the lack of Fsr-dependent gelE induction. When we constructed a V583 mutant with the same premature stop mutation in the fsrC gene the resulting strain was no longer able to degrade gelatin. We conclude that the reduced ability to transduce the quorum-sensing signal of the prematurely truncated FsrC protein is sufficient to explain the negative gelatinase phenotype. As the incongruent genotype and phenotype is detected in natural isolates, we believe that the silencing of the quorum-sensing system Fsr may be beneficial for some E. faecalis strains.
KeywordMeSH Terms
Quorum Sensing
Signal Transduction
164. Nam  KH, Kurinov  I, Ke  A,     ( 2011 )

Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.

The Journal of biological chemistry 286 (35)
PMID : 21697083  :   DOI  :   10.1074/jbc.M111.256263     PMC  :   PMC3162437    
Abstract >>
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 ? tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ?26 ? wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an �\/�] domain and an �\-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.
KeywordMeSH Terms
165. Laverde Gomez  JA, Hendrickx  AP, Willems  RJ, Top  J, Sava  I, Huebner  J, Witte  W, Werner  G,     ( 2011 )

Intra- and interspecies genomic transfer of the Enterococcus faecalis pathogenicity island.

PloS one 6 (4)
PMID : 21559082  :   DOI  :   10.1371/journal.pone.0016720     PMC  :   PMC3084688    
Abstract >>
Enterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibiotic resistance and pathogenicity traits. Enterococcus faecalis harbours a pathogenicity island (PAI) of 153 kb containing several virulence factors including the enterococcal surface protein (esp). Until now only internal fragments of the PAI or larger chromosomal regions containing it have been transferred. Here we demonstrate precise excision, circularization and horizontal transfer of the entire PAI element from the chromosome of E. faecalis strain UW3114. This PAI (ca. 200 kb) contained some deletions and insertions as compared to the PAI of the reference strain MMH594, transferred precisely and integrated site-specifically into the chromosome of E. faecalis (intergenic region) and Enterococcus faecium (tRNAlys). The internal PAI structure was maintained after transfer. We assessed phenotypic changes accompanying acquisition of the PAI and expression of some of its determinants. The esp gene is expressed on the surface of donor and both transconjugants. Biofilm formation and cytolytic activity were enhanced in E. faecalis transconjugants after acquisition of the PAI. No differences in pathogenicity of E. faecalis were detected using a mouse bacteraemia and a mouse peritonitis models (tail vein and intraperitoneal injection). A 66 kb conjugative pheromone-responsive plasmid encoding erm(B) (pLG2) that was transferred in parallel with the PAI was sequenced. pLG2 is a pheromone responsive plasmid that probably promotes the PAI horizontal transfer, encodes antibiotic resistance features and contains complete replication and conjugation modules of enterococcal origin in a mosaic-like composition. The E. faecalis PAI can undergo precise intra- and interspecies transfer probably with the help of conjugative elements like conjugative resistance plasmids, supporting the role of horizontal gene transfer and antibiotic selective pressure in the successful establishment of certain enterococci as nosocomial pathogens.
KeywordMeSH Terms
Genomic Islands
166. Liu  Y, Wang  Y, Wu  C, Shen  Z, Schwarz  S, Du  XD, Dai  L, Zhang  W, Zhang  Q, Shen  J,     ( 2012 )

First report of the multidrug resistance gene cfr in Enterococcus faecalis of animal origin.

Antimicrobial agents and chemotherapy 56 (3)
PMID : 22203597  :   DOI  :   10.1128/AAC.06091-11     PMC  :   PMC3294887    
Abstract >>
The multiresistance gene cfr was identified for the first time in an Enterococcus faecalis isolate of animal origin. The 32,388-bp plasmid pEF-01, which carried the cfr gene, was sequenced completely. Three copies of the insertion sequence IS1216 were identified in pEF-01, and the detection of a cfr- and IS1216-containing amplicon by inverse PCR suggests that IS1216 may play a role in the dissemination of cfr by a recombination process.
KeywordMeSH Terms
167.     ( 1997 )

Identification and characterization of cell wall-cell division gene clusters in pathogenic gram-positive cocci.

Journal of bacteriology 179 (17)
PMID : 9287029  :   DOI  :   10.1128/jb.179.17.5632-5635.1997     PMC  :   PMC179445    
Abstract >>
Clusters of peptidoglycan biosynthesis and cell division genes (DCW genes) were identified and sequenced in two gram-positive cocci, Staphylococcus aureus and Enterococcus faecalis. The results indicated some similarities in organization compared with previously reported bacterial DCW gene clusters, including the presence of penicillin-binding proteins at the left ends and ftsA and ftsZ cell division genes at the right ends of the clusters. However, there were also some important differences, including the absence of several genes, the comparative sizes of the div1B and ftsQ genes, and a wide range of amino acid sequence similarities when the genes of the gram-positive cocci were translated and compared to bacterial homologs.
KeywordMeSH Terms
Cytoskeletal Proteins
Hexosyltransferases
Peptidyl Transferases
168.     ( 1997 )

Enterococcal transposon Tn5384: evolution of a composite transposon through cointegration of enterococcal and staphylococcal plasmids.

Antimicrobial agents and chemotherapy 41 (9)
PMID : 9303373  :   PMC  :   PMC164024    
Abstract >>
Mechanisms for the possible transfer of antimicrobial resistance genes between staphylococci and enterococci remain poorly defined. We have previously reported the transfer between Enterococcus faecalis strains of a multiresistance chromosomal element (beta-lactamase positive and resistance to erythromycin, gentamicin, mercuric chloride, streptomycin, and tetracycline) which we have tentatively designated Tn5385. Tn5385 is a composite of several smaller transposable elements, including Tn5384, a 26-kb composite transposon conferring resistance to erythromycin, gentamicin, and mercuric chloride. Analyses of 7 kb within Tn5384 and flanking sequences within the larger element revealed sequences characteristic of staphylococcal beta-lactamase and small, mobilizable plasmids flanking a region with a sequence identical to those of the replication genes previously described for enterococcal and streptococcal broad-host-range plasmids. These diverse regions are linked by insertion sequences IS256 and IS257 in a manner which suggests a series of cointegration events as the genesis of the current relationship. Taken together, these data suggest that Tn5384 and the larger element within which it is incorporated (Tn5385) evolved at least in part as a result of cointegration between an enterococcal broad-host-range plasmid and staphylococcal beta-lactamase and small mobilizable plasmids. These results implicate broad-host-range plasmids in the transfer of resistance determinants from staphylococci to enterococci.
KeywordMeSH Terms
DNA Transposable Elements
169.     ( 1996 )

Comparative analysis of 18 sex pheromone plasmids from Enterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family.

Molecular & general genetics : MGG 252 (6)
PMID : 8917306  :   DOI  :   10.1007/bf02173969    
Abstract >>
A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3'-terminus of the surface exclusion gene, sep1, of sex pheromone plasmid pPD1 in Enterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.
KeywordMeSH Terms
170.     ( 1997 )

Multiplex PCR detection of vanA, vanB, vanC-1, and vanC-2/3 genes in enterococci.

Journal of clinical microbiology 35 (3)
PMID : 9041416  :   PMC  :   PMC229654    
Abstract >>
Vancomycin-resistant enterococci (VRE) are increasingly isolated from clinical specimens. One hundred clinical isolates of enterococci (E. casseliflavus/E. flavescens [n = 10], E. faecalis [n = 34], E. faecium [n = 43], E. avium [n = 1], E. gallinarum [n = 11], and E. raffinosus [n = 1]) were examined for the presence of vanA, vanB, vanC-1, and vanC-2/3 genes by a single multiplex PCR performed directly with colonies from blood agar plates. Six previously characterized VRE strains which carry either vanA, vanB, vanC-1, or vanC-2 genes were used as controls. To discriminate among van genes, the PCR amplicons were digested with MspI and were electrophoresed on agarose gels. Because of significant sequence homology between vanC-2 and vanC-3 genes, this assay is unable to discriminate these genes from each other; therefore, these are referred to as vanC-2/3 genes. PCR products were detected in 63 of the 100 clinical isolates. The restriction fragment length patterns were consistent with vanA for 10 strains, vanB for 30 strains, vanC-1 for 12 strains, vanC-2 for 6 strains, and vanA and vanC-1 for 1 strain. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanA and vanB were all > and = 64 micrograms/ml. The vancomycin MICs for the isolates with restriction fragment length patterns consistent with vanC-1 or vanC-2 were 4 to 8 micrograms/ml. The vancomycin MICs for the isolates from which no PCR amplicons were produced were 2 to 4 micrograms/ml. A PCR product was produced in four isolates (vancomycin MICs, 4 to > 256 micrograms/ml) with restriction fragment length patterns differing from those for the control vanA, vanB, vanC-1, and vanC-2 isolates. DNA sequencing of these amplicons revealed that two of the four isolates had nucleic acid sequences which were closely related to the published sequence for the vanB gene and two had nucleic acid sequences which were closely related to the published sequence for the vanC-2 and vanC-3 genes. Multiplex PCR-restriction fragment length polymorphism appears to be a useful and convenient method for rapidly detecting and discriminating genotypes for vancomycin-resistant Enterococcus spp. in the clinical laboratory. In instances in which unusual restriction fragment patterns of PCR amplicons occur, DNA sequencing can be performed to discriminate van genotypes.
KeywordMeSH Terms
Bacteriological Techniques
Carbon-Oxygen Ligases
Genes, Bacterial
171.     ( 1993 )

Characterization of the left 4 kb of conjugative transposon Tn916: determinants involved in excision.

Plasmid 30 (3)
PMID : 8302931  :   DOI  :   10.1006/plas.1993.1055    
Abstract >>
The rate-limiting step in movement of the conjugative transposon Tn916, originally identified in Enterococcus faecalis, is believed to be an excision event that generates a non-replicative circular intermediate. When present on a plasmid vector in Escherichia coli, Tn916 generally excises at a high frequency. It was reported previously that insertion of Tn5 in a region near the left end of Tn916 eliminated the ability to excise; and the mutation could be complemented in trans. In this communication the nucleotide sequence of 4 kb of Tn916 DNA connecting the recently sequenced tet(M) determinant (Su et al., 1992; Burdett, 1990) with the left end of the transposon. Ten open reading frames (ORFs) were deduced, two of which (ORF3 and ORF4) were encoded in-frame within a third (ORF2). Mutants with Tn5 insertions in the ORF1 or ORF2 (ORF3 and ORF4) were defective in excision, but could be complemented in vivo by a co-resident plasmid containing the ORF1 or ORF2 determinant, respectively. The data support the view that both ORF1 and ORF2 are essential for excision. ORF1 and ORF2 are essentially identical to determinants designated xis-Tn and int-Tn, respectively, in the closely related Tn1545. A Tn5 insertion in ORF5 eliminated conjugative transfer between E. faecalis strains. Functions for the remaining ORFs (ORF6 through ORF10) remain unknown; however, nucleotide sequences within ORF6 and ORF9 had significant homology with sequence downstream of other tet(M) determinants.
KeywordMeSH Terms
Conjugation, Genetic
DNA Transposable Elements
Plasmids
172.     ( 1996 )

Cloning and genetic organization of the bacteriocin 31 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI17.

Journal of bacteriology 178 (12)
PMID : 8655558  :   DOI  :   10.1128/jb.178.12.3585-3593.1996     PMC  :   PMC178130    
Abstract >>
The conjugative plasmid pYI17 (57.5 kb) isolated from Enterococcus faecalis YI717 confers a pheromone response on the host and encodes the bacteriocin 31 gene. Bacteriocin 31 is active against E. hirae 9790, E. faecium, and Listeria monocytogenes. pYI17 was mapped physically by restriction enzyme analysis and the relational clone method. Deletion mutant and sequence analyses of the EcoRI fragment B cloned from pYl17 revealed that a 1.0-kb fragment contained the bacteriocin gene (bacA) and an immunity gene (bacB). This fragment induced bacteriocin activity in E. faecalis OG1X and E. hirae 9790. The bacA gene is located on the pYI17 physical map between 3.37 and 3.57 kb, and bacB is located between 3.59 kb and 3.87 kb, bacA encodes 67 amino acids, and bacB encodes 94 amino acids. The deduced amino acid sequence of the bacA protein contained a series of hydrophobic residues typical of a signal sequence at its amino terminus. The predicted mature bacA protein (43 amino acids) showed sequence homology with the membrane-active class II bacteriocins of lactic acid bacteria. Analysis of Tn5 insertion mutants and the resulting transcripts indicated that these genes are transcribed as an operon composed of bacA, bacB, and an open reading frame located downstream of bacB designated ORF3.
KeywordMeSH Terms
Genes, Bacterial
Plasmids
173.     ( 1996 )

Characterization of Tn1547, a composite transposon flanked by the IS16 and IS256-like elements, that confers vancomycin resistance in Enterococcus faecalis BM4281.

Gene 172 (1)
PMID : 8654967  :   DOI  :   10.1016/0378-1119(96)00110-2    
Abstract >>
A 64-kb genetic element harboring a vanB vancomycin-resistance (VmR) gene cluster was shown to translocate from the chromosome of Enterococcus faecalis BM4281 into the hemolysin (Hly) plasmid, pIP964. Sequence analysis of the resulting junction fragments indicated that the VmR genes were carried by a composite transposon, Tn1547, bounded by two distantly related insertion sequences (IS), designated IS256-like and IS16, in a direct orientation. IS256-like (1324 bp) was identical to IS256, except for two nucleotide (nt) transitions in the putative transposase gene. IS16 (1466 bp) contained a large open reading frame (ORF) that was 61% identical to the gene encoding the putative transposase of IS256. There was 58% identity between the deduced amino acid (aa) sequences of the putative transposases of IS256-like (390 aa) and IS16 (395 aa). IS16 was delineated by imperfect inverted repeats (IR) (18 out of 26 bp) which were related (23/26 bp identity) to their respective imperfect IR counterparts in IS256. The difference between the IS was not a barrier for transposition of Tn1547 which generated an 8-bp duplication at the target site. Dissemination of VanB-type resistance among enterococci results from two mechanisms: (i) large conjugative elements translocate from chromosome to chromosome following inter-strain transfer; and (ii) as described in this report, the VmR genes transpose from replicon to replicon within the same strain as part of composite transposons that are internal to the conjugative elements.
KeywordMeSH Terms
DNA Transposable Elements
174.     ( 1995 )

Enterococcus faecalis plasmid pAD1 replication and maintenance.

Developments in biological standardization 85 (N/A)
PMID : 8586250  :  
Abstract >>
A determinant has been identified which is required for the stable inheritance of a replicon isolated from the pheromone-responsive E. faecalis plasmid pAD1. This determinant, called par, is encoded on a DNA fragment no larger than 457 bp and directs the production of two small RNAs of approximately 250 (RNAI) and 145 (RNAII) nucleotides. Mutations affecting par RNA production also affected plasmid stability, indicating that these RNAs play a central role in par function. Putative transcription start sites were identified within par by DNA sequence analysis, but the sizes of the observed RNAs and the effects of transposon inserts on RNA production are not entirely consistent with predictions based on the hypothetical RNAs produced from these start sites. The absence of clearly functional open reading frames and the presence of various sequence repeat elements within par suggest that these RNAs may not be translated but may perform some structural role in the partition apparatus. The isolated par determinant was capable of producing both RNAs and stabilizing a heterologous vector, indicating that no other pAD1-encoded determinants are required for par function.
KeywordMeSH Terms
175.     ( 1996 )

Identification and characterization of the genes of Enterococcus faecalis plasmid pCF10 involved in replication and in negative control of pheromone-inducible conjugation.

Plasmid 35 (1)
PMID : 8693026  :   DOI  :   10.1006/plas.1996.0005    
Abstract >>
The prgB gene of the Enterococcus faecalis pheromone-inducible conjugative plasmid pCF10 encodes the surface protein Asc10. This protein mediates cell aggregation and its expression results in high-frequency transfer of the plasmid from donor to recipient. To identify the minimum region necessary for negative regulation of prgB expression, target plasmids were constructed containing a recently identified positive control region and a prgB::lacZ transcriptional fusion; expression of prgB in cells carrying these plasmids was thus verified by beta-galactosidase assay. The target plasmids were used in genetic studies with compatible plasmids containing cloned pCF10 genes supplying putative negative control functions to define the minimum region of pCF10 required for shutdown of prgB expression in the absence of exogenous pheromone. The minimum segment required for negative control, as indentified by deletion analysis, was a 6.9-kb region extending from the 5' end of a gene called prgN, through a previously identified gene, prgX. The DNA in this region, which had not been previously characterized (2.85 kb), was sequenced, and several potential regulatory genes and plasmid replication genes were identified. Genetic analysis indicated that the prgN, -Y, and -X genes are involved in negative control; prgW may also play a role in negative control, since it appeared to be required for expression of prgY. prgX, or a closely adjacent DNA sequence, acted in cis. The region of pCF10 containing negative control genes was also shown to function as an autonomous replicon in E. faecalis.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
176.     ( 1996 )

Comparison of the beta-lactamase gene cluster in clonally distinct strains of Enterococcus faecalis.

Antimicrobial agents and chemotherapy 40 (5)
PMID : 8723460  :   PMC  :   PMC163285    
Abstract >>
Ten beta-lactamase-producing Enterococcus faecalis isolates were examined for the presence of the staphylococcal beta-lactamase repressor and antirepressor genes. Four isolates, previously shown to be unrelated to each other by pulsed-field gel electrophoresis analysis, were positive for both genes by PCR, although beta-lactamase production was not induced with methicillin. Six isolates, previously shown to be clonally related, were negative for both genes by PCR. The blaZ sequences of eight beta-lactamase-producing E. faecalis isolates were determined. Seven isolates from five distinct clones had sequences identical to that previously reported for E. faecalis HH22, regardless of whether the repressor or antirepressor was demonstrated by PCR. However, blaZ from one isolate differed from those of the other enterococci by 11 nucleotides; this isolate is part of the large clone, as defined by pulsed-field gel electrophoresis and multilocus enzyme analysis, that includes HH22. These findings suggest either that enterococci have acquired the bla gene cluster from more than one source or that the gene cluster has undergone considerable change since acquisition by this clone.
KeywordMeSH Terms
177.     ( 1993 )

Cloning and characterization of a region of the Enterococcus faecalis conjugative plasmid, pCF10, encoding a sex pheromone-binding function.

Journal of bacteriology 175 (16)
PMID : 8349565  :   DOI  :   10.1128/jb.175.16.5253-5259.1993     PMC  :   PMC204993    
Abstract >>
In order to investigate the mechanism by which peptide sex pheromones induce expression of the conjugation functions of certain Enterococcus faecalis plasmids, a biological assay was developed to measure the ability of cells carrying the conjugative plasmid pCF10 to bind the sex pheromone cCF10. The data indicated that pCF10 endows its host E. faecalis cell with the ability to specifically remove (apparently by irreversible binding) cCF10 activity from culture medium. The pCF10 DNA encoding this ability was localized to a 3.4-kb segment within a region involved in negative control of expression of conjugal transfer functions. This segment also encoded ability to bind the pheromone inhibitor peptide iCF10. DNA sequencing revealed three open reading frames, which have been denoted prgW (pheromone responsive gene W), prgZ, and prgY. The deduced product of prgW resembled regulatory proteins from other bacteria and eucaryotes, with a very high degree of identity within a putative DNA-binding domain. The prgY gene actually extended into an adjacent region of pCF10 and could encode a protein with significant similarity to a protein called TraB, believed to be involved in shutdown of pheromone cAD1 production by cells carrying the pheromone-inducible hemolysin plasmid pAD1, according to F.Y. An and D.B. Clewell (Abstr. Gen. Meet. Am. Soc. Microbiol. 1992, H70, 1992). The prgZ gene product showed significant relatedness to binding proteins encoded by oligopeptide permease (opp) operons in gram-positive and gram-negative bacteria and is highly similar to a pAD1-encoded protein, TraC, which is believed to mediate sex pheromone cAD1 binding (K. Tanimoto, F. Y. An, and D. B. Clewell, submitted for publication). A Tn5 insertion into prgZ abolished cCF10 binding ability.
KeywordMeSH Terms
178.     ( 1993 )

Identification, characterization, and nucleotide sequence of a region of Enterococcus faecalis pheromone-responsive plasmid pAD1 capable of autonomous replication.

Journal of bacteriology 175 (7)
PMID : 8384618  :   DOI  :   10.1128/jb.175.7.1900-1909.1993     PMC  :   PMC204257    
Abstract >>
A 5-kbp region of pAD1, previously shown to be capable of supporting replication, copy control, and stable inheritance of the plasmid, was cloned into a replicon probe vector and subjected to transposon insertional mutagenesis. Transposon inserts identifying essential replication, copy control, and stability functions were isolated. Deletion of stability functions not essential for replication resulted in delimitation of a basic replicon. The complete DNA sequence of this approximately 3-kbp region and the precise positions of several transposon inserts were determined, and the phenotypic effects of the transposon inserts were correlated with the physical locations of individual determinants. The following three genes, apparently involved in plasmid maintenance, were identified; repA, which encodes a protein required for replication; repB, which encodes a protein involved in copy control; and repC, which may be involved in stable inheritance. In addition, two clusters of repeats composed of a consensus sequence, TAGTARRR, were identified, one located between the divergently transcribed repA and repB genes and another located downstream of repC. The region between repA and repB contained 25 repeats divided into two subregions of 13 and 12 repeats separated by 78 bp. The region located downstream of repC contained only three repeats but may be essential for plasmid replication, since deletion of this determinant resulted in loss of ability to replicate in Enterococcus faecalis. We hypothesize that the repeat units represent protein-binding sites required for assembly of the replisome and control of plasmid copy number. Another region of unrelated repeat units that may also be involved in replication is located within the repA gene. Possible mechanisms of action of these determinants are discussed.
KeywordMeSH Terms
DNA Helicases
DNA-Binding Proteins
Proteins
Trans-Activators
179.     ( 1996 )

Sequences found on staphylococcal beta-lactamase plasmids integrated into the chromosome of Enterococcus faecalis CH116.

Plasmid 35 (2)
PMID : 8700969  :   DOI  :   10.1006/plas.1996.0010    
Abstract >>
We have previously reported the presence of the staphylococcal beta-lactamase gene in chromosomes of Enterococcus faecalis strains CH19 and CH116. CH116 also harbors a 26-kb mobile element, designated Tn5384, which confers resistance to erythromycin and gentamicin. Sequence analysis of the rightmost 9 kb of Tn5384 indicates that this element lies immediately upstream of the beta-lactamase determinant in E. faecalis CH116. This 9-kb region consists of sequences highly homologous to those previously described in staphylococcal beta-lactamase plasmids, including a beta-lactamase transposon indistinguishable from Tn552, an open reading frame encoding a deduced amino acid sequence 94% identical to a previously described potential staphylococcal invertase, an intact copy of staphylococcal insertion-like element IS257, and the major portion of the staphylococcal organomercurial lyase (merB) gene. These data are consistent with the hypothesis that several of the resistance genes encoded within the large transferable region of the CH116 chromosome were originally components of a staphylococcal beta-lactamase plasmid.
KeywordMeSH Terms
180.     ( 1994 )

Characterization of the determinant (traB) encoding sex pheromone shutdown by the hemolysin/bacteriocin plasmid pAD1 in Enterococcus faecalis.

Plasmid 31 (2)
PMID : 8029329  :   DOI  :   10.1006/plas.1994.1023    
Abstract >>
pAD1 is a hemolysin/bacteriocin plasmid originally identified in Enterococcus faecalis DS16. It encodes a mating response to a peptide sex pheromone, cAD1, secreted by recipient bacteria. Once pAD1 is acquired, production of the pheromone ceases--a trait related in part to a determinant designated traB. Here we report the nucleotide sequence of traB and the position of several transposon insertions resulting in the characteristic self-induction phenotype. The deduced product has a mass of 43.7 kDa with the C-terminal third consisting primarily of hydrophobic amino acid residues.
KeywordMeSH Terms
Bacteriocin Plasmids
Hemolysin Factors
181.     ( 1994 )

Nucleotide sequence of the 18-kb conjugative transposon Tn916 from Enterococcus faecalis.

Plasmid 32 (3)
PMID : 7899523  :   DOI  :   10.1006/plas.1994.1077    
Abstract >>
Conjugative transposon Tn916 from Enterococcus faecalis DS16 encodes tetracycline resistance (Tet M) as well as determinants necessary for its own movement. Determination of the nucleotide sequence of Tn916 has been completed. The element is 18,032 bp in length and has an overall G+C content of 38.8%. Twenty-four potential open reading frames (ORFs) were identified based on sequence analysis. Similarities of the ORFs to other known determinants, which were revealed by database searches, are discussed.
KeywordMeSH Terms
Conjugation, Genetic
DNA Transposable Elements
182. Korten  V, Huang  WM, Murray  BE,     ( 1994 )

Analysis by PCR and direct DNA sequencing of gyrA mutations associated with fluoroquinolone resistance in Enterococcus faecalis.

Antimicrobial agents and chemotherapy 38 (9)
PMID : 7811024  :   DOI  :   10.1128/aac.38.9.2091     PMC  :   PMC284689    
Abstract >>
A region of gyrA, the gene encoding subunit A of DNA gyrase, that is known to be associated with resistance was amplified and sequenced from 16 Enterococcus faecalis and Enterococcus faecium isolates. Six ciprofloxacin-resistant clinical isolates (MICs of ciprofloxacin, 32 to 64 micrograms/ml) and one multistep resistant laboratory mutant of E. faecalis (MIC of ciprofloxacin, 128 micrograms/ml) contained a change from serine to arginine or to isoleucine at codon 83 or a change from glutamic acid to lysine or to glycine at codon 87 (Escherichia coli GyrA coordinates); these changes have been associated with fluoroquinolone resistance in other species. No difference in the region studied was found in two ciprofloxacin-resistant E. faecium isolates (MICs, 32 micrograms/ml) or in four laboratory derived, spontaneous ciprofloxacin-resistant mutants of E. faecalis (MICs, 8 to 16 micrograms/ml), suggesting that other mechanisms may be responsible for fluoroquinolone resistance in some enterococci.
KeywordMeSH Terms
Mutation
183.     ( 1994 )

Cloning, sequencing and expression in Escherichia coli of the low-affinity penicillin binding protein of Enterococcus faecalis.

FEMS microbiology letters 123 (1��2��)
PMID : 7988905  :   DOI  :   10.1111/j.1574-6968.1994.tb07207.x    
Abstract >>
Low-affinity penicillin binding proteins are particular membrane proteins, in several Gram-positive bacteria, which are involved in beta-lactam antibiotic resistance. The structural gene for the low-affinity penicillin binding protein 5 (PBP5) of Enterococcus faecalis was cloned and sequenced. From the sequence of the 3378 bp, a 2040 bp coding region was identified. From biochemical analysis it emerges that E. faecalis PBP5 is a type II membrane protein with an uncleaved N-terminal and is composed of 679 amino acids with a molecular weight of 74055. This protein showed 48 and 33% of identity with Enterococcus hirae PBP5 and Staphylococcus aureus PBP2a, both low-affinity PBPs involved in beta-lactam resistance. Anti-PBP5 antibodies cross-reacted with a membrane protein present in other species of enterococci, but the entire gene fragment cloned hybridized only with DNAs of E. faecalis strains, thus suggesting that genes coding for low-affinity PBPs of enterococci are not strictly homologous. In this experiment digoxigenin-labelled E. faecalis DNA was used.
KeywordMeSH Terms
Bacterial Proteins
Hexosyltransferases
Peptidyl Transferases
184.     ( 1994 )

Determination of the gene sequence and the molecular structure of the enterococcal peptide antibiotic AS-48.

Journal of bacteriology 176 (20)
PMID : 7929005  :   DOI  :   10.1128/jb.176.20.6334-6339.1994     PMC  :   PMC196975    
Abstract >>
The structural gene of the enterococcal peptide antibiotic AS-48 (as-48) has been identified and cloned by using two degenerate 17-mer DNA oligonucleotides on the basis of the amino acid sequences of two peptides obtained by digestion of the antibiotic with Glu-C endoproteinase. That as-48 gene codes for a 105-amino-acid prepeptide, giving rise to a 70-amino-acid mature protein. Comparative analysis demonstrated that the 16-amino-acid sequence of one of the AS-48 Glu-C peptides, designated V8-5, was composed of a 12-amino-acid sequence corresponding to the C-terminal end sequence (from isoleucine +59 to tryptophan +70 [I+59 to W+70]) of the prepeptide and terminated in four residues forming the N terminus (M+1 to E+4) of a putative AS-48 propeptide. These data, combined with the characteristics of the gene sequence, strongly suggested that the antibiotic peptide was a 70-residue cyclic molecule. We propose that the AS-48 translated primary product is very likely submitted to a posttranslational modification during secretion (i) by an atypical or a typical signal peptidase that cleaves off a 35-residue or shorter signal peptide, respectively, from the prepeptide molecule and (ii) by the linkage of the methionine residue (M+1) to the C-terminal tryptophan residue (W+70) to obtain the cyclic peptide (a tail-head linkage).
KeywordMeSH Terms
Anti-Bacterial Agents
Bacterial Proteins
Peptides
185. Li  X, Weinstock  GM, Murray  BE,     ( 1995 )

Generation of auxotrophic mutants of Enterococcus faecalis.

Journal of bacteriology 177 (23)
PMID : 7592480  :   DOI  :   10.1128/jb.177.23.6866-6873.1995     PMC  :   PMC177555    
Abstract >>
A 22-kb segment of chromosomal DNA from Enterococcus faecalis OG1RF containing the pyrimidine biosynthesis genes pyrC and pyrD was previously detected as complementing Escherichia coli pyrC and pyrD mutations. In the present study, it was found that the E. faecalis pyrimidine biosynthetic genes in this clone (designated pKV48) are part of a larger cluster resembling that seen in Bacillus spp. Transposon insertions were isolated at a number of sites throughout the cluster and resulted in loss of the ability to complement E. coli auxotrophs. The DNA sequences of the entire pyrD gene of E. faecalis and selected parts of the rest of the cluster were determined, and computer analyses found these to be similar to genes from Bacillus subtilis and Bacillus caldolyticus pyrimidine biosynthesis operons. Five of the transposon insertions were introduced back into the E. faecalis chromosome, and all except insertions in pyrD resulted in pyrimidine auxotrophy. The prototrophy of pyrD knockouts was observed for two different insertions and suggests that E. faecalis is similar to Lactococcus lactis, which has been shown to possess two pyrD genes. A similar analysis was performed with the purL gene from E. faecalis, contained in another cosmid clone, and purine auxotrophs were isolated. In addition, a pool of random transposon insertions in pKV48, isolated in E. coli, was introduced into the E. faecalis chromosome en masse, and an auxotroph was obtained. These results demonstrate a new methodology for constructing defined knockout mutations in E. faecalis.
KeywordMeSH Terms
Genes, Bacterial
Mutagenesis, Insertional
186. Platteeuw  C, Michiels  F, Joos  H, Seurinck  J, de Vos  WM,     ( 1995 )

Characterization and heterologous expression of the tetL gene and identification of iso-ISS1 elements from Enterococcus faecalis plasmid pJH1.

Gene 160 (1)
PMID : 7628724  :   DOI  :   10.1016/0378-1119(95)00208-n    
Abstract >>
The tetracycline-resistance (TcR) determinant of the Enterococcus faecalis plasmid pJH1 has been identified and located on a 2.2-kb RsaI-EcoRI fragment. The fragment was cloned in Escherichia coli, and specified TcR in this host. The nucleotide (nt) sequence of the cloned fragment showed the presence of an open reading frame (ORF) of 1374 bp, designated tetL. The nt sequence of tetL from pJH1 was identical to that of the tetL present on pLS1 from Streptococcus agalactiae. Upstream of the pJH1 tetL, part of another ORF was found that, except for two single-nt substitutions, was identical to an iso-ISS1 element from Lactococcus lactis. Hybridization studies indicated the presence of several ISS1-like elements in plasmid pJH1, but not on the En. faecalis chromosome. To study its usefulness as a marker in Gram+ organisms, the pJH1 tetL was cloned on the broad-host-range plasmid pNZ124, resulting in pNZ280, that was found to give resistance to 40 micrograms Tc/ml in Lc. lactis and Bacillus subtilis.
KeywordMeSH Terms
DNA Transposable Elements
Drug Resistance, Microbial
Plasmids
187. Nakayama  J, Abe  Y, Ono  Y, Isogai  A, Suzuki  A,     ( 1995 )

Isolation and structure of the Enterococcus faecalis sex pheromone, cOB1, that induces conjugal transfer of the hemolysin/bacteriocin plasmids, pOB1 and pYI1.

Bioscience, biotechnology, and biochemistry 59 (4)
PMID : 7772836  :  
Abstract >>
A bacterial sex pheromone, cOB1, which induces conjugal transfer of the Enterococcus faecalis hemolysin-bacteriocin (Hly/Bac) plasmid, pOB1, was isolated from the culture broth of pOB1-free E. faecalis. Its structure was found to be a hydrophobic octapeptide, H-Val-Ala-Val-Leu-Val-Leu-Gly-Ala-OH. The cOB1 peptide induced the mating response of not only pOB1 but also another incompatibility group Hly/Bac plasmid, pYI1.
KeywordMeSH Terms
Conjugation, Genetic
Plasmids
188. Lowe  AM, Lambert  PA, Smith  AW,     ( 1995 )

Cloning of an Enterococcus faecalis endocarditis antigen: homology with adhesins from some oral streptococci.

Infection and immunity 63 (2)
PMID : 7822045  :   PMC  :   PMC173055    
Abstract >>
Serum from a patient with Enterococcus faecalis endocarditis was used to identify the gene efaA cloned in Lambda ZapII in Escherichia coli. Nucleotide sequence analysis revealed a 924-bp open reading frame encoding a protein with a predicted molecular weight of 34,768. The amino acid sequence of EfaA shows 55 to 60% homology to a group of streptococcal proteins, FimA from Streptococcus parasanguis, SsaB from Streptococcus sanguis, ScaA from Streptococcus gordonii, and PsaA from Streptococcus pneumoniae. Members of this group have been shown to be adhesins, and we hypothesize that EfaA may function as an adhesin in endocarditis.
KeywordMeSH Terms
Fimbriae Proteins
Genes, Bacterial
189. Fujimoto  S, Tomita  H, Wakamatsu  E, Tanimoto  K, Ike  Y,     ( 1995 )

Physical mapping of the conjugative bacteriocin plasmid pPD1 of Enterococcus faecalis and identification of the determinant related to the pheromone response.

Journal of bacteriology 177 (19)
PMID : 7559345  :   DOI  :   10.1128/jb.177.19.5574-5581.1995     PMC  :   PMC177367    
Abstract >>
The pheromone-responding conjugative bacteriocin plasmid pPD1 (59 kb) of Enterococcus faecalis was mapped physically by using a relational clone approach, and transposon analysis with Tn917 (Emr) or Tn916 (Tcr) facilitated the location of the bacteriocin-related genes in a segment of about 6.7 kb. Tn917 insertions within a 3-kb region resulted in constitutive clumping. The nucleotide sequence of the region that included the insertions giving rise to constitutive clumping was determined. The region of pPD1 spanned about 8 kb and was found to contain a number of open reading frames, some of which were named on the basis of homologies with two other pheromone-responding plasmids, pAD1 and pCF10. The genes were arranged in the sequence repB-repA-traC-traB-traA-ipd-traE-traF- orfY-sea-1 with all but repB and traA oriented in the same (left-to-right) direction. traC and traB corresponded, respectively, to traC and traB of pAD1 and to prgY and prgZ of pCF10.
KeywordMeSH Terms
Oligopeptides
Pheromones
190. Nakayama  J, Yoshida  K, Kobayashi  H, Isogai  A, Clewell  DB, Suzuki  A,     ( 1995 )

Cloning and characterization of a region of Enterococcus faecalis plasmid pPD1 encoding pheromone inhibitor (ipd), pheromone sensitivity (traC), and pheromone shutdown (traB) genes.

Journal of bacteriology 177 (19)
PMID : 7559344  :   DOI  :   10.1128/jb.177.19.5567-5573.1995     PMC  :   PMC177366    
Abstract >>
Bacteriocin plasmid pPD1 in Enterococcus faecalis encodes a mating response to recipient-produced sex pheromone cPD1. Once a recipient acquires pPD1, transconjugants apparently shut off cPD1 activity in broth culture and no longer behave as recipients for pPD1. This event is performed by synthesis of the pheromone inhibitor iPD1 and also by repression of cPD1 production, the so-called "pheromone shutdown." A 5.4-kb EcoRV-HincII segment of pPD1, which expressed iPD1 in Escherichia coli, was sequenced and found to be organized as traC-traB-traA-ipd; each open reading frame is analogous to that found in other pheromone plasmids, pAD1 and pCF10, and thus is designated in accordance with the nomenclature in pAD1. The ipd gene encodes a peptide consisting of 21 amino acids, in which the C-terminal eight residues correspond to iPD1. The putative TraC product has a strong similarity to oligopeptide-binding proteins found in other bacterial species, as do pheromone-binding proteins of pCF10 and pAD1. A strain carrying traC-disrupted pPD1 required a concentration of cPD1 fourfold higher than that needed by the wild-type strain for induction of sexual aggregation. These results suggest that the TraC product contributes to pheromone sensitivity as a pheromone-binding protein. A strain transformed with traB-disrupted pPD1 produced a high level of cPD1 similar to that produced by plasmid-free recipients and underwent self-induction. Thus, the TraB product contributes to cPD1 shutdown.
KeywordMeSH Terms
Fimbriae Proteins
191. Mori  M, Sakagami  Y, Narita  M, Isogai  A, Fujino  M, Kitada  C, Craig  RA, Clewell  DB, Suzuki  A,     ( 1984 )

Isolation and structure of the bacterial sex pheromone, cAD1, that induces plasmid transfer in Streptococcus faecalis.

FEBS letters 178 (1)
PMID : 6437872  :   DOI  :   10.1016/0014-5793(84)81248-x    
Abstract >>
The Streptococcus faecalis sex pheromone cAD1, which is involved in the conjugative transfer of the hemolysin plasmid pAD1, has been isolated and its structure determined. Its Mr is 818 and its amino acid sequence is H-Leu-Phe-Ser-Leu-Val-Leu-Ala-Gly-OH. A replicate of the pheromone synthesized by the liquid-phase method showed the same biological activity and chromatographic behavior as the isolated cAD1. Pheromone activity was detectable at a concentration of approximately 5 X 10(-11) M.
KeywordMeSH Terms
Plasmids
192. Suzuki  A, Mori  M, Sakagami  Y, Isogai  A, Fujino  M, Kitada  C, Craig  RA, Clewell  DB,     ( 1984 )

Isolation and structure of bacterial sex pheromone, cPD1.

Science (New York, N.Y.) 226 (4676)
PMID : 6436978  :   DOI  :   10.1126/science.6436978    
Abstract >>
The Streptococcus faecalis sex pheromone cPD1, which induces a mating response in cells harboring the conjugative plasmid pPD1, has been isolated and its structure determined. It was found to have a molecular weight of 912, and its amino acid sequence was H-Phe-Leu-Val-Met-Phe-Leu-Ser-Gly-OH. A synthetic octapeptide showed the same biological activity and chromatographic behavior as the isolated cPD1. Pheromone activity was detectable at a concentration of approximately 4 X 10(-11)M.
KeywordMeSH Terms
193. Poyart  C, Berche  P, Trieu-Cuot  P,     ( 1995 )

Characterization of superoxide dismutase genes from gram-positive bacteria by polymerase chain reaction using degenerate primers.

FEMS microbiology letters 131 (1)
PMID : 7557308  :   DOI  :   10.1016/0378-1097(95)00232-t    
Abstract >>
An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes, and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.
KeywordMeSH Terms
194. Nakayama  J, Ruhfel  RE, Dunny  GM, Isogai  A, Suzuki  A,     ( 1994 )

The prgQ gene of the Enterococcus faecalis tetracycline resistance plasmid pCF10 encodes a peptide inhibitor, iCF10.

Journal of bacteriology 176 (23)
PMID : 7545961  :   DOI  :   10.1128/jb.176.23.7405-7408.1994     PMC  :   PMC197135    
Abstract >>
Conjugative transfer of the Enterococcus faecalis tetracycline resistance plasmid pCF10 is stimulated by a peptide pheromone, cCF10. Once a recipient strain acquires pCF10 and thus becomes a pheromone-responsive donor, cCF10 activity is no longer detected in culture filtrates. Here we show that pCF10 encodes a peptide inhibitor, iCF10, secreted by donor cells; this inhibitor antagonizes the cCF10 activity in culture filtrates. In order to detect and quantitate iCF10, we developed a reverse-phase high-performance liquid chromatography assay in which the inhibitor peptide elutes separately from the pheromone; this type of assay enabled us to determine that lack of pheromone activity in donor culture filtrates was due to secretion of a mixture of iCF10 and cCF10, rather than abolition of cCF10 secretion. The gene encoding iCF10, prgQ, is located on the EcoRI-C fragment of pCF10. The open reading frame comprising the prgQ gene encodes a 23-amino-acid precursor that resembles a signal peptide. This precursor is cleaved to the mature heptapeptide iCF10 during the secretion process.
KeywordMeSH Terms
195. Trieu-Cuot  P, Courvalin  P,     ( 1983 )

Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III.

Gene 23 (3)
PMID : 6313476  :   DOI  :   10.1016/0378-1119(83)90022-7    
Abstract >>
We have cloned in Escherichia coli and sequenced a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The resistance gene was located by analysis of the initiation and termination codons in an open reading frame (ORF) of 792 bp. The deduced gene product, a 3'5''-aminoglycoside phosphotransferase of type III, has an Mr of 29,200. Comparison of its amino acid sequence with those of type I (Oka et al., 1981) and type II (Beck et al., 1982) 3' phosphotransferase, from transposable elements Tn903 and Tn5, respectively, indicated a statistically significant structural relationship between these enzymes from phylogenetically remote bacterial genera. The degree of homology observed indicate that phosphotransferase type III and type I genes have diverged from a common ancestor and that the phosphotransferase type II gene has emerged more recently from the type I evolutionary pathway.
KeywordMeSH Terms
R Factors
196. Weaver  KE, Tritle  DJ,     ( 1994 )

Identification and characterization of an Enterococcus faecalis plasmid pAD1-encoded stability determinant which produces two small RNA molecules necessary for its function.

Plasmid 32 (2)
PMID : 7531349  :   DOI  :   10.1006/plas.1994.1053    
Abstract >>
A determinant, designated par, essential for stable maintenance for an autonomously replicating fragment of the Enterococcus faecalis plasmid pAD1, was identified by transposon mutagenesis, and its DNA sequence was determined. The position of flanking transposon inserts with no effect on stability indicates that par is encoded on no more than approximately 720 bp of DNA. This region contains no large open reading frames (> 62 amino acids) but does contain a number of direct and inverted repeats and GC- and AT-rich boxes. Disruption of these elements by transposon insertion, or deletion of the entire cluster of elements, resulted in a loss of plasmid stability but did not affect replication or copy number in E. faecalis. Characterization of selected mutants suggested that some manipulations of par may interfere with essential plasmid replication functions and/or be lethal to the host cell. Northern blot analysis revealed that two small RNA molecules of approximately 250 and 145 nucleotides homologous to par are produced by cells containing the complete pAD1 replicon. Mutations affecting par that resulted in a decrease in plasmid stability also resulted in changes in the pattern of production of the par RNAs, suggesting that these RNAs are important for par function.
KeywordMeSH Terms
197. Perkins  JB, Youngman  PJ,     ( 1984 )

A physical and functional analysis of Tn917, a Streptococcus transposon in the Tn3 family that functions in Bacillus.

Plasmid 12 (2)
PMID : 6095351  :  
Abstract >>
The erythromycin-resistance (Emr)-conferring transposon Tn917, first isolated in the genus Streptococcus, has in previous work been shown to function efficiently in the spore-forming species Bacillus subtilis, where it has been developed as a tool for identifying and studying sporulation genes. In the present work, a physical analysis of Tn917 was undertaken, including detailed restriction mapping, chemical DNA sequencing, heteroduplex studies, and Southern hybridization analysis, as a first step in understanding the genetic organization of this useful insertion element. The location and transcriptional orientation of the transposon-borne erm gene (the gene responsible for the Emr phenotype) have been determined, and a partial sequence of DNA 5' to the coding sequence of this gene indicates that its inducibility is probably the result of "translational attenuation," a mechanism known to be responsible for the regulation of at least two other gram-positive erm genes. Restriction mapping and heteroduplex analysis have revealed extensive homology between Tn917 and the Staphylococcus transposon Tn551, throughout virtually their entire lengths, and DNA sequencing studies have revealed a remarkably high degree of sequence correspondence within the terminal inverted repeats of Tn917, Tn551 and the gram-negative transposon Tn3. Tn917 was also shown to generate a 5-bp duplication upon insertion, as do Tn3 and Tn551 (and all of the other Tn3-related elements studied thus far), strengthening the conclusion that these three transposons are members of a highly dispersed family of related insertion elements which populate both gram-positive and gram-negative genera.
KeywordMeSH Terms
DNA Transposable Elements
198. Mori  M, Tanaka  H, Sakagami  Y, Isogai  A, Fujino  M, Kitada  C, White  BA, An  FY, Clewell  DB, Suzuki  A,     ( 1986 )

Isolation and structure of the Streptococcus faecalis sex pheromone, cAM373.

FEBS letters 206 (1)
PMID : 3093276  :   DOI  :   10.1016/0014-5793(86)81342-4    
Abstract >>
The Streptococcus faecalis sex pheromone, cAM373, which induces a mating response of donor cells harboring plasmid pAM373 and is also produced by Staphylococcus aureus, was isolated and its structure determined. Supernatant from an overnight culture of a recipient strain was subjected to successive purification procedures, and 4.4 micrograms cAM373 was obtained. The isolated pheromone showed activity at a concentration as low as 5 X 10(-11) M. Sequence analysis indicated that cAM373 was a heptapeptide, H-Ala-Ile-Phe-Ile-Leu-Ala-Ser-OH, and that its Mr was 733. A synthetic replicate of the peptide showed the same biological activity and chromatographic behavior as the native cAM373.
KeywordMeSH Terms
199. Rehman  S, Li  YG, Schmitt  A, Lassinantti  L, Christie  PJ, Berntsson  RP,     ( 2019 )

Enterococcal PcfF Is a Ribbon-Helix-Helix Protein That Recruits the Relaxase PcfG Through Binding and Bending of the oriT Sequence.

Frontiers in microbiology 10 (N/A)
PMID : 31134011  :   DOI  :   10.3389/fmicb.2019.00958     PMC  :   PMC6514445    
Abstract >>
The conjugative plasmid pCF10 from Enterococcus faecalis encodes a Type 4 Secretion System required for plasmid transfer. The accessory factor PcfF and relaxase PcfG initiate pCF10 transfer by forming the catalytically active relaxosome at the plasmid's origin-of-transfer (oriT) sequence. Here, we report the crystal structure of the homo-dimeric PcfF, composed of an N-terminal DNA binding Ribbon-Helix-Helix (RHH) domain and a C-terminal stalk domain. We identified key residues in the RHH domain that are responsible for binding pCF10's oriT sequence in vitro, and further showed that PcfF bends the DNA upon oriT binding. By mutational analysis and pull-down experiments, we identified residues in the stalk domain that contribute to interaction with PcfG. PcfF variant proteins defective in oriT or PcfG binding attenuated plasmid transfer in vivo, but also suggested that intrinsic or extrinsic factors might modulate relaxosome assembly. We propose that PcfF initiates relaxosome assembly by binding oriT and inducing DNA bending, which serves to recruit PcfG as well as extrinsic factors necessary for optimal plasmid processing and engagement with the pCF10 transfer machine.
KeywordMeSH Terms
T4SS
X-ray crystallography
accessory factor
conjugation
protein structural and functional analysis
relaxosome
200. Brehm  J, Salmond  G, Minton  N,     ( 1987 )

Sequence of the adenine methylase gene of the Streptococcus faecalis plasmid pAM beta 1.

Nucleic acids research 15 (7)
PMID : 3104884  :   DOI  :   10.1093/nar/15.7.3177     PMC  :   PMC340918    
Abstract >>
N/A
KeywordMeSH Terms
Genes
Genes, Bacterial
Plasmids
201. Rushton-Green  R, Darnell  RL, Taiaroa  G, Carter  GP, Cook  GM, Morgan  XC,     ( 2019 )

Agricultural Origins of a Highly Persistent Lineage of Vancomycin-Resistant Enterococcus faecalis in New Zealand.

Applied and environmental microbiology 85 (13)
PMID : 31028029  :   DOI  :   10.1128/AEM.00137-19     PMC  :   PMC6581176    
Abstract >>
Enterococcus faecalis and Enterococcus faecium are human and animal gut commensals. Vancomycin-resistant enterococci (VRE) are important opportunistic pathogens with limited treatment options. Historically, the glycopeptide antibiotics vancomycin and avoparcin selected for the emergence of vancomycin resistance in human and animal isolates, respectively, resulting in global cessation of avoparcin use between 1997 and 2000. To better understand human- and animal-associated VRE strains in the postavoparcin era, we sequenced the genomes of 231 VRE isolates from New Zealand (NZ; 75 human clinical, 156 poultry) cultured between 1998 and 2009. E. faecium lineages and their antibiotic resistance carriage patterns strictly delineated between agricultural and human reservoirs, with bacitracin resistance ubiquitous in poultry but absent in clinical E. faecium strains. In contrast, one E. faecalis lineage (ST108) predominated in both poultry and human isolates in the 3 years following avoparcin discontinuation. Both phylogenetic and antimicrobial susceptibility (i.e., ubiquitous bacitracin resistance in both poultry and clinical ST108 isolates) analyses suggest an agricultural origin for the ST108 lineage. VRE isolate resistomes were carried on multiple, heterogeneous plasmids. In some isolate genomes, bacitracin, erythromycin, and vancomycin resistance elements were colocalized, indicating multiple potentially linked selection mechanisms.IMPORTANCE Historical antimicrobial use in NZ agriculture has driven the evolution of ST108, a VRE lineage carrying a range of clinically relevant antimicrobial resistances. The persistence of this lineage in NZ for over a decade indicates that coselection may be an important stabilizing mechanism for its persistence.
KeywordMeSH Terms
Enterococcus
VRE
antimicrobial
bacitracin
faecalis
faecium
genomics
phylogeny
vancomycin
Enterococcus
VRE
antimicrobial
bacitracin
faecalis
faecium
genomics
phylogeny
vancomycin
202. Chen  M, Pan  H, Lou  Y, Wu  Z, Zhang  J, Huang  Y, Yu  W, Qiu  Y,     ( 2018 )

Epidemiological characteristics and genetic structure of linezolid-resistant Enterococcus faecalis.

Infection and drug resistance 11 (N/A)
PMID : 30538507  :   DOI  :   10.2147/IDR.S181339     PMC  :   PMC6251436    
Abstract >>
The aim of this study was to investigate the mechanism of linezolid resistance and evaluate the risk factors for linezolid-resistant Enterococcus faecalis (LZR-Efa) infections. A total of 730 E. faecalis isolates were collected, and whole-genome sequencing and bioinformatics analysis were performed. Meanwhile, risk factors related to linezolid resistance were analyzed by binary logistic regression. Twenty-six LZR-Efa were isolated from various clinical samples, and 24 isolates were multidrug resistant. Four isolates were daptomycin nonsusceptible, while all LZR-Efa were susceptible to vancomycin. Thirteen different sequence types (STs) were identified, and the most prevalent type was ST16 (23.1%). The genes dfrE, lsaA, and emeA were identified in all isolates. A total of 23 E. faecalis were positive for optrA gene, and six amino acids mutations were identified among 18 LZR-Efa in OptrA. The 23S rRNA mutation was found in 16 LZR-Efa isolates. However, the presence of cfr was not identified. Furthermore, there were 41 virulence genes detected, and 10 genes (ace, bopD, cpsA, cpsB, ebpB, ebpC, efaA, fss1, fss2, and srtC) were found in all isolates. A total of nine isolates were positive for multiple virulent factors (ace, asa1, cylA, efaA, esp, and gelE). There was no difference in the number of virulence factors among different specimens (P=0.825). It is of note that all patients had not been prescribed linezolid or traveled abroad previously. Moreover, previous use of carbapenems was a risk factor for LZR-Efa infections. The main trends of LZR-Efa, with lower level of resistance, were sporadic mainly in the department of surgery. optrA and 23S rRNA were the main resistance mechanisms. In addition, carbapenems use was an independent predictor of LZR-Efa infections.
KeywordMeSH Terms
linezolid
resistance mechanism
risk factors
virulent factors
203. Moon  TM, D'Andréa  ?D, Lee  CW, Soares  A, Jakoncic  J, Desbonnet  C, Garcia-Solache  M, Rice  LB, Page  R, Peti  W,     ( 2018 )

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into �]-lactam resistance.

The Journal of biological chemistry 293 (48)
PMID : 30355734  :   DOI  :   10.1074/jbc.RA118.006052     PMC  :   PMC6290140    
Abstract >>
The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of �]-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 ?) and PBP5 (2.7 ?) in their apo and acyl-enzyme complexes with the �]-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three �]-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.
KeywordMeSH Terms
ESKAPE pathogen
Enterococcus
antibiotic action
antibiotic resistance
crystal structure
enzyme structure
penicillin-binding proteins
transpeptidase
β-lactam
ESKAPE pathogen
Enterococcus
antibiotic action
antibiotic resistance
crystal structure
enzyme structure
penicillin-binding proteins
transpeptidase
β-lactam
beta-Lactam Resistance
204. Zhou  W, Gao  S, Xu  H, Zhang  Z, Chen  F, Shen  H, Zhang  C,     ( 2019 )

Distribution of the optrA gene in Enterococcus isolates at a tertiary care hospital in China.

Journal of global antimicrobial resistance 17 (N/A)
PMID : 30641287  :   DOI  :   10.1016/j.jgar.2019.01.001    
Abstract >>
Linezolid-resistant Enterococcus have spread worldwide. This study investigated the prevalence of linezolid-non-susceptible Enterococcus (LNSE) and the potential mechanism and molecular epidemiology of LNSE isolates from Nanjing, China. Linezolid susceptibility of 2555 Enterococcus was retrospectively determined by Etest. Vancomycin and teicoplanin MICs were determined for LNSE by Etest. PCR and DNA sequencing were used to investigate the potential molecular mechanism. Clonal relatedness between LNSE isolates was analysed by MLST. WGS was also performed. A total of 27 Enterococcus isolates (24 Enterococcus faecalis, 3 Enterococcus faecium) with linezolid MICs of 4-48�gg/mL were identified, among which 20 E. faecalis and 3 E. faecium were positive for optrA. No mutations were found in genes encoding domain V of 23S rRNA or ribosomal proteins L3/L4; the cfr gene was not found. The 24 linezolid-non-susceptible E. faecalis were classified into eight STs (ST16, ST480, ST476, ST631, ST585, ST428, ST25 and ST689). The three linezolid-non-susceptible E. faecium were classified as ST17, ST400 and ST195. Comparison of the deduced OptrA amino acid sequences of the 23 optrA-positive isolates by PCR-based sequencing and WGS with that of the original OptrA from E. faecalis E349 revealed seven variants (KD, EDP, EDM, D, EDD, RDK and DP) in 16 isolates, with no mutations in the remaining 7 isolates. optrA was found downstream of fexA by searching the pE349 sequence based on WGS data. Emergence of LNSE with optrA-mediated resistance and clonal dissemination of ST16 E. faecalis in our hospital may pose a potential public-health threat.
KeywordMeSH Terms
Cfr gene
Enterococcus
Linezolid
Non-susceptible
OptrA
Resistance
WGS
Cfr gene
Enterococcus
Linezolid
Non-susceptible
OptrA
Resistance
WGS
205. Hung  WW, Chen  YH, Tseng  SP, Jao  YT, Teng  LJ, Hung  WC,     ( 2019 )

Using groEL as the target for identification of Enterococcus faecium clades and 7 clinically relevant Enterococcus species.

Journal of microbiology, immunology, and infection = Wei mian yu gan ran za zhi 52 (2)
PMID : 30473144  :   DOI  :   10.1016/j.jmii.2018.10.008    
Abstract >>
Accurate identification is important for effective treatment because Enterococcus species have talents to cope with various antibiotics either by intrinsic resistance or by acquisition of mobile genetic elements. The groEL gene is a permissive target in identification of bacteria. We aimed to develop simple assays based on groEL for identification of enterococci. We continued our previous work and determined groEL gene sequences of Enterococcus species isolated from clinical specimens. Phylogenetic analysis based on groEL revealed that each strain clustered well with their reference strains (bootstrap value 100%), in which Enterococcusfaecium and Enterococcusgallinarum could be split into two clades. The divergence of E. faecium was coincident with hospital-associated clade, known as clade A, and community-associated clade, known as clade B. A PCR-restriction fragment length polymorphism (PCR-RFLP) assay was therefore designed to differentiate the two E. faecium clades, based on the specific RsaI cutting sites present in the two clades. To differentiate 7 clinical relevant Enterococcus species, the multiplex PCR assay was designed to identify Enterococcusavium, Enterococcuscasseliflavus, Enterococcusfaecalis, E. faecium, E. gallinarum, Enterococcushirae and Enterococcusraffinosus. Specificity was tested with other Enterococcus species including Enterococcuscecorum, Enterococcusdurans and Enterococcusmundtii. None of these bacterial species generated products of similar size to those of the seven Enterococcus species. The simple PCR-RFLP and multiplex PCR assays on the basis of groEL gene provided an alternative way to identify Enterococcus species.
KeywordMeSH Terms
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Enterococcus
Multiplex PCR
PCR-RFLP
groEL
Phylogeny
206. Mori  M, Sakagami  Y, Ishii  Y, Isogai  A, Kitada  C, Fujino  M, Adsit  JC, Dunny  GM, Suzuki  A,     ( 1988 )

Structure of cCF10, a peptide sex pheromone which induces conjugative transfer of the Streptococcus faecalis tetracycline resistance plasmid, pCF10.

The Journal of biological chemistry 263 (28)
PMID : 3139658  :  
Abstract >>
The peptide pheromone, cCF10, which induces aggregation and high frequency plasmid transfer in Streptococcus faecalis cells carrying the tetracycline resistance plasmid, pCF10, was isolated and its structure determined. The molecular weight of cCF10 is 789, and its amino acid sequence is H-Leu-Val-Thr-Leu-Val-Phe-Val-OH. Pheromone activity, as determined by a clumping induction assay, was detectable at a concentration of 2.5 x 10(-11) M. A peptide of the same sequence as that of the cCF10 produced by S. faecalis cells was synthesized by the liquid-phase method. The synthetic pheromone showed biological activity and chromatographic behavior that was identical to that of the cCF10 of bacterial origin. When the response of S. faecalis cells to various concentrations of synthetic cCF10 was monitored by measuring both the frequency of plasmid transfer and the synthesis of pheromone-inducible antigens, an excellent correlation was observed between donor ability and the appearance of a 150-kilodalton protein that appears to be involved in formation of mating aggregates. The dose-response data in the range of concentrations where the amount of pheromone became limiting (10(-11)-10(-12) M) were consistent with the notion that as few as one or two molecules per donor cell may be sufficient to induce a mating response.
KeywordMeSH Terms
Conjugation, Genetic
R Factors
207. Darnell  RL, Nakatani  Y, Knottenbelt  MK, Gebhard  S, Cook  GM,     ( 2019 )

Functional characterization of BcrR: a one-component transmembrane signal transduction system for bacitracin resistance.

Microbiology (Reading, England) 165 (4)
PMID : 30777814  :   DOI  :   10.1099/mic.0.000781    
Abstract >>
Bacitracin is a cell wall targeting antimicrobial with clinical and agricultural applications. With the growing mismatch between antimicrobial resistance and development, it is essential we understand the molecular mechanisms of resistance in order to prioritize and generate new effective antimicrobials. BcrR is a unique membrane-bound one-component system that regulates high-level bacitracin resistance in Enterococcus faecalis. In the presence of bacitracin, BcrR activates transcription of the bcrABD operon conferring resistance through a putative ATP-binding cassette (ABC) transporter (BcrAB). BcrR has three putative functional domains, an N-terminal helix-turn-helix DNA-binding domain, an intermediate oligomerization domain and a C-terminal transmembrane domain. However, the molecular mechanisms of signal transduction remain unknown. Random mutagenesis of bcrR was performed to generate loss- and gain-of-function mutants using transcriptional reporters fused to the target promoter PbcrA. Fifteen unique mutants were isolated across all three proposed functional domains, comprising 14 loss-of-function and one gain-of-function mutant. The gain-of-function variant (G64D) mapped to the putative dimerization domain of BcrR, and functional analyses indicated that the G64D mutant constitutively expresses the PbcrA-luxABCDE reporter. DNA-binding and membrane insertion were not affected in the five mutants chosen for further characterization. Homology modelling revealed putative roles for two key residues (R11 and S33) in BcrR activation. Here we present a new model of BcrR activation and signal transduction, providing valuable insight into the functional characterization of membrane-bound one-component systems and how they can coordinate critical bacterial responses, such as antimicrobial resistance.
KeywordMeSH Terms
Enterococcus
antimicrobial resistance
membrane protein
regulator
208. Deshpande  LM, Castanheira  M, Flamm  RK, Mendes  RE,     ( 2018 )

Evolving oxazolidinone resistance mechanisms in a worldwide collection of enterococcal clinical isolates: results from the SENTRY Antimicrobial Surveillance Program.

The Journal of antimicrobial chemotherapy 73 (9)
PMID : 29878213  :   DOI  :   10.1093/jac/dky188    
Abstract >>
This study evaluated the oxazolidinone resistance mechanisms among a global collection of enterococcal clinical isolates. The epidemiology of optrA-carrying isolates and the optrA genetic context were determined. Enterococcal isolates (26 648) from the SENTRY Antimicrobial Surveillance Program (2008-16) were identified by MALDI-TOF MS and MICs were determined by broth microdilution. Isolates with linezolid MICs of ?4 mg/L were screened for resistance mechanisms. Isolates carrying optrA had their genome sequenced for genetic context and epidemiology information. Thirty-six Enterococcus faecalis and 66 Enterococcus faecium had linezolid MICs of ?4 mg/L (0.38% of surveillance enterococci). E. faecalis had a linezolid MIC range of 4-16 mg/L, while E. faecium displayed higher values (4-64 mg/L). Nine E. faecalis had G2576T mutations and optrA was detected in 26 (72.2%) isolates from the Asia-Pacific region, North America, Latin America and Europe; 3 isolates also produced Cfr [Thailand (1)] or Cfr(B) [Panama (2)]. All E. faecium isolates had G2576T alterations, while three isolates from the USA had concomitant presence of cfr(B). The optrA gene was plasmid- and chromosome-located in 22 and 3 E. faecalis, respectively. One isolate signalled hybridization on plasmid and chromosome. The genetic context of optrA varied. E. faecalis belonging to the same clonal complex were detected in distinct geographical regions. Also, genetically distinct isolates from Ireland had an identical optrA context, indicating plasmid dissemination. Alterations in 23S rRNA remained the main oxazolidinone resistance mechanism in E. faecium, while optrA prevailed in E. faecalis. These results demonstrate global dissemination of optrA and warrant surveillance for monitoring.
KeywordMeSH Terms
209. Yan  J, Xia  Y, Yang  M, Zou  J, Chen  Y, Zhang  D, Ma  L,     ( 2018 )

Quantitative Proteomics Analysis of Membrane Proteins in Enterococcus faecalis With Low-Level Linezolid-Resistance.

Frontiers in microbiology 9 (N/A)
PMID : 30100900  :   DOI  :   10.3389/fmicb.2018.01698     PMC  :   PMC6072972    
Abstract >>
Despite increasing reports of low-level linezolid-resistant enterococci worldwide, the mechanism of this resistance remains poorly understood. Previous transcriptome studies of low-level linezolid-resistant Enterococcus faecalis isolates have demonstrated a number of significantly up-regulated genes potentially involved in mediation of drug resistance. However, whether the transcriptome faithfully reflects the proteome remains unknown. In this study, we performed quantitative proteomics analysis of membrane proteins in an E. faecalis isolate (P10748) with low-level linezolid-resistance in comparison with two linezolid-susceptible strains 3138 and ATCC 29212, all of which have been previously investigated by whole transcriptome analysis. A total of 8,197 peptides associated with 1,170 proteins were identified in all three isolates with false discovery rate (FDR) at 1% and P < 0.05. There were 14 significantly up-regulated and 6 significantly down-regulated proteins in strain P10748 compared to strains 3138 and ATCC 29212, which were in general positively correlated with transcription levels revealed in previous transcriptome studies. Our analysis suggests that the low-level linezolid-resistance in E. faecalis is conferred primarily by the ATP-binding cassette protein OptrA through ribosomal protection and, possibly, also by the enterococcal surface protein (Esp) and other proteins through biofilm formation. The genetic transfer of optrA is potentially regulated by the surface exclusion protein Sea1, conjugal transfer protein TraB, replication protein RepA and XRE family transcription regulator protein. This report represents the first investigation of the mechanisms of linezolid-resistance in E. faecalis by a quantitative proteomics approach.
KeywordMeSH Terms
Enterococcus faecalis
linezolid
low-level resistance
membrane proteins
quantitative proteomics
210. Schmitt  A, Jiang  K, Camacho  MI, Jonna  VR, Hofer  A, Westerlund  F, Christie  PJ, Berntsson  RP,     ( 2018 )

PrgB promotes aggregation, biofilm formation, and conjugation through DNA binding and compaction.

Molecular microbiology 109 (3)
PMID : 29723434  :   DOI  :   10.1111/mmi.13980     PMC  :   PMC6158044    
Abstract >>
Gram-positive bacteria deploy type IV secretion systems (T4SSs) to facilitate horizontal gene transfer. The T4SSs of Gram-positive bacteria rely on surface adhesins as opposed to conjugative pili to facilitate mating. Enterococcus faecalis PrgB is a surface adhesin that promotes mating pair formation and robust biofilm development in an extracellular DNA (eDNA) dependent manner. Here, we report the structure of the adhesin domain of PrgB. The adhesin domain binds and compacts DNA in vitro. In vivo PrgB deleted of its adhesin domain does not support cellular aggregation, biofilm development and conjugative DNA transfer. PrgB also binds lipoteichoic acid (LTA), which competes with DNA binding. We propose that PrgB binding and compaction of eDNA facilitates cell aggregation and plays an important role in establishment of early biofilms in mono- or polyspecies settings. Within these biofilms, PrgB mediates formation and stabilization of direct cell-cell contacts through alternative binding of cell-bound LTA, which in turn promotes establishment of productive mating junctions and efficient intra- or inter-species T4SS-mediated gene transfer.
KeywordMeSH Terms
Conjugation, Genetic
211. Martin  P, Trieu-Cuot  P, Courvalin  P,     ( 1986 )

Nucleotide sequence of the tetM tetracycline resistance determinant of the streptococcal conjugative shuttle transposon Tn1545.

Nucleic acids research 14 (17)
PMID : 3020504  :   DOI  :   10.1093/nar/14.17.7047     PMC  :   PMC311716    
Abstract >>
The nucleotide sequence of the tetracycline resistance gene tetM encoded by streptococcal conjugative shuttle transposon Tn1545 has been determined. The resistance gene was identified as a coding sequence of 1917 base pairs corresponding to a protein with a Mr of 72,500 daltons. This value is in good agreement with that, 68,000 daltons, estimated by SDS-polyacrylamide gel electrophoresis of Escherichia coli minicell extracts. The tetM gene product does not exhibit any sequence homology with either the Gram-negative (tetA, tetB and tetC), or the Bacillus and Staphylococcus tetracycline resistance proteins. The average hydropathy value of the tetM gene product (-0.21) contrasts with those calculated for the other TET proteins which are markedly hydrophobic (0.76 to 0.93). Hybridization experiments performed with an intragenic tetM probe do not support the claim [Taylor, D. (1986), J. Bact. 165, 1037-1039)] that tetracycline resistance in Campylobacter is due to acquisition of tetM.
KeywordMeSH Terms
DNA Transposable Elements
Tetracycline
212. Cai  J, Schwarz  S, Chi  D, Wang  Z, Zhang  R, Wang  Y,     ( 2019 )

Faecal carriage of optrA-positive enterococci in asymptomatic healthy humans in Hangzhou, China.

Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 25 (5)
PMID : 30076974  :   DOI  :   10.1016/j.cmi.2018.07.025    
Abstract >>
To investigate the faecal carriage of optrA-positive enterococci among asymptomatic healthy humans in Hangzhou, China, and to characterize the genetic context of optrA. A total of 3458 stool samples from healthy individuals were collected and cultured on a selective medium containing 10 mg/L florfenicol and resulting enterococci were screened for the presence of optrA by PCR. OptrA variants were determined by amino acid sequence comparison with the original OptrA from Enterococcus faecalis E349. Whole genome sequencing and PCR mapping were performed to obtain and analyse the genetic environment of optrA. Similar optrA carriage rates (?3.5%) were detected in samples from adults (55/1558) and children (66/1900). Linezolid resistance rates for E. faecalis, Enterococcus faecium and other Enterococcus species were 58.5% (38/65), 42.3% (11/26) and 0% (0/31), respectively. Nineteen OptrA variants exhibiting different linezolid MICs were identified. Isolates carrying wild-type OptrA and variants RDK, KLDP, KD, D, RDKP, and EDP generally demonstrated linezolid MICs ?8 mg/L. The OptrA variants, with fexA upstream and erm(A) downstream, were flanked by IS1216E at one or both ends. The fexA-optrA(wild-type) was located downstream of a Tn554 transposon, and was inserted into the radC gene. The EDM variant was detected in 31/73 enterococci with linezolid MICs ?4 mg/L. Despite the variable genetic context, Tn558-araC-optrA(EDM)-erm(A)-met was the most common gene array. This study revealed a correlation between linezolid MIC, genetic context and OptrA variant. Intestinal colonization of healthy individuals by optrA-positive enterococci is a concern, and active epidemiological surveillance of optrA is warranted.
KeywordMeSH Terms
Enterococcus
Genetic context
Intestinal colonization
OptrA variant
Oxazolidinone resistance
Enterococcus
Genetic context
Intestinal colonization
OptrA variant
Oxazolidinone resistance
Enterococcus
Genetic context
Intestinal colonization
OptrA variant
Oxazolidinone resistance
Drug Resistance, Bacterial
213. Kohler  V, Goessweiner-Mohr  N, Aufschnaiter  A, Fercher  C, Probst  I, Pavkov-Keller  T, Hunger  K, Wolinski  H, Büttner  S, Grohmann  E, Keller  W,     ( 2018 )

TraN: A novel repressor of an Enterococcus conjugative type IV secretion system.

Nucleic acids research 46 (17)
PMID : 30060171  :   DOI  :   10.1093/nar/gky671     PMC  :   PMC6158623    
Abstract >>
The dissemination of multi-resistant bacteria represents an enormous burden on modern healthcare. Plasmid-borne conjugative transfer is the most prevalent mechanism, requiring a type IV secretion system that enables bacteria to spread beneficial traits, such as resistance to last-line antibiotics, among different genera. Inc18 plasmids, like the Gram-positive broad host-range plasmid pIP501, are substantially involved in propagation of vancomycin resistance from Enterococci to methicillin-resistant strains of Staphylococcus aureus. Here, we identified the small cytosolic protein TraN as a repressor of the pIP501-encoded conjugative transfer system, since deletion of traN resulted in upregulation of transfer factors, leading to highly enhanced conjugative transfer. Furthermore, we report the complex structure of TraN with DNA and define the exact sequence of its binding motif. Targeting this protein-DNA interaction might represent a novel therapeutic approach against the spreading of antibiotic resistances.
KeywordMeSH Terms
Conjugation, Genetic
214. Shaw  JH, Clewell  DB,     ( 1985 )

Complete nucleotide sequence of macrolide-lincosamide-streptogramin B-resistance transposon Tn917 in Streptococcus faecalis.

Journal of bacteriology 164 (2)
PMID : 2997130  :   PMC  :   PMC214320    
Abstract >>
Streptococcus faecalis transposon Tn917 was cloned in Escherichia coli on plasmid vector pBR325. The erythromycin resistance determinant of Tn917 was not expressed in the E. coli background. The nucleotide sequence of Tn917 was determined and found to be 5,257 base pairs in length. Six open reading frames (ORFs) were identified and designated 1 through 6 (5' to 3'); all were on the same DNA strand. A region exhibiting strong homology with known promoters was identified upstream from ORF1. ORFs 1 to 3 were virtually identical to the previously sequenced erythromycin resistance determinant on Streptococcus sanguis plasmid pAM77. At the 3' point, where the homology between Tn917 and pAM77 ends, was a 20-base-pair region about 80% homologous with a component of the res site of Tn3. The amino acid sequence of ORF4 showed homology with other site-specific recombination enzymes, including approximately 30% homology with the resolvase of Tn3. Contained within Tn917 was a directly oriented 73-base-pair duplication of the left terminus. The Tn917 sequence revealed that antibiotic-enhanced transposition might be due to extension of transcription from the resistance-related genes (in ORFs 1 to 3) into transposition genes (in ORFs 4 to 6). Transcription analyses resulted in data consistent with this interpretation.
KeywordMeSH Terms
DNA Transposable Elements
Macrolides
215. Rubio-Cosials  A, Schulz  EC, Lambertsen  L, Smyshlyaev  G, Rojas-Cordova  C, Forslund  K, Karaca  E, Bebel  A, Bork  P, Barabas  O,     ( 2018 )

Transposase-DNA Complex Structures Reveal Mechanisms for Conjugative Transposition of Antibiotic Resistance.

Cell 173 (1)
PMID : 29551265  :   DOI  :   10.1016/j.cell.2018.02.032     PMC  :   PMC5871717    
Abstract >>
Conjugative transposition drives the emergence of multidrug resistance in diverse bacterial pathogens, yet the mechanisms are poorly characterized. The Tn1549 conjugative transposon propagates resistance to the antibiotic vancomycin used for severe drug-resistant infections. Here, we present four high-resolution structures of the conserved Y-transposase of Tn1549 complexed with circular transposon DNA intermediates. The structures reveal individual transposition steps and explain how specific DNA distortion and cleavage mechanisms enable DNA strand exchange with an absolute minimum homology requirement. This appears to uniquely allow Tn916-like conjugative transposons to bypass DNA homology and insert into diverse genomic sites, expanding gene transfer. We further uncover a structural regulatory mechanism that prevents premature cleavage of the transposon DNA before a suitable target DNA is found and generate a peptide antagonist that interferes with the transposase-DNA structure to block transposition. Our results reveal mechanistic principles of conjugative transposition that could help control the spread of antibiotic resistance genes.
KeywordMeSH Terms
DNA complex
Tn1549 transposon
Tn916-like transposon family
antibiotic resistance
conjugative transposition
crystallography
gene transfer
multidrug-resistant bacteria
tyrosine recombinase
vancomycin
216. Rice  LB, Desbonnet  C, Tait-Kamradt  A, Garcia-Solache  M, Lonks  J, Moon  TM, D'Andréa  ?D, Page  R, Peti  W,     ( 2018 )

Structural and Regulatory Changes in PBP4 Trigger Decreased �]-Lactam Susceptibility in Enterococcus faecalis.

mBio 9 (2)
PMID : 29615500  :   DOI  :   10.1128/mBio.00361-18     PMC  :   PMC5885037    
Abstract >>
Enterococcus faecalis strains resistant to penicillin and ampicillin are rare and have been associated with increases in quantities of low-affinity penicillin-binding protein 4 (PBP4) or with amino acid substitutions in PBP4. We report an E. faecalis strain (LS4828) isolated from a prosthetic knee joint that was subjected to long-term exposure to aminopenicillins. Subsequent cultures yielded E. faecalis with MICs of penicillins and carbapenems higher than those for wild-type strain E. faecalis JH2-2. Sequence analysis of the pbp4 gene of LS4828 compared to that of JH2-2 revealed two point mutations with amino acid substitutions (V223I, A617T) and deletion of an adenine from the region upstream of the predicted pbp4 -35 promoter sequence (UP region). Purified PBP4 from LS4828 exhibited less affinity for Bocillin FL than did PBP4 from JH2-2, which was recapitulated by purified PBP4 containing only the A617T mutation. Differential scanning fluorimetry studies showed that the LS4828 and A617T variants are destabilized compared to wild-type PBP4. Further, reverse transcription-PCR indicated increased transcription of pbp4 in LS4828 and Western blot analysis with polyclonal PBP4 antibody revealed greater quantities of PBP4 in LS4828 than in JH2-2 lysates and membrane preparations. Placing the promoter regions from LS4828 or JH2-2 upstream of a green fluorescent protein reporter gene confirmed that the adenine deletion was associated with increased transcription. Together, these data suggest that the reduced susceptibility to �]-lactam antibiotics observed in E. faecalis LS4828 results from a combination of both increased expression and remodeling of the active site, resulting in reduced affinity for penicillins and carbapenems.IMPORTANCEEnterococcus faecalis is an important cause of community-acquired and nosocomial infections and creates therapeutic dilemmas because of its frequent resistance to several classes of antibiotics. We report an E. faecalis strain with decreased ampicillin and imipenem susceptibility isolated after prolonged courses of aminopenicillin therapy for a prosthetic joint infection. Its reduced susceptibility is attributable to a combination of increased quantities of low-affinity PBP4 and an amino acid substitution in proximity to the active site that destabilizes the protein. Our findings provide a cautionary tale for clinicians who elect to "suppress" infections in prosthetic joints and offer novel insights into the interaction of �]-lactam antibiotics with low-affinity PBP4. These insights will help inform future efforts to develop therapeutics capable of inhibiting clinical enterococcal strains.
KeywordMeSH Terms
Enterococcus
antibiotic resistance
penicillin-binding proteins
beta-Lactam Resistance
217. Shin  E, Mduma  S, Keyyu  J, Fyumagwa  R, Lee  Y,     ( 2017 )

An Investigation of Enterococcus Species Isolated from the African Buffalo (Syncerus caffer) in Serengeti National Park, Tanzania.

Microbes and environments 32 (4)
PMID : 29081464  :   DOI  :   10.1264/jsme2.ME17025     PMC  :   PMC5745028    
Abstract >>
We isolated Enterococcus species that colonized in the African buffalo (Syncerus caffer) in order to investigate their genetic relatedness and antimicrobial susceptibility. A total of 219 isolates were obtained and a 16S rRNA gene sequence analysis showed they were classified into Enterococcus avium, E. casseliflavus, E. faecalis, E. faecium, E. hirae, or E. mundtii. Multilocus sequence typing of E. faecalis and E. faecium isolates indicated that some of the isolates showed an evolutionary distance that was far from the primary founders. The antimicrobial susceptibility of the enterococcal isolates suggested that the significant transmission of antimicrobial resistance via human intervention had not yet occurred.
KeywordMeSH Terms
African buffalo
Enterococcus species
MLST
antimicrobial susceptibility
phylogeny
African buffalo
Enterococcus species
MLST
antimicrobial susceptibility
phylogeny
218.     ( 2013 )

Cold denaturation of a protein dimer monitored at atomic resolution.

Nature chemical biology 9 (4)
PMID : 23396077  :   DOI  :   10.1038/nchembio.1181     PMC  :   PMC5521822    
Abstract >>
Protein folding and unfolding are crucial for a range of biological phenomena and human diseases. Defining the structural properties of the involved transient species is therefore of prime interest. Using a combination of cold denaturation with NMR spectroscopy, we reveal detailed insight into the unfolding of the homodimeric repressor protein CylR2. Seven three-dimensional structures of CylR2 at temperatures from 25 �XC to -16 �XC reveal a progressive dissociation of the dimeric protein into a native-like monomeric intermediate followed by transition into a highly dynamic, partially folded state. The core of the partially folded state seems critical for biological function and misfolding.
KeywordMeSH Terms
219.     ( 2013 )

Presence of the vanC1 gene in a vancomycin-resistant Enterococcus faecalis strain isolated from ewe bulk tank milk.

Journal of medical microbiology 62 (Pt 3)
PMID : 23161771  :   DOI  :   10.1099/jmm.0.052274-0    
Abstract >>
N/A
KeywordMeSH Terms
Vancomycin Resistance
220.     ( 2013 )

Transferable multiresistance plasmids carrying cfr in Enterococcus spp. from swine and farm environment.

Antimicrobial agents and chemotherapy 57 (1)
PMID : 23070165  :   DOI  :   10.1128/AAC.01605-12     PMC  :   PMC3535926    
Abstract >>
Seventy-seven porcine Enterococcus isolates with florfenicol MICs of ?16 �gg of were/ml screened for the presence of the multiresistance gene cfr, its location on plasmids, and its genetic environment. Three isolates-Enterococcus thailandicus 3-38 (from a porcine rectal swab collected at a pig farm), Enterococcus thailandicus W3, and Enterococcus faecalis W9-2 (the latter two from sewage at a different farm), carried the cfr gene. The SmaI pulsed-field gel electrophoresis patterns of the three isolates differed distinctly. In addition, E. faecalis W9-2 was assigned to a new multilocus sequence type ST469. Mating experiments and Southern blot analysis indicated that cfr is located on conjugative plasmids pW3 (?75 kb) from E. thailandicus W3, p3-38 (?72 kb) from E. thailandicus 3-38, and pW9-2 (?55 kb) from E. faecalis W9-2; these plasmids differed in their sizes, additional resistance genes, and the analysis of the segments encompassing the cfr gene. Sequence analysis revealed that all plasmids harbored a 4,447-bp central region, in which cfr was bracketed by two copies of the novel insertion sequence ISEnfa4 located in the same orientation. The sequences flanking the central regions of these plasmids, including the partial tra gene regions and a �s-�`-�a toxin-antitoxin module, exhibited >95% nucleotide sequence identity to the conjugative plasmid pAM�]1 from E. faecalis. Conjugative plasmids carrying cfr appear to play an important role in the dissemination and maintenance of the multiresistance gene cfr among enterococcal isolates and possibly other species of Gram-positive bacteria.
KeywordMeSH Terms
Conjugation, Genetic
Plasmids
221.     ( 2013 )

Characterization of plasmid pML21 of Enterococcus faecalis ML21 from koumiss.

Current microbiology 66 (2)
PMID : 23090644  :   DOI  :   10.1007/s00284-012-0255-8    
Abstract >>
N/A
KeywordMeSH Terms
Plasmids
222.     ( 2012 )

Use of tuf as a target for sequence-based identification of Gram-positive cocci of the genus Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus.

Annals of clinical microbiology and antimicrobials 11 (N/A)
PMID : 23181410  :   DOI  :   10.1186/1476-0711-11-31     PMC  :   PMC3533577    
Abstract >>
Accurate identification of isolates belonging to genus Enterococcus, Streptococcus, coagulase-negative Staphylococcus, and Lactococcus at the species level is necessary to provide a better understanding of their pathogenic potential, to aid in making clinical decisions, and to conduct epidemiologic investigations,especially when large blind samples must be analyzed. It is useful to simultaneously identify species in different genera using a single primer pair. We developed a primer pair based on the tuf gene (encoding elongation factor) sequence to identify 56 Gram-positive cocci isolates. The target sequences were amplified from all 56 samples. The sequencing results and the phylogenetic tree derived from the partial tuf gene sequences identified the isolates as three enterococcal species, two lactococcal species, two staphylococcal species, and six streptococcal species, as well as eight isolates that were novel species of the genus Streptococcus. Partial gene sequence analysis of the sodA, dnaK, and 16S RNA genes confirmed the results obtained by tuf gene sequencing. Based on the uniform amplification of the tuf gene from all samples and the ability to identify all isolates at both the genus and species levels, we conclude that the primer pair developed in this research provides a powerful tool for identifying these organisms in clinical laboratories where large blind samples are used.
KeywordMeSH Terms
223.     ( 2012 )

Structural and functional characterization of VanG D-Ala:D-Ser ligase associated with vancomycin resistance in Enterococcus faecalis.

The Journal of biological chemistry 287 (45)
PMID : 22969085  :   DOI  :   10.1074/jbc.M112.405522     PMC  :   PMC3488035    
Abstract >>
d-Alanyl:d-lactate (d-Ala:d-Lac) and d-alanyl:d-serine ligases are key enzymes in vancomycin resistance of Gram-positive cocci. They catalyze a critical step in the synthesis of modified peptidoglycan precursors that are low binding affinity targets for vancomycin. The structure of the d-Ala:d-Lac ligase VanA led to the understanding of the molecular basis for its specificity, but that of d-Ala:d-Ser ligases had not been determined. We have investigated the enzymatic kinetics of the d-Ala:d-Ser ligase VanG from Enterococcus faecalis and solved its crystal structure in complex with ADP. The overall structure of VanG is similar to that of VanA but has significant differences mainly in the N-terminal and central domains. Based on reported mutagenesis data and comparison of the VanG and VanA structures, we show that residues Asp-243, Phe-252, and Arg-324 are molecular determinants for d-Ser selectivity. These residues are conserved in both enzymes and explain why VanA also displays d-Ala:d-Ser ligase activity, albeit with low catalytic efficiency in comparison with VanG. These observations suggest that d-Ala:d-Lac and d-Ala:d-Ser enzymes have evolved from a common ancestral d-Ala:d-X ligase. The crystal structure of VanG showed an unusual interaction between two dimers involving residues of the omega loop that are deeply anchored in the active site. We constructed an octapeptide mimicking the omega loop and found that it selectively inhibits VanG and VanA but not Staphylococcus aureus d-Ala:d-Ala ligase. This study provides additional insight into the molecular evolution of d-Ala:d-X ligases and could contribute to the development of new structure-based inhibitors of vancomycin resistance enzymes.
KeywordMeSH Terms
Protein Structure, Tertiary
Vancomycin Resistance
224.     ( 2012 )

Structure and mode of peptide binding of pheromone receptor PrgZ.

The Journal of biological chemistry 287 (44)
PMID : 22948145  :   DOI  :   10.1074/jbc.M112.386334     PMC  :   PMC3481316     DOI  :   10.1074/jbc.M112.386334     PMC  :   PMC3481316    
Abstract >>
We present the crystal structure of the pheromone receptor protein PrgZ from Enterococcus faecalis in complex with the heptapeptide cCF10 (LVTLVFV), which is used in signaling between conjugative recipient and donor cells. Comparison of PrgZ with homologous oligopeptide-binding proteins (AppA and OppA) explains the high specificity of PrgZ for hydrophobic heptapeptides versus the promiscuity of peptide binding in the homologous proteins.
KeywordMeSH Terms
Enterococcus faecalis
Enterococcus faecalis
225.     ( 2013 )

The 2.5 ? structure of the enterococcus conjugation protein TraM resembles VirB8 type IV secretion proteins.

The Journal of biological chemistry 288 (3)
PMID : 23188825  :   DOI  :   10.1074/jbc.M112.428847     PMC  :   PMC3548508    
Abstract >>
Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gram-negative (G-) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G+), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G+ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 ? resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G-) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G+). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G+ and G- bacteria, we propose a new classification scheme of VirB8-like proteins.
KeywordMeSH Terms
Conjugation, Genetic
226.     ( 1998 )

Induction of ermAMR from a clinical strain of Enterococcus faecalis by 16-membered-ring macrolide antibiotics.

Journal of bacteriology 180 (21)
PMID : 9791136  :   PMC  :   PMC107645    
Abstract >>
We cloned the MLSB resistance determinant by PCR from a clinical isolate of Enterococcus faecalis 373, which is induced more strongly by a 16-membered-ring macrolide, tylosin, than by erythromycin. To elucidate the molecular basis of resistance of E. faecalis 373, we analyzed the cloned gene, designated ermAMR, by site-directed mutagenesis and reporter gene assay. Our results showed that an arginine-to-cysteine change in the seventh codon of the putative leader peptide endowed tylosin with resistance inducibility and that TAAA duplication enabled the control region to express the downstream methylase gene at a drastically increased level.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
227.     ( 2012 )

Different genetic supports for the tet(S) gene in Enterococci.

Antimicrobial agents and chemotherapy 56 (11)
PMID : 22908170  :   DOI  :   10.1128/AAC.00758-12     PMC  :   PMC3486547    
Abstract >>
The diversity of tet(S) genetic contexts of 13 enterococci from human, animal, and environmental samples from different geographical areas is reported. The tet(S) gene was linked to either CTn6000 variants of chromosomal location or composite platforms flanked by IS1216 located on plasmids (?40 to 115 kb). The comparative analysis of all tet(S) genetic elements available in the GenBank databases suggests that CTn6000 might be the origin of a variety of tet(S)-carrying platforms that were mobilized to different plasmids.
KeywordMeSH Terms
DNA Transposable Elements
Genes, Bacterial
Plasmids
228.     ( 1999 )

The Enterococcus faecalis pyr operon is regulated by autogenous transcriptional attenuation at a single site in the 5' leader.

Journal of bacteriology 181 (4)
PMID : 9973361  :   PMC  :   PMC93512    
Abstract >>
The 5' end of the Enterococcus faecalis pyr operon specifies, in order, the promoter, a 5' untranslated leader, the pyrR gene encoding the regulatory protein for the operon, a 39-nucleotide (nt) intercistronic region, the pyrP gene encoding a uracil permease, a 13-nt intercistronic region, and the pyrB gene encoding aspartate transcarbamylase. The 5' leader RNA is capable of forming stem-loop structures involved in attenuation control of the operon. No attenuation regions, such as those found in the Bacillus subtilis pyr operon, are present in the pyrR-pyrP or pyrP-pyrB intercistronic regions. Several lines of evidence demonstrate that the E. faecalis pyr operon is repressed by uracil via transcriptional attenuation at the single 5' leader termination site and that attenuation is mediated by the PyrR protein.
KeywordMeSH Terms
5' Untranslated Regions
Bacterial Proteins
Operon
229.     ( 1999 )

Infection-derived Enterococcus faecalis strains are enriched in esp, a gene encoding a novel surface protein.

Infection and immunity 67 (1)
PMID : 9864215  :   PMC  :   PMC96296    
Abstract >>
We report the identification of a new cell wall-associated protein of Enterococcus faecalis. Studies on the distribution of the gene encoding this novel surface protein, Esp, reveal a significant (P < 0.001) enrichment in infection-derived E. faecalis isolates. Interestingly, the esp gene was not identified in any of 34 clinical E. faecium isolates or in 4 other less pathogenic enterococcal species tested. Analysis of the structural gene among various E. faecalis isolates reveals the existence of alternate forms of expression of the Esp protein. The deduced primary structure of the Esp protein from strain MMH594, inferred to be 1,873 amino acids (aa) with a predicted mass of approximately 202 kDa, reveals a core region consisting of repeat units that make up 50% of the protein. Esp bears global organizational similarity to the Rib and C alpha proteins of group B streptococci. Identity among Esp, Rib, and C alpha proteins is strikingly localized to a stretch of 13 aa within repeats of similar length. The high degree of conservation of this 13-residue sequence suggests that it plays an important role in the natural selection for this trait among infection-derived E. faecalis and group B streptococcal isolates.
KeywordMeSH Terms
Antigens, Bacterial
Genes, Bacterial
230.     ( 1998 )

In vivo relations between pAMbeta1-encoded type I topoisomerase and plasmid replication.

Molecular microbiology 28 (5)
PMID : 9663686  :   DOI  :   10.1046/j.1365-2958.1998.00862.x    
Abstract >>
A number of large extrachromosomal elements encode prokaryotic type I topoisomerases of unknown functions. Here, we analysed the topoisomerase Topbeta encoded by the Gram-positive broad-host-range plasmid pAMbeta1. We show that this enzyme possesses the DNA relaxation activity of type I topoisomerases. Interestingly, it is active only on plasmids that use DNA polymerase I to initiate replication, such as pAMbeta1, and depends on the activity of this polymerase. This is the first example, to our knowledge, of prokaryotic type I topoisomerase that is specific for a given type of replicon. During pAMbeta1 replication in Bacillus subtilis cells, Topbeta promotes premature arrest of DNA polymerase I, approximately 190bp downstream of the replication initiation point. We propose that Topbeta acts on the early replication intermediates of pAMbeta1, which contain D-loops formed by DNA polymerase I-mediated strand displacement. The possible role of the resulting DNA Pol I arrest in plasmid replication is discussed.
KeywordMeSH Terms
DNA Replication
DNA, Bacterial
Plasmids
231.     ( 1998 )

Conservation of the major cold shock protein in lactic acid bacteria.

Current microbiology 37 (5)
PMID : 9767713  :  
Abstract >>
Primers designed from consensus regions of the major cold shock gene of different bacterial species were used in PCR amplification of Lactic Acid Bacteria (LAB). An appropriately-sized PCR product was obtained from Lactococcus lactis subsp. lactis LL43-1 and MG1363; Lactococcus lactis subsp. cremoris LC10-1, LC11-1, and LC12-1; Streptococcus thermophilus ST1-1; Enterococcus faecalis EF1-1; Lactobacillus acidophilus LA1-1; Lactobacillus helveticus LH1-1; Pediococcus pentosaceus PP1-1; and Bifidobacterium animalis BA1-1. The PCR products were cloned and sequenced. The deduced amino acid sequences displayed high sequence similarity with the major cold shock proteins of Escherichia coli and Bacillus subtilis and the human Y-box factor. The amino acid residues of the cold shock domain implicated in nucleic acid binding in several unrelated species were also highly conserved in the LAB strains. It is possible, therefore, that this protein in LAB may also act as a transcriptional enhancer to other cold shock genes and/or act as an RNA chaperone unwinding tightly folded RNA molecules.
KeywordMeSH Terms
Cold Temperature
232.     ( 1998 )

A cluster of genes involved in polysaccharide biosynthesis from Enterococcus faecalis OG1RF.

Infection and immunity 66 (9)
PMID : 9712783  :   PMC  :   PMC108521    
Abstract >>
Our previous work identified a cosmid clone containing a 43-kb insert from Enterococcus faecalis OG1RF that produced a nonprotein antigen in Escherichia coli. In the present work, we studied this clone in detail. Periodate treatment of lysates of the clone confirmed that the antigen was carbohydrate in nature. Analysis of DNA sequences and transposon insertion mutants suggested that the insert contained a multicistronic gene cluster. Database comparison showed that the cluster contained genes similar to genes involved in the biosynthesis of dTDP-rhamnose, glycosyltransferases, and ABC transporters involved in the export of sugar polymers from both gram-positive and gram-negative organisms. Insertions in several genes within the cluster abolished the immunoreactivity of the clone. This is the first report on a gene cluster of E. faecalis involved in the biosynthesis of an antigenic polysaccharide.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
233.     ( 1999 )

Detection of a streptomycin/spectinomycin adenylyltransferase gene (aadA) in Enterococcus faecalis.

Antimicrobial agents and chemotherapy 43 (1)
PMID : 9869582  :   PMC  :   PMC89037    
Abstract >>
Genes encoding streptomycin/spectinomycin adenylyltransferases [ANT(3")(9)] have been reported to exist in gram-negative organisms and Staphylococcus aureus. During a study of high-level aminoglycoside resistance in enterococci, we encountered an isolate of Enterococcus faecalis that was streptomycin resistant but did not appear to contain the 6'-adenylyltransferase gene (aadE) when examined by PCR with specific primers. Phosphocellulose paper binding assays indicated the presence of an ANT(3")(9) enzyme. Streptomycin and spectinomycin MICs of 4,000 and 8,000 microg/ml, respectively, were observed for the isolate. PCR primers corresponding to a highly conserved region of the aadA gene were used to amplify a specific 284-bp product. The product hybridized with a digoxigenin-labeled PCR product from E. coli C600(pHP45Omega) known to contain the aadA gene. The aadA gene was transferred via filter matings from the E. faecalis donor to E. faecalis JH2-2. PCR primers designed for analysis of integrons were used to amplify a 1-kb product containing the aadA gene, which was cloned into the vector pCRII and transformed into Escherichia coli DH5-alpha competent cells. D-Rhodamine dye terminator cycle sequencing was used to determine the gene sequence, which was compared to previously reported sequences of aadA genes. We found the aadA gene in E. faecalis to be identical to the aadA genes reported by Sundstr om et al. for E. coli plasmid R6-5 (L. Sundstr?m, P. R?dstr?m, G. Swedberg, and O. Sk?ld, Mol. Gen. Genet. 213:191-201, 1988), by Fling et al. for the aadA within transposon Tn7 (M. E. Fling, J. Kopf, and C. Richards, Nucleic Acids Res. 13:7095-7106, 1985), and by Hollingshead and Vapnek for E. coli R538-1 (S. Hollingshead and D. Vapnek, Plasmid 13:17-30, 1985). Previous reports of the presence of the aadA gene in enterococci appear to be erroneous and probably describe an aadE gene, since the isolates were reported to be susceptible to spectinomycin.
KeywordMeSH Terms
234.     ( 1999 )

Characterization of dihydrofolate reductase genes from trimethoprim-susceptible and trimethoprim-resistant strains of Enterococcus faecalis.

Antimicrobial agents and chemotherapy 43 (1)
PMID : 9869579  :   PMC  :   PMC89034    
Abstract >>
Enterococci are usually susceptible in vitro to trimethoprim; however, high-level resistance (HLR) (MICs, >1,024 microg/ml) has been reported. We studied Enterococcus faecalis DEL, for which the trimethoprim MIC was >1,024 microg/ml. No transfer of resistance was achieved by broth or filter matings. Two different genes that conferred trimethoprim resistance when they were cloned in Escherichia coli (MICs, 128 and >1,024 microg/ml) were studied. One gene that coded for a polypeptide of 165 amino acids (MIC, 128 microg/ml for E. coli) was identical to dfr homologs that we cloned from a trimethoprim-susceptible E. faecalis strain, and it is presumed to be the intrinsic E. faecalis dfr gene (which causes resistance in E. coli when cloned in multiple copies); this gene was designated dfrE. The nucleotide sequence 5' to this dfr gene showed similarity to thymidylate synthetase genes, suggesting that the dfr and thy genes from E. faecalis are located in tandem. The E. faecalis gene that conferred HLR to trimethoprim in E. coli, designated dfrF, codes for a predicted polypeptide of 165 amino acids with 38 to 64% similarity with other dihydrofolate reductases from gram-positive and gram-negative organisms. The nucleotide sequence 5' to dfrF did not show similarity to the thy sequences. A DNA probe for dfrF hybridized under high-stringency conditions only to colony lysates of enterococci for which the trimethoprim MIC was >1,024 microg/ml; there was no hybridization to plasmid DNA from the strain of origin. To confirm that this gene causes trimethoprim resistance in enterococci, we cloned it into the integrative vector pAT113 and electroporated it into RH110 (E. faecalis OG1RF::Tn916DeltaEm) (trimethoprim MIC, 0.5 microg/ml), which resulted in RH110 derivatives for which the trimethoprim MIC was >1, 024 microg/ml. These results indicate that dfrF is an acquired but probably chromosomally located gene which is responsible for in vitro HLR to trimethoprim in E. faecalis.
KeywordMeSH Terms
235.     ( 1998 )

Site-specific DNA binding using a variation of the double stranded RNA binding motif.

Nature structural biology 5 (7)
PMID : 9665166  :   DOI  :   10.1038/799    
Abstract >>
The integrase family of site-specific recombinases catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria and yeast. The solution structure of the DNA binding domain of the integrase protein from the conjugative transposon Tn916 has been determined using NMR spectroscopy. The structure provides the first insights into distal site DNA binding by a site-specific integrase and reveals that the N-terminal domain is structurally similar to the double stranded RNA binding domain (dsRBD). The results of chemical shift mapping experiments suggest that the integrase protein interacts with DNA using residues located on the face of its three stranded beta-sheet. This surface differs from the proposed RNA binding surface in dsRBDs, suggesting that different surfaces on the same protein fold can be used to bind DNA and RNA.
KeywordMeSH Terms
236.     ( 1997 )

Detection and speciation of bacteria through PCR using universal major cold-shock protein primer oligomers.

Journal of industrial microbiology & biotechnology 19 (4)
PMID : 9439003  :  
Abstract >>
The detection of bacteria using PCR is a well-established diagnostic technique. However, conventional PCR requires the use of DNA primer oligomers that are specific to the target organism and, as a consequence, a sample can only be tested for the presence of that specific target. A significant advantage would be to probe a sample for the presence of any bacteria, followed by identification. To achieve this it is necessary to identify a DNA sequence common to all bacteria. Here we demonstrate that such a sequence may be that encoding the major cold-shock proteins. Using two universal PCR primer oligomers from conserved regions of these gene homologues, we have amplified a 200 base-pair DNA sequence from more than 30 diverse Gram-positive and Gram-negative bacteria, including representatives from the genera Aeromonas, Bacillus, Citrobacter, Enterobacter, Enterococcus, Escherichia, Klebsiella, Lactobacillus, Lactococcus, Listeria, Pediococcus, Photobacterium, Proteus, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia. Sequence analysis of the amplified products confirmed a high level of DNA homology. Significantly, however, there are sufficient nucleotide variations to allow the unique allocation of each amplified sequence to its parental bacterium.
KeywordMeSH Terms
DNA Primers
237.     ( 1998 )

Carbamate kinase from Enterococcus faecalis and Enterococcus faecium--cloning of the genes, studies on the enzyme expressed in Escherichia coli, and sequence similarity with N-acetyl-L-glutamate kinase.

European journal of biochemistry 253 (1)
PMID : 9578487  :   DOI  :   10.1046/j.1432-1327.1998.2530280.x    
Abstract >>
Carbamate kinase (CK) catalyzes the reversible reaction NH2COO- + ATP <--> NHCOOPO3(2-) + ADP, serving to synthesize ATP from carbamoyl phosphate in those microorganisms that derive energy from anaerobic arginine degradation via the arginine dihydrolase pathway. We report here the cloning and sequencing of the CK gene from Enterococcus faecalis and Enterococcus faecium and we demonstrate that the amino acid sequence of CK is identical in the two species. The enzyme, expressed and isolated from Escherichia coli using simple purification procedures, was used to generate crystals suitable for X-ray studies and to investigate the utilization by CK of bicarbonate and other carbamate analogs. CK had a bicarbonate-dependent ATPase activity and, therefore, is able to synthesize carboxyphosphate, an unstable compound that is an intermediate in the reactions catalyzed by carbamoyl-phosphate synthetase (CPS) and by biotin carboxylase. Other functional similarities with CPS include the utilization of acetate by CK with a similarly high Km and the similar Km values of CK for carbamate and of CPS for bicarbonate. Enterococcal CK was inhibited by adenosine(5')pentaphospho(5')adenosine (Ap5A) and Ap6A and, less powerfully, by Ap4A, whereas Ap3A is essentially non-inhibitory. Thus, inhibition by Ap5A seems not to be a valid criterion to differentiate between CK and CPS, for the two enzymes can be inhibited by Ap5A. All these results support the relatedness of CK and CPS. Finally, we used limited proteolysis: (a) to localize the epitopes for monoclonal antibodies obtained against CK; (b) to demonstrate the importance of the C-terminus for enzyme activity; and (c) to show that Arg158 is highly exposed and may be essential for activity. Comparison of the sequence of CK with known protein sequences demonstrates considerable similarity of CK with bacterial N-acetylglutamate kinases, strongly suggesting that these two enzymes may share a similar structure and the same catalytic mechanism.
KeywordMeSH Terms
Genes, Bacterial
238.     ( 1998 )

Analysis of the gene cluster involved in production and immunity of the peptide antibiotic AS-48 in Enterococcus faecalis.

Molecular microbiology 27 (2)
PMID : 9484890  :   DOI  :   10.1046/j.1365-2958.1998.00682.x    
Abstract >>
A region of 7.8 kb of the plasmid pMB2 from Enterococcus faecalis S-48 carrying the information necessary for production and immunity of the peptide antibiotic AS-48 has been cloned and sequenced. It contains the as-48A structural gene plus five open reading frames (as-48B, as-48C, as-48C1, as-48D and as-48D1). Besides As-48D, all the predicted gene products are basic hydrophobic proteins with potential membrane-spanning domains (MSDs). None of them shows any homology with protein sequences stored in databanks, except for As-48D, which shows similarity to the C-terminal domain of ABC transporters and contains a highly conserved ATP-binding site. The gene products of as-48B, as-48C, as-48C1 and as-48D are thought to be involved in AS-48 production and secretion. The only gene able to provide resistance to AS-48 by itself is as-48D1. Immunity also seems to be enhanced at least by the products of as-48B, as-48C1 and as-48D genes. Transcription analysis using probes derived from the different ORFs revealed two large (3.5 and 2.7kb) mRNAs, suggesting that the different genes are organized in two constitutive operons.
KeywordMeSH Terms
Anti-Bacterial Agents
Bacterial Proteins
Genes, Bacterial
Multigene Family
Peptides
239.     ( 1998 )

Transfer of Tn5385, a composite, multiresistance chromosomal element from Enterococcus faecalis.

Journal of bacteriology 180 (3)
PMID : 9457879  :   PMC  :   PMC106943    
Abstract >>
Tn5385 is a ca. 65-kb element integrated into the chromosomes of clinical Enterococcus faecalis strains CH19 and CH116. It confers resistance to erythromycin, gentamicin, mercuric chloride, streptomycin, tetracycline-minocycline, and penicillin via beta-lactamase production. Tn5385 is a composite structure containing regions previously found in staphylococcal and enterococcal plasmids. Several transposons and transposon-like elements within Tn5385 have been identified, including conjugative transposon Tn5381, composite transposon Tn5384, and elements indistinguishable from staphylococcal transposons Tn4001 and Tn552. The divergent regions of Tn5385 are linked by a series of insertion sequence (IS) elements (IS256, IS257, and IS1216) of staphylococcal and enterococcal origin. The ends of Tn5385 consist of directly repeated copies of enterococcal IS1216. Within the chromosomes of strains CH19 and CH116, Tn5385 has interrupted an open reading frame with substantial homology to previously described alkyl hydrogen peroxide reductase genes. Segments of this open reading frame in both CH19 and CH116 have been deleted, but the amount of deleted DNA differs for the two insertions. Transfer of Tn5385 from both donors into E. faecalis recipients occurs at a low frequency. Two types of transconjugants have been identified. In one type, the target alkyl hydrogen peroxide reductase open reading frame has been deleted, and sequences flanking Tn5385 in the respective donors are carried over to the transconjugants. These data suggest that the mechanism of Tn5385 insertion into the recipient chromosome in these transconjugants was recombination across flanking regions in the donors and homologous sequences in the recipients. The second type of transconjugant appears to have resulted from excision of Tn5385 from the CH19 chromosome by recombination across the terminal IS1216 elements and insertion into the recipient chromosome by recombination across Tn5381 (within Tn5385) and a previously transferred Tn5381 copy in the recipient chromosome. These data confirm that Tn5385 is a composite structure with genetic material from diverse genera and suggest that it is a functional transposon. They also suggest that chromosomal recombination is a mechanism of genetic exchange in enterococci.
KeywordMeSH Terms
Chromosomes, Bacterial
DNA Transposable Elements
DNA, Bacterial
240.     ( 1997 )

Cloning and genetic and sequence analyses of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pPD1.

Journal of bacteriology 179 (24)
PMID : 9401046  :   DOI  :   10.1128/jb.179.24.7843-7855.1997     PMC  :   PMC179750    
Abstract >>
The pheromone-responsive conjugative plasmid pPD1 (59 kb) of Enterococcus faecalis encodes the bacteriocin 21 (bac21) determinant. Cloning, transposon insertion mutagenesis and sequence analysis of the bac21 determinant showed that an 8.5-kb fragment lying between kb 27.1 and 35.6 of the pPD1 map is required for complete expression of the bacteriocin. The 8.5-kb fragment contained nine open reading frames (ORFs), bacA to bac1, which were oriented in the same (upstream-to-downstream) direction. Transposon insertions into the bacA to bacE ORFs, which are located in the proximal half of bac21, resulted in defective bacteriocin expression. Insertions into the bacF to bac1 ORFs, which are located in the distal half of bac21, resulted in reduced bacteriocin expression. Deletion mutant analysis of the cloned 8.5-kb fragment revealed that the deletion of segments between kb 31.6 and 35.6 of the pPD1 map, which contained the distal region of the determinant encoding bacF to bac1, resulted in reduced bacteriocin expression. The smallest fragment (4.5 kb) retaining some degree of bacteriocin expression contained the bacA to bacE sequences located in the proximal half of the determinant. The cloned fragment encoding the 4.5-kb proximal region and a Tn916 insertion mutant into pPD1 bacB trans-complemented intracellularly to give complete expression of the bacteriocin. bacA encoded a 105-residue sequence with a molecular mass of 11.1 kDa. The deduced BacA protein showed 100% homology to the broad-spectrum antibiotic peptide AS-48, which is encoded on the E. faecalis conjugative plasmid pMB2 (58 kb). bacH encoded a 195-residue sequence with a molecular mass of 21.9 kDa. The deduced amino acid sequence showed significant homology to the C-terminal region of HlyB (31.1% identical residues), a protein located in the Escherichia coli alpha-hemolysin operon that is a representative bacterial ATP-binding cassette export protein.
KeywordMeSH Terms
Bacterial Proteins
Peptides
241.     ( 1998 )

Alterations in the GyrA subunit of DNA gyrase and the ParC subunit of DNA topoisomerase IV associated with quinolone resistance in Enterococcus faecalis.

Antimicrobial agents and chemotherapy 42 (2)
PMID : 9527801  :   PMC  :   PMC105429    
Abstract >>
The gyrA and parC genes of 31 clinical isolates of Enterococcus faecalis, including fluoroquinolone-resistant isolates, were partially sequenced and analyzed for target alterations. Topoisomerase IV may be a primary target in E. faecalis, but high-level fluoroquinolone resistance was associated with simultaneous alterations in both GyrA and ParC.
KeywordMeSH Terms
242.     ( 1997 )

Cloning and genetic analysis of the UV resistance determinant (uvr) encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pAD1.

Journal of bacteriology 179 (23)
PMID : 9393713  :   DOI  :   10.1128/jb.179.23.7468-7475.1997     PMC  :   PMC179699    
Abstract >>
The conjugative pheromone-responsive plasmid pAD1 (59.6 kb) of Enterococcus faecalis encodes a UV resistance determinant (uvr) in addition to the hemolysin-bacteriocin determinant. pAD1 enhances the UV resistance of wild-type E. faecalis FA2-2 and E. faecalis UV202, which is a UV-sensitive derivative of E. faecalis JH2-2. A 2.972-kb fragment cloned from between 27.7 and 30.6 kb of the pAD1 map conferred UV resistance function on UV202. Sequence analysis showed that the cloned fragment contained three open reading frames designated uvrA, uvrB, and uvrC. The uvrA gene is located on the pAD1 map between 28.1 and 29.4 kb. uvrB is located between 30.1 and 30.3 kb, and uvrC is located between 30.4 and 30.6 kb on the pAD1 map. The uvrA, uvrB, and uvrC genes encode sequences of 442, 60, and 74 amino acids, respectively. The deduced amino acid sequence of the uvrA-encoded protein showed 20% homology of the identical residues with the E. coli UmuC protein. Tn917 insertion mutagenesis and deletion mutant analysis of the cloned fragment showed that uvrA conferred UV resistance. A palindromic sequence, 5'-GAACNGTTC-3', which is identical to the consensus sequence found within the putative promoter region of the Bacillus subtilis DNA damage-inducible genes, was located within the promoter region of uvrA. Two uvrA transcripts of different lengths (i.e., 1.54 and 2.14 kb) which terminate at different points downstream of uvrA were detected in UV202 carrying the deletion mutant containing uvrA. The longer transcript, 2.14 kb, was not detected in UV202 carrying the deletion mutant containing both uvrA and uvrB, which suggests that uvrB encodes a terminator for the uvrA transcript. The uvrA transcript was not detected in any significant quantity in UV202 carrying the cloned fragment containing uvrA, uvrB, and uvrC; on the other hand, the 1.54-kb uvrA transcript was detected in the strain exposed to mitomycin C, which suggests that the UvrC protein functions as a regulator of uvrA.
KeywordMeSH Terms
DNA Helicases
Endodeoxyribonucleases
Escherichia coli Proteins
Genes, Bacterial
Ultraviolet Rays

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