( 2002 )
Isocitrate lyase of the facultative intracellular pathogen Rhodococcus equi.
PMID : 11882714 : DOI : 10.1099/00221287-148-3-793
Isocitrate lyase is the first enzyme of the glyoxylate shunt which is required for the assimilation of fatty acids and acetate. The intracellular pathogen Rhodococcus equi contains high activities of this enzyme following growth on acetate and lactate, indicating that it plays an important role in the metabolism of these substrates. The gene encoding isocitrate lyase (aceA) was cloned and sequenced. It specifies a 46846 Da protein, which was shown to be functional by expressing it in Escherichia coli. A gene similar to fadB, encoding 3-hydroxyacyl-CoA dehydrogenase, was located 90 bp downstream from aceA. Northern hybridization and RT-PCR experiments showed that aceA and fadB are cotranscribed into a 2.8 kb transcript. A smaller 1.6 kb aceA transcript was also observed which was 2.5-fold more abundant than the aceA-fadB transcript. It is proposed that a stable hairpin structure with a free energy (DeltaG) of -28.5 kcal x mol(-1) and located in the 90 bp aceA-fadB intergenic region is involved in stabilizing the aceA transcript.
( 2001 )
Identification and mutagenesis by allelic exchange of choE, encoding a cholesterol oxidase from the intracellular pathogen Rhodococcus equi.
PMID : 11466283 : DOI : 10.1128/JB.183.16.4796-4805.2001 PMC : PMC99534
The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10(-2) and 10(-3). Twelve percent of the recombinants were double-crossover choE mutants. The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.
( 2001 )
Virulence plasmid of Rhodococcus equi contains inducible gene family encoding secreted proteins.
PMID : 11159951 : DOI : 10.1128/IAI.69.2.650-656.2001 PMC : PMC97935
Rhodococcus equi causes severe pyogranulomatous pneumonia in foals. This facultative intracellular pathogen produces similar lesions in immunocompromised humans, particularly in AIDS patients. Virulent strains of R. equi bear a large plasmid that is required for intracellular survival within macrophages and for virulence in foals and mice. Only two plasmid-encoded proteins have been described previously; a 15- to 17-kDa surface protein designated virulence-associated protein A (VapA) and an antigenically related 20-kDa protein (herein designated VapB). These two proteins are not expressed by the same R. equi isolate. We describe here the substantial similarity between VapA and VapB. Moreover, we identify three additional genes carried on the virulence plasmid, vapC, -D, and -E, that are tandemly arranged downstream of vapA. These new genes are members of a gene family and encode proteins that are approximately 50% homologous to VapA, VapB, and each other. vapC, -D, and -E are found only in R. equi strains that express VapA and are highly conserved in VapA-positive isolates from both horses and humans. VapC, -D, and -E are secreted proteins coordinately regulated by temperature with VapA; the proteins are expressed when R. equi is cultured at 37 degrees C but not at 30 degrees C, a finding that is compatible with a role in virulence. As secreted proteins, VapC, -D, and -E may represent targets for the prevention of rhodococcal pneumonia. An immunologic study using VapA-specific antibodies and recombinant Vap proteins revealed no evidence of cross-reactivity despite extensive sequence similarity over the carboxy terminus of all four proteins.
( 2000 )
DNA sequence and comparison of virulence plasmids from Rhodococcus equi ATCC 33701 and 103.
PMID : 11083803 : DOI : 10.1128/iai.68.12.6840-6847.2000 PMC : PMC97788
The virulence plasmids of the equine virulent strains Rhodococcus equi ATCC 33701 and 103 were sequenced, and their genetic structure was analyzed. p33701 was 80,610 bp in length, and p103 was 1 bp shorter; their sequences were virtually identical. The plasmids contained 64 open reading frames (ORFs), 22 of which were homologous with genes of known function and 3 of which were homologous with putative genes of unknown function in other species. Putative functions were assigned to five ORFs based on protein family characteristics. The most striking feature of the virulence plasmids was the presence of a 27,536-bp pathogenicity island containing seven virulence-associated protein (vap) genes, including vapA. These vap genes have extensive homology to vapA, which encodes a thermoregulated and surface-expressed protein. The pathogenicity island contained a LysR family transcriptional regulator and a two-component response regulator upstream of six of the vap genes. The vap genes were present as a cluster of three (vapA, vapC, and vapD), as a pair (vapE and vapF), or individually (vapG; vapH). A region of extensive direct repeats of unknown function, possibly associated with thermoregulation, was present immediately upstream of the clustered and the paired genes but not the individual vap genes. There was extensive homology among the C-terminal halves of all vap genes but not generally among the N-terminal halves. The remainder of the plasmid consisted of a large region which appears to be associated with conjugation functions and a large region which appears to be associated with replication and partitioning functions.
( 2000 )
The iron dependent regulatory protein IdeR (DtxR) of Rhodococcus equi.
PMID : 11004391 : DOI : 10.1111/j.1574-6968.2000.tb09310.x
This paper reports the presence of an ideR gene, which encodes an iron-dependent regulatory protein, in Rhodococcus erythropolis and in the intracellular pathogen Rhodococcus equi. The ideR gene of the latter encoded a protein of 230 amino acids with a molecular mass of 25619. The alpha-helices forming the helix-turn-helix motif of the R. equi protein were identical to those of the DtxR protein of Corynebacterium diphtheriae, which is an IdeR homologue. This indicates that the two proteins bind to the same DNA binding site. This was confirmed following expression of IdeR in Escherichia coli, which showed that the IdeR protein could repress transcription of the tox promoter of C. diphtheriae in an iron dependent manner. An open reading frame specifying a 283-amino acid polypeptide similar to galE encoding UDP-galactose 4-epimerase was present downstream of the ideR gene.
( 1999 )
Identification of mycobacterial species by comparative sequence analysis of the RNA polymerase gene (rpoB).
PMID : 10325313 : PMC : PMC84932
For the differentiation and identification of mycobacterial species, the rpoB gene, encoding the beta subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 44 reference strains of mycobacteria and clinical isolates (107 strains) by PCR. The nucleotide sequences were directly determined (306 bp) and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Comparative sequence analysis of rpoB DNAs provided the basis for species differentiation within the genus Mycobacterium. Slowly and rapidly growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. Pathogenic Mycobacterium kansasii was easily differentiated from nonpathogenic M. gastri; this differentiation cannot be achieved by using 16S rRNA gene (rDNA) sequences. By being grouped into species-specific clusters with low-level sequence divergence among strains of the same species, all of the clinical isolates could be easily identified. These results suggest that comparative sequence analysis of amplified rpoB DNAs can be used efficiently to identify clinical isolates of mycobacteria in parallel with traditional culture methods and as a supplement to 16S rDNA gene analysis. Furthermore, in the case of M. tuberculosis, rifampin resistance can be simultaneously determined.
El Azhari N,
( 2007 )
pcaH, a molecular marker for estimating the diversity of the protocatechuate-degrading bacterial community in the soil environment.
PMID : 17427160 : DOI : 10.1002/ps.1357
Microorganisms degrading phenolic compounds play an important role in soil carbon cycling as well as in pesticide degradation. The pcaH gene encoding a key ring-cleaving enzyme of the beta-ketoadipate pathway was selected as a functional marker. Using a degenerate primer pair, pcaH fragments were cloned from two agricultural soils. Restriction fragment length polymorphism (RFLP) screening of 150 pcaH clones yielded 68 RFLP families. Comparison of 86 deduced amino acid sequences displayed 70% identity to known PcaH sequences. Phylogenetic analysis results in two major groups mainly related to PcaH sequences from Actinobacteria and Proteobacteria phyla. This confirms that the developed primer pair targets a wide diversity of pcaH sequences, thereby constituting a suitable molecular marker to estimate the response of the pca community to agricultural practices.
( 2007 )
A novel endoglycoceramidase hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides.
PMID : 17244618 : DOI : 10.1074/jbc.M608445200
Enzymes capable of hydrolyzing the beta-glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids has been found in microorganisms and invertebrates and designated endoglycoceramidase (EC 22.214.171.124) or ceramide glycanase. Here we report the molecular cloning, characterization, and homology modeling of a novel endoglycoceramidase that hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. The novel enzyme was purified from a culture supernatant of Rhodococcus equi, and the gene encoding 488 deduced amino acids was cloned using peptide sequences of the purified enzyme. Eight residues essential for the catalytic reaction in microbial and animal endoglycoceramidases were all conserved in the deduced amino acid sequence of the novel enzyme. Homology modeling of the enzyme using endocellulase E1 as a template revealed that the enzyme displays a (beta/alpha)8 barrel structure in which Glu234 at the end of beta-strand 4 and Glu341 at the end of beta-strand 7 could function as an acid/base catalyst and a nucleophile, respectively. Site-directed mutagenesis of these glutamates resulted in a complete loss of the activity without a change in their CD spectra. The recombinant enzyme hydrolyzed the beta-galactosidic linkage between oligosaccharides and ceramides of 6-gala series glycosphingolipids that were completely resistant to hydrolysis by the enzymes reported so far. In contrast, the novel enzyme did not hydrolyze ganglio-, globo-, or lactoseries glycosphingolipids. The enzyme is therefore systematically named "oligogalactosyl-N-acylsphingosine 1,1'-beta-galactohydrolase" or tentatively designated "endogalactosylceramidase."
( 2006 )
A nucleotide mutation associated with fluoroquinolone resistance observed in gyrA of in vitro obtained Rhodococcus equi mutants.
PMID : 16563665 : DOI : 10.1016/j.vetmic.2006.01.015
In this study, the quinolone resistance-determining region (QRDR) in gyrA and gyrB of in vitro fluoroquinolone-resistant Rhodococcus equi mutants was sequenced. These mutants were selected from four R. equi strains on blood agar plates containing ciprofloxacin or enrofloxacin. Each mutant became 8- to 64 or greater-fold resistant to fluoroquinolones compared with their parent strains. From the results of sequence analysis of QRDR in gyrA and gyrB, a nucleotide mutation of codon GAC for GGC in gyrA was detected in all mutants, but no mutation was observed in gyrB. This mutation leads to amino acid substitution of Asp for Gly in putative GyrA in R. equi. The position of this substitution corresponds to position 87 of GyrA in Escherichia coli. Our results suggest that the mutation of QRDR in gyrA, which was observed in in vitro fluoroquinolone-resistant R. equi mutants in this study, is closely associated with fluoroquinolone resistance.
( 2005 )
The iron-regulated iupABC operon is required for saprophytic growth of the intracellular pathogen Rhodococcus equi at low iron concentrations.
PMID : 15866930 : DOI : 10.1128/JB.187.10.3438-3444.2005 PMC : PMC1112021
Rhodococcus equi is a facultative intracellular pathogen which proliferates rapidly in both manure-enriched soil and alveolar macrophages. Although both environments are characterized by extremely low concentrations of free iron, very little is known regarding the strategies employed by R. equi to thrive under these conditions. This paper reports the characterization of an R. equi transposome mutant that fails to grow at low iron concentrations. The transposome was shown to be inserted into iupA, the first gene of the iupABC operon encoding an ABC transport system highly similar to siderophore uptake systems. Disruption of the iupA gene also resulted in a failure of R. equi to utilize heme and hemoglobin as a source of iron. Introduction of the iupABC operon in trans restored the wild-type phenotype of the mutant strain. iupABC transcripts were 180-fold more abundant in R. equi grown in iron-depleted medium than in organisms grown in iron-replete medium. Proliferation of the iupABC mutant strain in macrophages was comparable to that of the wild-type strain. Furthermore, the iupABC mutant was not attenuated in mice, showing that the iupABC operon is not required for virulence.
( 2005 )
Isocitrate lyase activity is required for virulence of the intracellular pathogen Rhodococcus equi.
PMID : 16177351 : DOI : 10.1128/IAI.73.10.6736-6741.2005 PMC : PMC1230931
Rhodococcus equi is an important pathogen of foals, causing severe pyogranulomatous pneumonia. Virulent R. equi strains grow within macrophages, a process which remains poorly characterized. A potential source of carbon for intramacrophage R. equi is membrane lipid-derived fatty acids, which following beta oxidation are assimilated via the glyoxylate bypass. To assess the importance of isocitrate lyase, the first enzyme of the glyoxylate bypass, in virulence of a foal isolate of R. equi, a mutant was constructed by a strategy of single homologous recombination using a suicide plasmid containing an internal fragment of the R. equi aceA gene encoding isocitrate lyase. Complementation of the resulting mutant with aceA showed that the mutant was specific for this gene. Assessment of virulence in a mouse macrophage cell line showed that the mutant was killed, in contrast to the parent strain. Studies in the liver of intravenously infected mice showed enhanced clearance of the mutant. When four 3-week-old foals were infected intrabronchially, the aceA mutant was completely attenuated, in contrast to the parent strain. In conclusion, the aceA gene was shown to be essential for virulence of R. equi, suggesting that membrane lipids may be an important source of carbon for phagocytosed R. equi.
( 2004 )
The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence.
PMID : 15504593 : DOI : 10.1016/j.vetmic.2004.08.005
An 81 kb virulence plasmid containing a pathogenicity island (PI) plays a crucial role in the pathogenesis of Rhodococcus equi pneumonia in foals but its specific function in virulence and regulation of plasmid-encoded virulence genes is unclear. Using a LacZ selection marker developed for R. equi in this study, in combination with an apramycin resistance gene, an efficient two-stage homologous recombination targeted gene mutation procedure was used to mutate three virulence plasmid genes, a LysR regulatory gene homologue (ORF4), a ResD-like two-component response regulator homologue (ORF8), and a gene (ORF10) of unknown function that is highly expressed by R. equi inside macrophages, as well as the chromosomal gene operon, phoPR. Virulence testing by liver clearance after intravenous injection in mice showed that the ORF4 and ORF8 mutants were fully attenuated, that the phoPR mutant was hypervirulent, and that virulence of the ORF10 mutant remained unchanged. A virulence plasmid DNA microarray was used to compare the plasmid gene expression profile of each of the four gene-targeted mutants against the parental R. equi strain. Changes were limited to PI genes and gene induction was observed for all mutants, suggesting that expression of virulence plasmid genes is dominated by a negative regulatory network. The finding of attenuation of ORF4 and ORF8 mutants despite enhanced transcription of vapA suggests that factors other than VapA are important for full expression of virulence. ORF1, a putative Lsr antigen gene, was strongly and similarly induced in all mutants, implying a common regulatory pathway affecting this gene for all four mutated genes. ORF8 is apparently the centre of this common pathway. Two distinct highly correlated gene induction patterns were observed, that of the ORF4 and ORF8 mutants, and that of the ORF10 and phoPR mutants. The gene induction pattern distinguishing these two groups paralleled their virulence in mice.
La Scola B,
( 2004 )
rpoB gene sequencing for identification of Corynebacterium species.
PMID : 15364970 : DOI : 10.1128/JCM.42.9.3925-3931.2004 PMC : PMC516356
The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species.
( 2004 )
The immunogenicity of Rhodococcus equi GroEL2-based vaccines in a murine model.
PMID : 15127846 : DOI : 10.1016/j.vetimm.2003.11.001
Rhodococcus equi is a significant intracellular bacterial pathogen in foals. However, at present there is no commercially available vaccine for the prevention of R. equi-induced disease in these animals. Studies have shown that GroEL based vaccines can afford protection against some intracellular pathogens. In this study, the R. equi gene encoding the heat shock protein GroEL2 was cloned and sequenced, with a view to using it as a vaccine candidate. The promoter region of the gene contained two copies of controlling inverted repeat of chaperone expression (CIRCE) motifs, which are well-recognised transcriptional regulators of bacterial heat shock proteins. The R. equi GroEL2 was expressed in E. coli BL21 DE3 with a C-terminal His-tag and sequenced to confirm its identity. The R. equi purified His-tagged GroEL2 protein and a groEL2-based DNA vaccine were used in separate experiments to immunise BALB/c mice. The recombinant protein-based vaccine elicited a mixed Th1/Th2 response whereas the DNA vaccine was found to elicit a predominantly Th1 biased immune response. However, when vaccinated mice were challenged intravenously with 1.5 x 10(7) R. equi neither vaccine elicited enhanced bacterial clearance from the spleen or liver in this model. The reasons for this apparent lack of success are discussed.
Gueneau de Novoa P,
( 2004 )
The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts.
PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836
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.
( 2010 )
Mutant selection window and characterization of allelic diversity for ciprofloxacin-resistant mutants of Rhodococcus equi.
PMID : 20498313 : DOI : 10.1128/AAC.01670-09 PMC : PMC2916315
The mutant prevention concentration (MPC) for ciprofloxacin was determined for two Rhodococcus equi strains. The MPC for both strains was 32 mug/ml, which is above the peak serum concentration of ciprofloxacin obtainable by oral administration in humans. Nine single nucleotide changes corresponding to eight amino acid substitutions in the quinolone resistance-determining regions of DNA gyrase subunits A and B were characterized. Only mutants with amino acid changes in Ser-83 of GyrA were highly resistant (>or=64 microg/ml). Our results suggest that ciprofloxacin monotherapy against R. equi infection may result in the emergence of ciprofloxacin-resistant mutants.
( 2010 )
Analysis of plasmid diversity in 96 Rhodococcus equi strains isolated in Normandy (France) and sequencing of the 87-kb type I virulence plasmid.
PMID : 20707817 : DOI : 10.1111/j.1574-6968.2010.02070.x
To characterize the potential epidemiological relationship between the origin of Rhodococcus equi strains and the type of their virulence plasmids, we performed a comparative analysis of virulence plasmid types encountered in 96 R. equi strains isolated from (1) autopsied horses, (2) organic samples (horse faeces, manure and straw) and (3) environmental samples. Our results revealed no clear epidemiological link between virulence plasmid type and the origin of R. equi strains isolated from horse-related environments. To understand this result, we determined the nucleotide sequence of the second most frequently isolated virulence plasmid type: a 87-kb type I (pVAPA116) plasmid and compared it with the previously sequenced (and most commonly encountered) 85-kb type I (pVAPA1037) plasmid. Our results show that the divergence between these two plasmids is mainly due to the presence of three allelic exchange loci, resulting in the deletion of two genes and the insertion of three genes in pVAPA116 compared with pVAPA1037. In conclusion, it appears that the divergence between the two sequenced rhodococcal virulence plasmids is not associated with the vap pathogenicity island and may result from an evolutionary process driven by a mobility-related invertase/resolvase invA-like gene.
( 2010 )
secA1 gene sequence polymorphisms for species identification of Nocardia species and recognition of intraspecies genetic diversity.
PMID : 20810768 : DOI : 10.1128/JCM.01113-10 PMC : PMC3020853
Sequence analysis of the Nocardia essential secretory protein SecA1 gene (secA1) for species identification of 120 American Type Culture Collection (ATCC) and clinical isolates of Nocardia (16 species) was studied in comparison with 5'-end 606-bp 16S rRNA gene sequencing. Species determination by both methods was concordant for all 10 ATCC strains. secA1 gene sequencing provided the same species identification as 16S rRNA gene analysis for 94/110 (85.5%) clinical isolates. However, 40 (42.6%) isolates had sequences with <99.0% similarity to archived secA1 sequences for the species, including 29 Nocardia cyriacigeorgica (96.6 to 98.9% similarity) and 4 Nocardia veterana (91.5 to 98.9% similarity) strains. Discrepant species identification was obtained for 16 (14.5%) clinical isolates, including 13/23 Nocardia nova strains (identified as various Nocardia species by secA1 sequencing) and 1 isolate each of Nocardia abscessus (identified as Nocardia asiatica), Nocardia elegans (Nocardia africana), and Nocardia transvalensis (Nocardia blacklockiae); both secA1 gene sequence analysis and deduced amino acid sequence analysis determined the species to be different from those assigned by 16S rRNA gene sequencing. The secA1 locus showed high sequence diversity (66 sequence or genetic types versus 40 16S rRNA gene sequence types), which was highest for N. nova (14 secA1 sequence types), followed by Nocardia farcinica and N. veterana (n = 7 each); there was only a single sequence type among eight Nocardia paucivorans strains. The secA1 locus has potential for species identification as an adjunct to 16S rRNA gene sequencing but requires additional deduced amino acid sequence analysis. It may be a suitable marker for phylogenetic/subtyping studies.
( 2010 )
Molecular detection and phylogenetic analysis of the alkane 1-monooxygenase gene from Gordonia spp.
PMID : 20047814 : DOI : 10.1016/j.syapm.2009.11.003
The alkB gene encodes for alkane 1-monooxygenase, which is a key enzyme responsible for the initial oxidation of inactivated alkanes. This functional gene can be used as a marker to assess the catabolic potential of bacteria in bioremediation. In the present study, a pair of primers was designed based on the conserved regions of the AlkB amino acid sequences of Actinobacteria, for amplifying the alkB gene from the genus Gordonia (20 Gordonia strains representing 13 species). The amplified alkB genes were then sequenced and analyzed. In the phylogenetic tree based on the translated AlkB amino acid sequences, all the Gordonia segregated clearly from other closely related genera. The sequence identity of the alkB gene in Gordonia ranged from 58.8% to 99.1%, which showed higher sequence variation at the inter-species level compared with other molecular markers, such as the 16S rRNA gene (93.1-99.8%), gyrB gene (77.5-97.3%) or catA gene (72.4-99.5%). The genetic diversity of four selected loci also showed that the alkB gene might have evolved faster than rrn operons, as well as the gyrB or catA genes, in Gordonia. All the available actinobacterial alkB gene sequences derived from the whole genome shotgun sequencing projects are phylogenetically characterized here for the first time, and they exclude the possibility of horizontal gene transfer of the alkB gene in these bacterial groups.
( 2008 )
Evolution of the Rhodococcus equi vap pathogenicity island seen through comparison of host-associated vapA and vapB virulence plasmids.
PMID : 18606735 : DOI : 10.1128/JB.00468-08 PMC : PMC2519538
The pathogenic actinomycete Rhodococcus equi harbors different types of virulence plasmids associated with specific nonhuman hosts. We determined the complete DNA sequence of a vapB(+) plasmid, typically associated with pig isolates, and compared it with that of the horse-specific vapA(+) plasmid type. pVAPB1593, a circular 79,251-bp element, had the same housekeeping backbone as the vapA(+) plasmid but differed over an approximately 22-kb region. This variable region encompassed the vap pathogenicity island (PAI), was clearly subject to selective pressures different from those affecting the backbone, and showed major genetic rearrangements involving the vap genes. The pVAPB1593 PAI harbored five different vap genes (vapB and vapJ to -M, with vapK present in two copies), which encoded products differing by 24 to 84% in amino acid sequence from the six full-length vapA(+) plasmid-encoded Vap proteins, consistent with a role for the specific vap gene complement in R. equi host tropism. Sequence analyses, including interpolated variable-order motifs for detection of alien DNA and reconstruction of Vap family phylogenetic relationships, suggested that the vap PAI was acquired by an ancestor plasmid via lateral gene transfer, subsequently evolving by vap gene duplication and sequence diversification to give different (host-adapted) plasmids. The R. equi virulence plasmids belong to a new family of actinobacterial circular replicons characterized by an ancient conjugative backbone and a horizontally acquired niche-adaptive plasticity region.
( 2017 )
Comparative Genomics of Rhodococcus equi Virulence Plasmids Indicates Host-Driven Evolution of the vap Pathogenicity Island.
PMID : 28369330 : DOI : 10.1093/gbe/evx057 PMC : PMC5434932
The conjugative virulence plasmid is a key component of the Rhodococcus equi accessory genome essential for pathogenesis. Three host-associated virulence plasmid types have been identified the equine pVAPA and porcine pVAPB circular variants, and the linear pVAPN found in bovine (ruminant) isolates. We recently characterized the R. equi pangenome (Anastasi E, et al. 2016. Pangenome and phylogenomic analysis of the pathogenic actinobacterium Rhodococcus equi. Genome Biol Evol. 8:3140-3148.) and we report here the comparative analysis of the virulence plasmid genomes. Plasmids within each host-associated type were highly similar despite their diverse origins. Variation was accounted for by scattered single nucleotide polymorphisms and short nucleotide indels, while larger indels-mostly in the plasticity region near the vap pathogencity island (PAI)-defined plasmid genomic subtypes. Only one of the plasmids analyzed, of pVAPN type, was exceptionally divergent due to accumulation of indels in the housekeeping backbone. Each host-associated plasmid type carried a unique PAI differing in vap gene complement, suggesting animal host-specific evolution of the vap multigene family. Complete conservation of the vap PAI was observed within each host-associated plasmid type. Both diversity of host-associated plasmid types and clonality of specific chromosomal-plasmid genomic type combinations were observed within the same R. equi phylogenomic subclade. Our data indicate that the overall strong conservation of the R. equi host-associated virulence plasmids is the combined result of host-driven selection, lateral transfer between strains, and geographical spread due to international livestock exchanges.
( 2015 )
An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi.
PMID : 25895973 : DOI : 10.1128/IAI.00376-15 PMC : PMC4468562
We report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted "bovine-type" allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species.
( 2015 )
Novel transferable erm(46) determinant responsible for emerging macrolide resistance in Rhodococcus equi.
PMID : 26377866 : DOI : 10.1093/jac/dkv279
The objective of this study was to identify the molecular mechanism of macrolide resistance in the actinomycete Rhodococcus equi, a major equine pathogen and zoonotic agent causing opportunistic infections in people. Macrolide-resistant (n = 62) and macrolide-susceptible (n = 62) clinical isolates of R. equi from foals in the USA were studied. WGS of 18 macrolide-resistant and 6 macrolide-susceptible R. equi was performed. Representative sequences of all known macrolide resistance genes identified to date were used to search the genome assemblies for putative homologues. PCR was used to screen for the presence of the identified resistance determinant in the rest of the isolates. Mating experiments were performed to verify mobility of the gene. A novel erm gene, erm(46), was identified in all sequenced resistant isolates, but not in susceptible isolates. There was complete association between macrolide resistance and the presence of erm(46) as detected by PCR screening of all 124 clinical isolates of R. equi. Expression of erm(46) in a macrolide-susceptible strain of R. equi induced high-level resistance to macrolides, lincosamides and streptogramins B, but not to other classes of antimicrobial agents. Transfer of erm(46) to macrolide-susceptible R. equi was confirmed. The transfer frequency ranged from 3 �� 10(-3) to 1 �� 10(-2). This is the first molecular characterization of resistance to macrolides, lincosamides and streptogramins B in R. equi. Resistance was due to the presence of a novel erm(46) gene mobilizable likely by conjugation, which has spread among equine isolates of R. equi in the USA.
( 2014 )
Structure of Rhodococcus equi virulence-associated protein B (VapB) reveals an eight-stranded antiparallel �]-barrel consisting of two Greek-key motifs.
PMID : 25005079 : DOI : 10.1107/S2053230X14009911 PMC : PMC4089522
Members of the virulence-associated protein (Vap) family from the pathogen Rhodococcus equi regulate virulence in an unknown manner. They do not share recognizable sequence homology with any protein of known structure. VapB and VapA are normally associated with isolates from pigs and horses, respectively. To contribute to a molecular understanding of Vap function, the crystal structure of a protease-resistant VapB fragment was determined at 1.4 ? resolution. The structure was solved by SAD phasing employing the anomalous signal of one endogenous S atom and two bound Co ions with low occupancy. VapB is an eight-stranded antiparallel �]-barrel with a single helix. Structural similarity to avidins suggests a potential binding function. Unlike other eight- or ten-stranded �]-barrels found in avidins, bacterial outer membrane proteins, fatty-acid-binding proteins and lysozyme inhibitors, Vaps do not have a next-neighbour arrangement but consist of two Greek-key motifs with strand order 41238567, suggesting an unusual or even unique topology.
( 2014 )
vanO, a new glycopeptide resistance operon in environmental Rhodococcus equi isolates.
PMID : 24342631 : DOI : 10.1128/AAC.01880-13 PMC : PMC3957834
We describe here the sequence and gene organization of a new glycopeptide resistance operon (vanO) in Rhodococcus equi from soil. The vanO operon has low homology to enterococcal van operons and harbors a vanHOX cluster transcribed in the direction opposite that of the vanS-vanR regulatory system and composed of three open reading frames with unknown function. This finding has clinical interest, since glycopeptides are used to treat R. equi infections and resistance has been reported in clinical isolates.
( 2014 )
Characterization of Rhodococcus equi isolates from submaxillary lymph nodes of wild boars (Sus scrofa), red deer (Cervus elaphus) and roe deer (Capreolus capreolus).
PMID : 24878324 : DOI : 10.1016/j.vetmic.2014.04.020
Rhodococcus equi is a soil saprophyte and an opportunistic pathogen causing infections in animals, and rarely in humans. The presence of R. equi in tissues and faeces of some wild animal species was demonstrated previously. In this study we characterized R. equi isolates from submaxillary lymph nodes of free-living wild boars (n=23), red deer (n=2) and roe deer (n=2). This is the first description of R. equi strains isolated from tissues of the Cervidae. All isolates were initially recognized as R. equi based on the phenotypic properties. Their identification was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, detection of the choE gene and by sequence analysis of the 16S rRNA and rpoB genes. The presence of three plasmidic genes (traA, vapA and vapB) associated with R. equi virulence was investigated by PCR. In 16 wild boar isolates the traA and vapB genes were detected and they were located on virulence plasmids type 5, 7 or 11. The isolates from cervids and the remaining wild boar isolates were classified as avirulent based on a genotype traA(-)/vapA(-)B(-). In summary, these results confirm that wild boars can be a source of intermediately virulent R. equi strains, and indicate that red deer and roe deer can be a reservoir of avirulent R. equi strains.
( 1997 )
Cloning and characterization of the genes of the CeqI restriction-modification system.
PMID : 9304804 :
Two genes from Corynebacterium equii, a Gram-positive bacterium producing the CeqI restriction-modification enzymes were cloned and sequenced. In vivo restriction experiments, DNA and amino acid sequence data suggest that the two genes code for the endonuclease and the methyltransferase enzymes. However, when the two genes are expressed in E. coli, practically no enzyme activity can be detected in the supernatants of sonicated cells. Based on the DNA sequence data CeqI restriction endonuclease (an EcoRV izoschizomer) consists of 270 amino acid residues with a predicted molecular mass of 31.6 kDa, in good agreement with the previously measured 32 +/- 2 kDa. The methyltransferase is 517 residues long (approx. 60 kDa). The two genes are in opposite orientation and overlap by 37 base pairs on the chromosome. The deduced amino acid sequence of the putative endonuclease gene revealed long stretches of hydrophobic amino acids, that may form the structural basis of the unusual aggregation properties of the restriction endonuclease. The amino acid sequence of the methylase shows homologies with other type II methyltransferases.
( 1997 )
Monooxygenase-like sequence of a Rhodococcus equi gene conferring increased resistance to rifampin by inactivating this antibiotic.
PMID : 8980786 : PMC : PMC163691
A DNA clone from Rhodococcus equi conferring low-level rifampin resistance through the ability to inactivate this antibiotic via its decomposition was identified. The iri (inactivation of rifampin) gene consisted of an open reading frame of 1,437 bp encoding a 479-amino-acid sequence strongly resembling those of monooxygenases acting upon phenolic compounds or involved in polyketide antibiotic synthesis. When expressed in Escherichia coli, the gene conferred resistance to a > 50-micrograms/ml concentration of the drug.
( 1995 )
A physical map of the 85 kb virulence plasmid of Rhodococcus equi 103.
PMID : 8521357 : PMC : PMC1263770
A physical map of the 85 kb virulence plasmid pOTS from Rhodococcus equi 103 was constructed. The restriction map contains 2 AsnI, 5 BglII, 9 EcoRI, 4 HindIII, and 3 XbaI sites. The positions of the EcoRI and HindIII of pOTS are identical to that of the 85 kb virulence plasmid of R. equi ATCC 33701 reported recently by others. EcoRI restriction fragment sizes were similar in the 85 kb plasmids isolated from 4 horse derived R. equi but, except apparently for the 28.3 and possibly 2.0 and 1.5 kb fragments, were different in an 80.1 kb plasmid isolated from a pig source R. equi.
( 1995 )
Sequence of the Rhodococcus equi gene encoding the virulence-associated 15-17-kDa antigens.
PMID : 7698659 : DOI : 10.1016/0378-1119(95)00009-u
The nucleotide sequence of the Rhodococcus equi gene encoding the virulence-associated 15-17-kDa antigens, located on plasmid pREAT701, has been determined. The gene encodes a 19-kDa protein of 189 amino acids, with an Ala-rich leader signal sequence (SS). At least five SS peptidase cleavage sites were found in this region. The molecular diversity of 15-17-kDa antigens might be attributed to the multiple SS peptidase cleavage sites.
( 1995 )
Molecular characterization of a lipid-modified virulence-associated protein of Rhodococcus equi and its potential in protective immunity.
PMID : 7704843 : PMC : PMC1263734
Virulent strains of Rhodococcus equi produce plasmid-mediated 15- and 17-kDa proteins, which are thermoregulated and apparently surface-expressed. We demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that R. equi produce three antigenically-related virulence-associated proteins, a diffuse 18-22-kDa, a 17.5-kDa and a 15-kDa protein. Phase partitioning of whole cells of R. equi strain 103 with Triton X-114 (TX-114) and labelling with [3H]-labelled palmitic acid showed that the two higher molecular weight proteins are hydrophobic and lipid modified. The 15-kDa protein did not partition into TX-114 and was not lipid modified. Cloning and expression of a fragment of the R. equi virulence plasmid in Escherichia coli showed that the three proteins were expressed from a single gene. Sequence analysis of this gene (designated vapA) revealed a 570-bp open reading frame encoding a polypeptide of 189 amino acids with a calculated molecular mass of 19,175 Da. The mature, nonlipid modified protein had a calculated mass of 16,246 Da. The 17.5- and 18-22-kDa forms of the protein are therefore due to lipid modification. No significant sequence homology of the vapA gene with other reported nucleotide sequences were found. Opsonization of virulent R. equi with an IgG1 mouse monoclonal antibody (MAb103) to the VapA protein significantly enhanced uptake in the murine macrophage cell line IC-21. Intraperitoneal injection of mice with Mab103 enhanced initial clearance from the liver of mice challenged intravenously with R. equi. Immunization of mice with the lipid-modified VapA purified by SDS-PAGE fractionation or with acetone precipitated VapA protein following TX-114 extraction resulted in significantly enhanced clearance from the liver and spleen following intravenous challenge. The VapA protein of R. equi appears therefore to be a protective immunogen.
( 2018 )
Detection of vapN in Rhodococcus equi isolates cultured from humans.
PMID : 29300774 : DOI : 10.1371/journal.pone.0190829 PMC : PMC5754133
Rhodococcus equi can cause severe infections in people, particularly in immunocompromised individuals. The R. equi virulence plasmids (vap) encoding vapA and vapB are linked to development of infections in domestic animals. Recently, a novel virulence plasmid, vapN, was identified in isolates cultured from cattle, but its prevalence or significance in human R. equi infections has not been extensively studied. To determine the prevalence of vapN in a diverse collection of human-derived isolates from different countries, 65 R. equi isolates collected by various institutions from 1984 to 2002 were screened for the presence of vapN and other virulence plasmids through polymerase chain reaction (PCR) using redesigned primer sets. Of the isolates that carried plasmids, 43% (16/37) were vapN-positive and fewer were vapB or vapA-positive (30 and 16%, respectively). This is the first report of vapN carriage in R. equi isolated from human infections. One isolate (H-30) carried vapN but did not amplify the conjugal plasmid transfer gene traA associated with carriage of vap, which could be explained by sequence variation within the traA gene. Another isolate (H-55) amplified traA, but did not amplify vapA, B, or N (traA+ vapABN-) with previously described primer sets or those developed for this study. The H-55 traA sequence had 98% identity to traA sequences in vapA plasmids, which suggests that it may carry a variant of previously characterized virulence plasmids or a novel virulence plasmid. Carriage of vapN in R. equi isolates derived from people is not uncommon and more research is needed to determine its significance in the epidemiology and pathogenesis of human R. equi infections.
( 2013 )
Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.
PMID : 23078612 : DOI : 10.1111/cmi.12050 PMC : PMC3864644
Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for �]-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi.
( 1997 )
Development of a Rhodococcus equi-Escherichia coli plasmid shuttle vector.
PMID : 9435020 : DOI : 10.1006/plas.1997.1311
Isolates of Rhodococcus equi from pneumonic foals possess an 85- or 90-kb virulence-associated plasmid. A prominent, thermoregulated surface antigen, VapA, encoded by these plasmids is thought to be important in virulence. A 135-kb fragment containing the origin of replication of R. equi strain 103 virulence-associated plasmid (pOTS) was identified, sequenced, and its location identified. A simple R. equi-Escherichia coli shuttle plasmid (pRE-1) derived from the E. coli plasmid pACYC177 and the pOTS ori was developed. The plasmid transformed readily and was stable in either host and expressed kanamycin resistance but not beta-lactamase in R. equi. An improved 5.9-kb vector, pRE-7, was developed from pRE-1 and pBluescript. Subcloning of vapA into the multiple cloning site of the beta-galactosidase gene of pRE-7 resulted in weak expression of the gene both in E. coli and R. equi. The shuttle vector may be useful in examining regulation of virulence gene expression in R. equi.