| 1. |
Nicoloff H,
Bringel F,
( 2003 ) ISLpl1 is a functional IS30-related insertion element in Lactobacillus plantarum that is also found in other lactic acid bacteria. PMID : 14532059 : DOI : 10.1128/aem.69.10.6032-6040.2003 PMC : PMC201200 Abstract >>
We describe the first functional insertion sequence (IS) element in Lactobacillus plantarum. ISLpl1, an IS30-related element, was found on the pLp3 plasmid in strain FB335. By selection of spontaneous mutants able to grow in the presence of uracil, it was demonstrated that the IS had transposed into the uracil phosphoribosyltransferase-encoding gene upp on the FB335 chromosome. The plasmid-carried IS element was also sequenced, and a second potential IS element was found: ISLpl2, an IS150-related element adjacent to ISLpl1. When Southern hybridization was used, the copy number and genome (plasmid versus chromosome) distribution data revealed different numbers and patterns of ISLpl1-related sequences in different L. plantarum strains as well as in Pediococcus strains. The ISLpl1 pattern changed over many generations of the strain L. plantarum NCIMB 1406. This finding strongly supports our hypothesis that ISLpl1 is a mobile element in L. plantarum. Database analysis revealed five quasi-identical ISLpl1 elements in Lactobacillus, Pediococcus, and Oenococcus strains. Three of these elements may be cryptic IS, since point mutations or 1-nucleotide deletions were found in their transposase-encoding genes. In some cases, ISLpl1 was linked to genes involved in cold shock adaptation, bacteriocin production, sugar utilization, or antibiotic resistance. ISLpl1 is transferred among lactic acid bacteria (LAB) and may play a role in LAB genome plasticity and adaptation to their environment.
|
2. |
Nakayama J,
Akkermans AD,
De Vos WM,
( 2003 ) High-throughput PCR screening of genes for three-component regulatory system putatively involved in quorum sensing from low-G + C gram-positive bacteria. PMID : 12723594 : DOI : 10.1271/bbb.67.480 Abstract >>
Quorum sensing of gram-positive bacteria is often regulated by three-component regulatory system composed of autoinducing peptide, sensor kinase and response regulator. We used PCR to study a gene cassette encoding this three-component regulatory system. Degenerate primers were designed from consensus amino acid sequences in the HPK10 subfamily, mostly involved in quorum sensing. Products amplified from genomic DNA of Lactobacillus, Enterococcus, and Clostridium species were cloned and sequenced; their deduced amino acid sequences were similar to those of members of the HPK10 subfamily. Complete genes for the putative gene cassette were cloned by inverse PCR from L. paracasei E93490 and L. plantarum WCFS6. Phylogenetic analysis grouped the cloned putative HPKs into the HPK10 subfamily. These results indicated the usefulness of this high-throughput gene screening and suggested that the three-component regulatory gene cassette are widely present.
|
3. |
Whittaker MM,
Barynin VV,
Igarashi T,
Whittaker JW,
( 2003 ) Outer sphere mutagenesis of Lactobacillus plantarum manganese catalase disrupts the cluster core. Mechanistic implications. PMID : 12631270 : DOI : 10.1046/j.1432-1033.2003.03459.x Abstract >>
X-ray crystallography of the nonheme manganese catalase from Lactobacillus plantarum (LPC) [Barynin, V.V., Whittaker, M.M., Antonyuk, S.V., Lamzin, V.S., Harrison, P.M., Artymiuk, P.J. & Whittaker, J.W. (2001) Structure9, 725-738] has revealed the structure of the dimanganese redox cluster together with its protein environment. The oxidized [Mn(III)Mn(III)] cluster is bridged by two solvent molecules (oxo and hydroxo, respectively) together with a micro 1,3 bridging glutamate carboxylate and is embedded in a web of hydrogen bonds involving an outer sphere tyrosine residue (Tyr42). A novel homologous expression system has been developed for production of active recombinant LPC and Tyr42 has been replaced by phenylalanine using site-directed mutagenesis. Spectroscopic and structural studies indicate that disruption of the hydrogen-bonded web significantly perturbs the active site in Y42F LPC, breaking one of the solvent bridges and generating an 'open' form of the dimanganese cluster. Two of the metal ligands adopt alternate conformations in the crystal structure, both conformers having a broken solvent bridge in the dimanganese core. The oxidized Y42F LPC exhibits strong optical absorption characteristic of high spin Mn(III) in low symmetry and lower coordination number. MCD and EPR measurements provide complementary information defining a ferromagnetically coupled electronic ground state for a cluster containing a single solvent bridge, in contrast to the diamagnetic ground state found for the native cluster containing a pair of solvent bridges. Y42F LPC has less than 5% of the catalase activity and much higher Km for H2O2 (approximately 1.4 m) at neutral pH than WT LPC, although the activity is slightly restored at high pH where the cluster is converted to a diamagnetic form. These studies provide new insight into the contribution of the outer sphere tyrosine to the stability of the dimanganese cluster and the role of the solvent bridges in catalysis by dimanganese catalases.
|
4. |
Daming R,
Yinyu W,
Zilai W,
Jun C,
Hekui L,
Jingye Z,
( 2003 ) Complete DNA sequence and analysis of two cryptic plasmids isolated from Lactobacillus plantarum. PMID : 12826059 : Abstract >>
The complete nucleotide sequence of two cryptic plasmids isolated from Lactobacillus plantarum strain AS1.2986 has been determined. The smaller plasmid, designated pLP2000, encodes a 37.0kDa Rep protein and has a 17bp sequence repeated 10 times. Sequence analysis of the larger plasmid, designated pLP9000, revealed nine putative open reading frames (ORFs). Based on sequence similarity, ORF1 codes for a putative magnesium transporter protein that shows similarities to CorA from plasmid pCIS3 (Lactococcus lactis). None of the nine ORFs shows similarity to any known Rep protein. Southern blot analysis indicates these two plasmids both replicate via a rolling circle (RC) mechanism.
|
5. |
Gevers D,
Danielsen M,
Huys G,
Swings J,
( 2003 ) Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage. PMID : 12571056 : DOI : 10.1128/aem.69.2.1270-1275.2003 PMC : PMC143591 Abstract >>
The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc(r)) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)(5)-PCR DNA fingerprinting technique, the Tc(r) lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc(r) lactic acid bacterial isolates displaying unique (GTG)(5)-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.
|
6. |
Maldonado A,
Ruiz-Barba JL,
Jiménez-Díaz R,
( 2003 ) Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8. PMID : 12514019 : DOI : 10.1128/aem.69.1.383-389.2003 PMC : PMC152457 Abstract >>
A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named alpha and beta, which were separated by C(2)-C(18) reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8 alpha and PLNC8 beta, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (alpha) and 4,000 Da (beta). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature alpha and beta peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative -35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.
|
7. |
Dobson CM,
Deneer H,
Lee S,
Hemmingsen S,
Glaze S,
Ziola B,
( 2002 ) Phylogenetic analysis of the genus Pediococcus, including Pediococcus claussenii sp. nov., a novel lactic acid bacterium isolated from beer. PMID : 12508860 : DOI : 10.1099/00207713-52-6-2003 Abstract >>
Pediococci are found in foods and on plants and as beer-spoilage agents. The goal of the present study was to use the DNA sequences of the first three variable regions of the 165 rRNA gene, the 16S-23S rRNA internally transcribed spacer region sequence and approximately a third of the 60 kDa heat-shock protein gene to elucidate phylogenetic groupings within the genus Pediococcus. Phylogenetic trees were created with sequence data from 31 Pediococcus and three Lactobacillus isolates. Complete 16S rRNA gene sequences from selected Pediococcus isolates were also examined. The results were interpreted in relation to the currently accepted Pediococcus species. We found that, where previously done, speciation of many Pediococcus isolates is inaccurate. Also, one grouping of seven isolates did not include any currently recognized Pediococcus species type isolate. Our phylogenetic analyses support the conclusion that these seven isolates, all of brewing spoilage origin, belong to a novel species, for which the name Pediococcus claussenii sp. nov. is proposed (type strain P06(T0 = ATCC BAA-344(T) = DSM 14800(T)). Phylogenetic analysis has therefore helped to resolve problems surrounding species identification of Pediococcus isolates.
|
8. |
Chavagnat F,
Haueter M,
Jimeno J,
Casey MG,
( 2002 ) Comparison of partial tuf gene sequences for the identification of lactobacilli. PMID : 12480101 : DOI : 10.1111/j.1574-6968.2002.tb11472.x Abstract >>
Comparative analysis of partial tuf sequences was evaluated for the identification and differentiation of lactobacilli. Comparison of the amino acid sequences allowed differentiation between species and also between the subspecies of Lactobacillus delbrueckii. The nucleotide sequence comparison allowed differentiation between other subspecies and between some strains. Lactobacilli from several collections and isolates from dairy samples were clearly identified by comparison of short tuf sequences with those of the type strains. In evaluating the taxonomy of the Lactobacillus casei-related taxa, different tuf amino acid signatures are in favour of a classification into three distinct species. The type strain designation for the L. casei species is discussed.
|
9. |
Silvestroni A,
Connes C,
Sesma F,
De Giori GS,
Piard JC,
( 2002 ) Characterization of the melA locus for alpha-galactosidase in Lactobacillus plantarum. PMID : 12406739 : DOI : 10.1128/aem.68.11.5464-5471.2002 PMC : PMC129937 Abstract >>
Alpha-galactosides are abundant sugars in legumes such as soy. Because of the lack of alpha-galactosidase (alpha-Gal) in the digestive tract, humans are unable to digest these sugars, which consequently induce flatulence. To develop the consumption of the otherwise highly nutritional soy products, the use of exogenous alpha-Gal is promising. In this framework, we characterized the melA gene for alpha-Gal in Lactobacillus plantarum. The melA gene encodes a cytoplasmic 84-kDa protein whose enzymatically active form occurs as oligomers. The melA gene was cloned and expressed in Escherichia coli, yielding an active alpha-Gal. We show that melA is transcribed from its own promoter, yielding a monocistronic mRNA, and that it is regulated at the transcriptional level, i.e., it is induced by melibiose but is not totally repressed by glucose. Posttranscriptional regulation by the carbon source could also occur. Upstream of melA, a putative galactoside transporter, designated RafP, was identified that shows high homology to LacS, the unique transporter for both alpha- and beta-galactosides in Streptococcus thermophilus. rafP is also expressed as a monocistronic mRNA. Downstream of melA, the lacL and lacM genes were identified that encode a heterodimeric beta-galactosidase. A putative galM gene identified in the same cluster suggests the presence of a galactose operon. These results indicate that the genes involved in galactoside catabolism are clustered in L. plantarum ATCC 8014. This first genetic characterization of melA and of its putative associated transporter, rafP, in a lactobacillus opens doors to various applications both in the manufacture of soy-derived products and in probiotic and nutraceutical issues.
|
10. |
Danielsen M,
( 2002 ) Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure. PMID : 12383727 : Abstract >>
The 10,877bp tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 was completely sequenced. The sequence revealed a composite structure containing DNA from up to four different sources. The replication region had homology to other plasmids of lactic acid bacteria while the tetracycline resistance region, containing a tet(M) gene, had high homology to sequences from Clostridium perfringens and Staphylococcus aureus. Within the tetracycline resistance region a Lactobacillus IS-element was found. The remaining part of the plasmid contained three open reading frames with unknown functions. The composite structure with several truncated genes suggests a recent assembly of the plasmid. This is the first sequence of an antibiotic resistance plasmid isolated from L. plantarum.
|
11. |
Van Reenen CA,
Chikindas ML,
Van Zyl WH,
Dicks LM,
( 2003 ) Characterization and heterologous expression of a class IIa bacteriocin, plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae. PMID : 12423916 : Abstract >>
Lactobacillus plantarum 423 produces a small heat-stable antimicrobial protein designated plantaricin 423. This protein is bactericidal for many Gram-positive foodborne pathogens and spoilage bacteria, including Listeria spp., Staphylococcus spp., Pediococcus spp., Lactobacillus spp., etc. The DNA sequence of the plantaricin 423-encoding region on plasmid pPLA4 revealed a four open reading frame (ORF) operon structure similar to pediocin PA-1/AcH from Pediococcus acidilactici and coagulin from Bacillus coagulans I(4). The first ORF, plaA, encodes a 56-amino acid prepeptide consisting of a 37-amino acid mature molecule, with a 19-amino acid N-terminal leader peptide. The second ORF, plaB, encodes a putative immunity protein with protein sequence similarities to several bacteriocin immunity proteins. The plaC and plaD genes are virtually identical to pedC and pedD of the pediocin PA-1 operon, as well as coaC and coaD of the coagulin operon. Plantaricin 423 was cloned on a shuttle vector under the control of a yeast promoter and heterologously produced in Saccharomyces cerevisiae.
|
12. |
Torriani S,
Felis GE,
Dellaglio F,
( 2001 ) Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers. PMID : 11472918 : DOI : 10.1128/AEM.67.8.3450-3454.2001 PMC : PMC93042 Abstract >>
In this study, we succeeded in differentiating Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum by means of recA gene sequence comparison. Short homologous regions of about 360 bp were amplified by PCR with degenerate consensus primers, sequenced, and analyzed, and 322 bp were considered for the inference of phylogenetic trees. Phylograms, obtained by parsimony, maximum likelihood, and analysis of data matrices with the neighbor-joining model, were coherent and clearly separated the three species. The validity of the recA gene and RecA protein as phylogenetic markers is discussed. Based on the same sequences, species-specific primers were designed, and a multiplex PCR protocol for the simultaneous distinction of these bacteria was optimized. The sizes of the amplicons were 318 bp for L. plantarum, 218 bp for L. pentosus, and 107 bp for L. paraplantarum. This strategy permitted the unambiguous identification of strains belonging to L. plantarum, L. pentosus, and L. paraplantarum in a single reaction, indicating its applicability to the speciation of isolates of the L. plantarum group.
|
13. |
Muscariello L,
Marasco R,
De Felice M,
Sacco M,
( 2001 ) The functional ccpA gene is required for carbon catabolite repression in Lactobacillus plantarum. PMID : 11425700 : DOI : 10.1128/AEM.67.7.2903-2907.2001 PMC : PMC92959 Abstract >>
We report the characterization of the ccpA gene of Lactobacillus plantarum, coding for catabolite control protein A. The gene is linked to the pepQ gene, encoding a proline peptidase, in the order ccpA-pepQ, with the two genes transcribed in tandem from the same strand as distinct transcriptional units. Two ccpA transcription start sites corresponding to two functional promoters were found, expression from the upstream promoter being autogenously regulated through a catabolite-responsive element (cre) sequence overlapping the upstream +1 site. During growth on ribose, the upstream promoter showed maximal expression, while growth on glucose led to transcription from the downstream promoter. In a ccpA mutant strain, the gene was transcribed mainly from the upstream promoter in both repressing and non repressing conditions. Expression of two enzyme activities, beta-glucosidase and beta-galactosidase, was relieved from carbon catabolite repression in the ccpA mutant strain. In vivo footprinting analysis of the catabolite-controlled bglH gene regulatory region in the ccpA mutant strain showed loss of protection of the cre under repressing conditions.
|
14. |
Holo H,
Jeknic Z,
Daeschel M,
Stevanovic S,
Nes IF,
( 2001 ) Plantaricin W from Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics. PMID : 11238971 : DOI : 10.1099/00221287-147-3-643 Abstract >>
Plantaricin W (Plw) is a new two-peptide bacteriocin, from Lactobacillus plantarum, which inhibits a large number of Gram-positive bacteria. The two peptides, Plwalpha (comprising 29 residues) and Plwbeta (comprising 32 residues), were isolated from the culture supernatants and characterized. The individual peptides had low antimicrobial activity but acted synergistically, and synergism was seen at all mixing ratios tested. The data indicate that the two peptides work in a 1:1 ratio. Chemical analyses showed that both peptides are lantibiotics, but two unmodified cysteines and one serine residue were present in Plwalpha, and Plwbeta contained one cysteine residue. The Plw structural genes were sequenced and shown to encode prepeptides with sequence similarities to two other two-peptide lantibiotics, namely staphylococcin C55 and lacticin 3147. The conserved residues are mainly serines, threonines and cysteines that can be involved in intramolecular thioether bond formation in the C-terminal parts of the molecules. This indicates that these bacteriocins are members of a new family of lantibiotics with common bridging patterns, and that the ring structures play an important functional role. Based on the data a structural model is presented in which each peptide has a central lanthionine and two overlapping thioether bridges close to their C-termini.
|
15. |
Kiatpapan P,
Kobayashi H,
Sakaguchi M,
Ono H,
Yamashita M,
Kaneko Y,
Murooka Y,
( 2001 ) Molecular characterization of Lactobacillus plantarum genes for beta-ketoacyl-acyl carrier protein synthase III (fabH) and acetyl coenzyme A carboxylase (accBCDA), which are essential for fatty acid biosynthesis. PMID : 11133475 : DOI : 10.1128/AEM.67.1.426-433.2001 PMC : PMC92595 Abstract >>
Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified six potential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotide sequence analysis suggested that fabH encoded beta-ketoacyl-acyl carrier protein synthase III, that the accB, accC, accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the beta and alpha subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes was different from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coli accB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in E. coli. The gene product of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the five genes in L. plantarum were regulated polycistronically in an acc operon.
|
16. |
Doi K,
Nishiyama K,
Eguchi T,
( 2000 ) Characterization of a phage resistance plasmid, pLKS, of silage-making Lactobacillus plantarum NGRI0101. PMID : 10830488 : DOI : 10.1271/bbb.64.751 Abstract >>
Phage contamination has resulted in abnormal fermentation in silage. We isolated a phage-resistant strain, Lactobacillus plantarum NGRI0101 from silage. The strain carried two plasmids, pLKL (6.8 kb) and pLKS (2.0 kb). By curing and retransformation of the plasmids, we clarified that pLKS has phage resistant activity, characterized as no adsorption inhibition. pLKS has 2,025 bp and three orfs, orfl23, orf132, and orf918. The predicted amino acid sequence of the orf918 product showed high similarity to those of Rep proteins of Pediococcus halophilus plasmid pUCL287 and Lactobacillus acidophilus plasmid pLA103. The replication origin (ori) was upstream from orf918. There was no gene similar to typical phage resistant genes encoded by known plasmids. The phage resistance of L. plantarum NGRI0101 may possibly be due to a plasmid-encoded abortive infection.
|
17. |
Salatiello I,
Marasco R,
( 2000 ) A physical and functional analysis of the newly-identified bglGPT operon of Lactobacillus plantarum. PMID : 10802183 : DOI : 10.1111/j.1574-6968.2000.tb09116.x Abstract >>
A newly-identified bglGPT operon of Lactobacillus plantarum was isolated and expressed in Escherichia coli. The sequence analysis of the cloned DNA fragment showed three open reading frames encoding (i) a 237-amino acid protein (BglG), (ii) a 577-amino acid protein (BglP) and (iii) a 486-amino acid protein (BglT). BglG, BglP and BglT were shown to be homologous to the BglG family of transcriptional antiterminators, to permeases of the phosphoenolpyruvate-dependent phosphotransferase system and to beta-glucosidases, respectively. Complementation of E. coli mutant strains showed that BglP and BglT are a permease and a beta-glucosidase active on the beta-glucosides, 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-glucoside, respectively. BglG was also shown to promote expression of a bglG-lacZ gene fusion in an E. coli bglG(-) background. A ribonucleic antiterminator sequence, the antiterminator-responsive cis-element and a 'catabolite responsive element', were found downstream of the transcriptional start point. Transcription of the operon was repressed 10-fold in L. plantarum cells grown on glucose as compared to ribose.
|
18. |
Derzelle S,
Hallet B,
Francis KP,
Ferain T,
Delcour J,
Hols P,
( 2000 ) Changes in cspL, cspP, and cspC mRNA abundance as a function of cold shock and growth phase in Lactobacillus plantarum. PMID : 10960094 : DOI : 10.1128/jb.182.18.5105-5113.2000 PMC : PMC94658 Abstract >>
An inverse PCR strategy based on degenerate primers has been used to identify new genes of the cold shock protein family in Lactobacillus plantarum. In addition to the two previously reported cspL and cspP genes, a third gene, cspC, has been cloned and characterized. All three genes encode small 66-amino-acid proteins with between 73 and 88% identity. Comparative Northern blot analyses showed that the level of cspL mRNA increases up to 17-fold after a temperature downshift, whereas the mRNA levels of cspC and cspP remain unchanged or increase only slightly (about two- to threefold). Cold induction of cspL mRNA is transient and delayed in time as a function of the severity of the temperature downshift. The cold shock behavior of the three csp mRNAs contrasts with that observed for four unrelated non-csp genes, which all showed a sharp decrease in mRNA level, followed in one case (bglH) by a progressive recovery of the transcript during prolonged cold exposure. Abundance of the three csp mRNAs was also found to vary during growth at optimal temperature (28 degrees C). cspC and cspP mRNA levels are maximal during the lag period, whereas the abundance of the cspL transcript is highest during late-exponential-phase growth. The differential expression of the three L. plantarum csp genes can be related to sequence and structural differences in their untranslated regions. It also supports the view that the gene products fulfill separate and specific functions, under both cold shock and non-cold shock conditions.
|
19. |
Remiger A,
Ehrmann MA,
( 2000 ) A gene cluster encoding plantaricin 1.25beta and other bacteriocin-like peptides in Lactobacillus plantarum TMW1.25. PMID : 10684981 : DOI : 10.1016/s0167-4781(00)00003-8 Abstract >>
Plantaricin 1.25beta is a thermostable class two bacteriocin produced by Lactobacillus plantarum TMW1.25 isolated from sausage fermentation. It is co-produced with several other bacteriocin-like peptides. Using oligonucleotides derived from previously determined peptide sequences, a 3.8 kb DNA fragment could be amplified. A neighboring 1.8 kb fragment was amplified using ligation-anchored single-specific-primer PCR. Sequencing of the complete 5.6 kb stretch revealed that the structural gene for plantaricin 1.25beta, plnB, was located downstream of another bacteriocin gene, plnC. Seven other open reading frames were detected, including plnK encoding a bacteriocin-like peptide, but not including any putative immunity genes. Interestingly, the gene cluster contained an IS30-like insertion sequence, designated IS125, as well as an ISS1 homolog.
|
20. |
Chen S,
Hao Z,
( 1999 ) Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum. PMID : 10543781 : PMC : PMC91639 Abstract >>
An Mn(2+) and Cd(2+) uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd(2+) sensitivity as well as energy-dependent Cd(2+) uptake activity. Both transcription and translation of mntA were induced by Mn(2+) starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd(2+) uptake systems have been identified in L. plantarum: one is a high-affinity Mn(2+) and Cd(2+) uptake system that is expressed in Mn(2+)-starved cells, and the other is a nonsaturable Cd(2+) uptake system that is expressed in Cd(2+)-sufficient cells (Z. Hao, H. R. Reiske, and D. B. Wilson, Appl. Environ. Microbiol. 65:592-99, 1999). MntA was not detected in an Mn(2+)-dependent mutant of L. plantarum which had lost high-affinity Mn(2+) and Cd(2+) uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn(2+) and Cd(2+) transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn(2+)- and Cd(2+)-binding sites of MntA are discussed. A second clone containing a low-affinity Cd(2+) transport system was also isolated.
|
21. |
Diancourt L,
Passet V,
Chervaux C,
Garault P,
Smokvina T,
Brisse S,
( 2007 ) Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination. PMID : 17704267 : DOI : 10.1128/AEM.01095-07 PMC : PMC2075077 Abstract >>
Robust genotyping methods for Lactobacillus casei are needed for strain tracking and collection management, as well as for population biology research. A collection of 52 strains initially labeled L. casei or Lactobacillus paracasei was first subjected to rplB gene sequencing together with reference strains of Lactobacillus zeae, Lactobacillus rhamnosus, and other species. Phylogenetic analysis showed that all 52 strains belonged to a single compact L. casei-L. paracasei sequence cluster, together with strain CIP107868 (= ATCC 334) but clearly distinct from L. rhamnosus and from a cluster with L. zeae and CIP103137(T) (= ATCC 393(T)). The strains were genotyped using amplified fragment length polymorphism, multilocus sequence typing based on internal portions of the seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG, and tandem repeat variation (multilocus variable-number tandem repeats analysis [MLVA] using nine loci). Very high concordance was found between the three methods. Although amounts of nucleotide variation were low for the seven genes (pi ranging from 0.0038 to 0.0109), 3 to 12 alleles were distinguished, resulting in 31 sequence types. One sequence type (ST1) was frequent (17 strains), but most others were represented by a single strain. Attempts to subtype ST1 strains by MLVA, ribotyping, clustered regularly interspaced short palindromic repeat characterization, and single nucleotide repeat variation were unsuccessful. We found clear evidence for homologous recombination during the diversification of L. casei clones, including a putative intragenic import of DNA into one strain. Nucleotides were estimated to change four times more frequently by recombination than by mutation. However, statistical congruence between individual gene trees was retained, indicating that recombination is not frequent enough to disrupt the phylogenetic signal. The developed multilocus sequence typing scheme should be useful for future studies of L. casei strain diversity and evolution.
|
22. |
Scheirlinck I,
Van der Meulen R,
Van Schoor A,
Vancanneyt M,
De Vuyst L,
Vandamme P,
Huys G,
( 2007 ) Influence of geographical origin and flour type on diversity of lactic acid bacteria in traditional Belgian sourdoughs. PMID : 17675431 : DOI : 10.1128/AEM.00894-07 PMC : PMC2075033 Abstract >>
A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.
|
23. |
Van der Meulen R,
Grosu-Tudor S,
Mozzi F,
Vaningelgem F,
Zamfir M,
de Valdez GF,
De Vuyst L,
( 2007 ) Screening of lactic acid bacteria isolates from dairy and cereal products for exopolysaccharide production and genes involved. PMID : 17716765 : DOI : 10.1016/j.ijfoodmicro.2007.07.014 Abstract >>
A total of 174 lactic acid bacteria (LAB) strains isolated from dairy and cereal products were screened for the production of exopolysaccharides (EPS). Therefore, a rapid screening method was developed based on ultrafiltration and gel permeation chromatography. Furthermore, a screening through the polymerase chain reaction (PCR) was performed with primer pairs targeting different genes involved in EPS production. Nine isolates produced a homopolysaccharide of the glucan type, whereas only one strain produced a heteropolysaccharide. The production of a glucan by a strain of Lactococcus lactis and the production of a heteropolysaccharide by a strain of Lactobacillus curvatus are reported for the first time. The PCR screening revealed many positive strains. For three of the ten EPS-producing strains, no corresponding genes could be detected. Furthermore, a lot of strains possessed one or more eps genes but did not produce an EPS. Therefore, a screening on the molecular level should always be accompanied by another screening method that is able to distinguish true EPS producer strains from non-producing ones. Statistical analysis did not reveal any relationship between the type and origin of the strains, the presence or absence of a capsular polysaccharide or EPS, and the presence or absence of eps genes.
|
24. |
Arena ME,
Fiocco D,
Manca de Nadra MC,
Pardo I,
Spano G,
( 2007 ) Characterization of a Lactobacillus plantarum strain able to produce tyramine and partial cloning of a putative tyrosine decarboxylase gene. PMID : 17657538 : DOI : 10.1007/s00284-006-0647-8 Abstract >>
The aim of this article was to analyze the ability of wine Lactobacillus plantarum strains to form tyramine. Preliminary identification of L. plantarum strains was performed by amplification of the recA gene. Primers pREV and PlanF, ParaF and PentF were used respectively as reverse and forward primers in the polymerase chain reaction tests as previously reported. Furthermore, the gene encoding for the tyrosine decarboxylase (TDC) was partially cloned from one strain identified as L. plantarum. The strain was further analyzed by 16S rDNA sequence and confirmed as belonging to L. plantarum species. The tyrosine decarboxylase activity was investigated and tyramine was determined by the high-performance liquid chromatography method. Moreover, a negative effect of sugars such as glucose and fructose and L: -malic acid on tyrosine decarboxylase activity was observed. The results suggest that, occasionally, L. plantarum is able to produce tyramine in wine and this ability is apparently confined only to L. plantarum strains harboring the tdc gene.
|
25. |
Naterstad K,
Rud I,
Kvam I,
Axelsson L,
( 2007 ) Characterisation of the gap operon from Lactobacillus plantarum and Lactobacillus sakei. PMID : 17294332 : DOI : 10.1007/s00284-006-0013-x Abstract >>
Glycolysis constitutes the primary energy-generating pathway of most species of lactic acid bacteria. The metabolism ultimately results in massive lactic acid production, which is responsible for the major preservative effect of these organisms. This study reports the identification, sequencing, and characterisation of the central glycolytic operon, the gap operon, from Lactobacillus plantarum NC8 and L. sakei Lb790. The structure of the operons of the two Lactobacillus strains were similar and organised in the order cggR-gap-pgk-tpi-eno, encoding a putative central glycolytic gene regulator and the four glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, triosephosphate isomerase, and enolase, respectively. This operon structure has not been reported in any other bacterial species so far. Transcriptional analysis revealed three major transcripts, the mono-cistronic gap and eno and the tetra-cistronic gap-pgk-tpi-eno.
|
26. |
Van Reenen CA,
Van Zyl WH,
Dicks LM,
( 2006 ) Expression of the immunity protein of plantaricin 423, produced by Lactobacillus plantarum 423, and analysis of the plasmid encoding the bacteriocin. PMID : 17056693 : DOI : 10.1128/AEM.01428-06 PMC : PMC1694273 Abstract >>
Plantaricin 423 is a class IIa bacteriocin produced by Lactobacillus plantarum isolated from sorghum beer. It has been previously determined that plantaricin 423 is encoded by a plasmid designated pPLA4, which is now completely sequenced. The plantaricin 423 operon shares high sequence similarity with the operons of coagulin, pediocin PA-1, and pediocin AcH, with small differences in the DNA sequence encoding the mature bacteriocin peptide and the immunity protein. Apart from the bacteriocin operon, no significant sequence similarity could be detected between the DNA or translated sequence of pPLA4 and the available DNA or translated sequences of the plasmids encoding pediocin AcH, pediocin PA-1, and coagulin, possibly indicating a different origin. In addition to the bacteriocin operon, sequence analysis of pPLA4 revealed the presence of two open reading frames (ORFs). ORF1 encodes a putative mobilization (Mob) protein that is homologous to the pMV158 superfamily of mobilization proteins. Highest sequence similarity occurred between this protein and the Mob protein of L. plantarum NCDO 1088. ORF2 encodes a putative replication protein that revealed low sequence similarity to replication proteins of plasmids pLME300 from Lactobacillus fermentum and pYIT356 from Lactobacillus casei. The immunity protein of plantaricin 423 contains 109 amino acids. Although plantaricin 423 shares high sequence similarity with the pediocin PA-1 operon, no cross-reactivity was recorded between the immunity proteins of plantaricin 423 and pediocin PA-1.
|
27. |
Goffin P,
Muscariello L,
Lorquet F,
Stukkens A,
Prozzi D,
Sacco M,
Kleerebezem M,
Hols P,
( 2006 ) Involvement of pyruvate oxidase activity and acetate production in the survival of Lactobacillus plantarum during the stationary phase of aerobic growth. PMID : 17012588 : DOI : 10.1128/AEM.00659-06 PMC : PMC1694206 Abstract >>
In addition to the previously characterized pyruvate oxidase PoxB, the Lactobacillus plantarum genome encodes four predicted pyruvate oxidases (PoxC, PoxD, PoxE, and PoxF). Each pyruvate oxidase gene was individually inactivated, and only the knockout of poxF resulted in a decrease in pyruvate oxidase activity under the tested conditions. We show here that L. plantarum has two major pyruvate oxidases: PoxB and PoxF. Both are involved in lactate-to-acetate conversion in the early stationary phase of aerobic growth and are regulated by carbon catabolite repression. A strain devoid of pyruvate oxidase activity was constructed by knocking out the poxB and poxF genes. In this mutant, acetate production was strongly affected, with lactate remaining the major end product of either glucose or maltose fermentation. Notably, survival during the stationary phase appeared to be dramatically improved in the poxB poxF double mutant.
|
28. |
Jeong DW,
Lee JH,
Kim KH,
Lee HJ,
( 2006 ) A food-grade expression/secretion vector for Lactococcus lactis that uses an alpha-galactosidase gene as a selection marker. PMID : 16943039 : DOI : 10.1016/j.fm.2005.06.003 Abstract >>
A new food-grade expression/secretion vector for lactococci, pFMN30, was developed using an alpha-galactosidase gene (melA) of Lactobacillus plantarum as a selection marker. The 4.9-kb pFMN30 is a derivative of the lactococcal vector pMG36e containing a broad-host-range replicon of pWV01. In Lactococcus lactis, transformants carrying the vector were easily detectable by the appearance of a blue colony on a X-alpha-gal-containing medium and also by the growth on a medium containing melibiose as a sole carbon source. The expression/secretion vector was equipped with the controllable and strong nisA promoter. In addition, usp45 signal peptide was inserted for the efficient secretion of a foreign protein outside cells. The vector pFMN30 was used for the expression and secretion of alpha-amylase as a reporter gene, lacking a signal sequence derived from Bacillus licheniformis in L. lactis. These results show that the food-grade expression/secretion vector constructed in the present study could be used for the production of foreign proteins in L. lactis for the production food materials and also for the medicinal purposes.
|
29. |
Renouf V,
Claisse O,
Miot-Sertier C,
Lonvaud-Funel A,
( 2006 ) Lactic acid bacteria evolution during winemaking: use of rpoB gene as a target for PCR-DGGE analysis. PMID : 16942997 : DOI : 10.1016/j.fm.2005.01.019 Abstract >>
Evolution of the microbial population during winemaking is crucial. Winemakers are more and more attentive to microbial aspects during fermentation. During aging, microbial stabilization is preponderant to avoid development of spoilage yeast and bacteria. Therefore, it is necessary to improve methods to study the evolution of micro-organisms and for early detection of undesirable strain. The aim of this study was to develop a culture-independent method for identifying lactic acid bacteria (LAB) and to monitoring predominant species. The benefits of PCR-DGGE for the analysis of microbial changes during winemaking were clearly demonstrated. Targeting rpoB gene allowed a reliable discrimination of each species. The primers were able to avoid the interspecies heterogeneity problem caused by the use of the 16S rRNA gene. This method was applied to study the influence of different oenological practices on LAB population and their evolution during winemaking.
|
30. |
Huys G,
D'Haene K,
Swings J,
( 2006 ) Genetic basis of tetracycline and minocycline resistance in potentially probiotic Lactobacillus plantarum strain CCUG 43738. 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.
|
31. |
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. 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.
|
32. |
Kaneko Y,
Kobayashi H,
Kiatpapan P,
Nishimoto T,
Napitupulu R,
Ono H,
Murooka Y,
( 2000 ) Development of a host-vector system for Lactobacillus plantarum L137 isolated from a traditional fermented food produced in the Philippines. PMID : 16232699 : Abstract >>
Lactobacillus plantarum NC13, a strain derived from the L. plantarum strain L137 isolated from a traditional fermented food produced in the Philippines, contains 9 of the 15 plasmids in the parental strain. To construct a shuttle vector between L. plantarum and Escherichia coli for genetic manipulation of L137 and its derivatives, recombinant plasmids were prepared by using the 9-plasmid DNA mixture and an E. coli vector, pBluescript II SK+. The resultant recombinant plasmids were re-transferred to L. plantarum NCL21, an NC13-derived strain cured of 3 of the 9 plasmids, and 3 recombinant plasmids were obtained. The smallest plasmid, pRN14, contained a small cryptic plasmid, pLTK2, which is one of the plasmids in L. plantarum L137. Thus, the complete nucleotide sequence of pLTK2 was determined. The pLTK2 is 2295 bp in length, and has a major open reading frame of 951 bp. An encoded sequence of 317-amino acids showed extensive similarity with genes encoding replication protein (repA). A putative replication origin in pLTK2 also showed high homology to those of other gram-positive bacterial plasmids that replicate by the rolling circle mechanism. The shuttle vector pRN14 contained the erythromycin resistance gene and the ColE1 and pLTK2 replication origins. Transformation of L. plantarum strains with pRN14 by electroporation was optimized to give a transformation efficiency of 2 x 10(4) transformants/ mug plasmid. Plasmid pRN14 was stably maintained in strain NCL21, as well as in L. casei K95-5.
|
33. |
de Las Rivas B,
Marcobal Ae,
Gómez A,
Muñoz R,
( 2005 ) Characterization of ISLpl4, a functional insertion sequence in Lactobacillus plantarum. PMID : 16278055 : DOI : 10.1016/j.gene.2005.09.005 Abstract >>
A Lactobacillus plantarum strain, CECT 4645, was found to have insertions of a sequence (985 bp in length) at least in eight loci in its genome. The prototype copy (Lp1) of this insertion sequence (named ISLpl4) has one open reading frame encoding a putative protein that is 292 amino acids in length with significant levels of similarity with IS982 family transposases. Perfect 16-bp inverted repeats were found at its termini. Upon transposition, generates 8-bp direct repeats of the target sequence, but no consensus sequences could be identified at either insertion site. The ISLpl4 pattern changed over many generations on the CECT 4645 strain. This finding strongly supports our hypothesis that ISLpl4 is a functional element in L. plantarum. Some of these elements may be cryptic, since point mutation or 1-nucleotide deletions were found in their transposase encoding genes. ISLpl4 copies have been detected in Leuconostoc mesenteroides, Oenococcus oeni, and Lactobacillus sakei. An ISLpl4 copy of O. oeni contained a +1 nucleotide insertion on its transposase encoding gene and, by using an experimental system, we were able to demonstrate that this specific sequence originates a +1 programmed translational frameshifting. Although the frameshifting process reported here operates at a low rate, this description might represent the first case of a functional +1 frameshifting among IS.
|
34. |
Spano G,
Rinaldi A,
Ugliano M,
Moio L,
Beneduce L,
Massa S,
( 2005 ) A beta-glucosidase gene isolated from wine Lactobacillus plantarum is regulated by abiotic stresses. PMID : 15752331 : DOI : 10.1111/j.1365-2672.2004.02521.x Abstract >>
Little genetic information exists on the ability of wine lactic acid bacteria (LAB) to hydrolyse glycoconjugates during malolactic fermentation. We tried to fill this important gap by characterizing a gene codifying for a putative beta-glucosidase enzyme from wine Lactobacillus plantarum and from a commercial strain of Oenococcus oeni. The coding region of the putative beta-glucosidase gene is 1400 nucleotides long and started with an ATG codon. The gene is widespread among LAB and the highest identity was observed between the nucleotide of L. plantarum, Lactobacillus pentosus, Lactobacillus paraplantarum and O. oenibeta-glucosidase gene. The protein sequence deduced from the isolated genes has a calculated molecular mass of 61.19 kDa. Furthermore, the expression of the beta-glucosidase gene in L. plantarum strain was analysed, under several stress, by reverse transcriptase (RT)-PCR and Northern-blot analysis. The gene was apparently regulated by abiotic stresses such as temperature, ethanol and pH. The beta-glucosidase gene is widespread among LAB and its expression is probably regulated by a wide range of abiotic stresses. The inhibitory effect of temperature and ethanol on the L. plantarumbeta-glucosidase gene may be useful to explain the differences found in beta-glucosidase activity reported in wines by several authors.
|
35. |
Hörmann S,
Vogel RF,
Ehrmann M,
( 2006 ) Construction of a new reporter system to study the NaCl-dependent dnaK promoter activity of Lactobacillus sanfranciscensis. PMID : 16133335 : DOI : 10.1007/s00253-005-0114-7 Abstract >>
A reporter system was developed to study gene expression in Lactobacillus sanfranciscensis. It was based on the Escherichia coli/Lactobacillus shuttle vector pLP3537 and the melA gene encoding alpha-galactosidase originating from Lactobacillus plantarum. melA was functionally expressed in E. coli and L. sanfranciscensis, and activity was easily monitored in vivo as well as in vitro by applying an optimized enzyme assay. The reporter system was validated by demonstrating the induction of the dnaK operon of L. sanfranciscensis by NaCl stress. The complete operon, which was composed of hrcA, grpE, dnaK, and dnaJ, was sequenced. A 299-bp sequence upstream of this operon, including a putative sigmaA-type promoter and a single conserved Controlling Inverted Repeat of Chaperone Expression element, was amplified. This amplicon was cloned directly upstream of melA. Both reporter enzyme activity and Northern hybridization analyses of dnaK and melA revealed a transcriptional induction, reaching its maximum when the culture was exposed to 0.75 M NaCl.
|
36. |
Nicoloff H,
Elagöz A,
Arsène-Ploetze F,
Kammerer B,
Martinussen J,
Bringel F,
( 2005 ) Repression of the pyr operon in Lactobacillus plantarum prevents its ability to grow at low carbon dioxide levels. PMID : 15743958 : DOI : 10.1128/JB.187.6.2093-2104.2005 PMC : PMC1064029 Abstract >>
Carbamoyl phosphate is a precursor for both arginine and pyrimidine biosynthesis. In Lactobacillus plantarum, carbamoyl phosphate is synthesized from glutamine, ATP, and carbon dioxide by two sets of identified genes encoding carbamoyl phosphate synthase (CPS). The expression of the carAB operon (encoding CPS-A) responds to arginine availability, whereas pyrAaAb (encoding CPS-P) is part of the pyrR1BCAaAbDFE operon coding for the de novo pyrimidine pathway repressed by exogenous uracil. The pyr operon is regulated by transcription attenuation mediated by a trans-acting repressor that binds to the pyr mRNA attenuation site in response to intracellular UMP/phosphoribosyl pyrophosphate pools. Intracellular pyrimidine triphosphate nucleoside pools were lower in mutant FB335 (carAB deletion) harboring only CPS-P than in the wild-type strain harboring both CPS-A and CPS-P. Thus, CPS-P activity is the limiting step in pyrimidine synthesis. FB335 is unable to grow in the presence of uracil due to a lack of sufficient carbamoyl phosphate required for arginine biosynthesis. Forty independent spontaneous FB335-derived mutants that have lost regulation of the pyr operon were readily obtained by their ability to grow in the presence of uracil and absence of arginine; 26 harbored mutations in the pyrR1-pyrB loci. One was a prototroph with a deletion of both pyrR1 and the transcription attenuation site that resulted in large amounts of excreted pyrimidine nucleotides and increased intracellular UTP and CTP pools compared to wild-type levels. Low pyrimidine-independent expression of the pyr operon was obtained by antiterminator site-directed mutagenesis. The resulting AE1023 strain had reduced UTP and CTP pools and had the phenotype of a high-CO2-requiring auxotroph, since it was able to synthesize sufficient arginine and pyrimidines only in CO2-enriched air. Therefore, growth inhibition without CO2 enrichment may be due to low carbamoyl phosphate pools from lack of CPS activity.
|
37. |
Spano G,
Beneduce L,
Perrotta C,
Massa S,
( 2005 ) Cloning and characterization of the hsp 18.55 gene, a new member of the small heat shock gene family isolated from wine Lactobacillus plantarum. PMID : 15748987 : DOI : 10.1016/j.resmic.2004.09.014 Abstract >>
Using a molecular approach based on PCR, RT-PCR and northern blot analysis, a new member of the small heat shock family of wine, Lactobacillus plantarum, was cloned and characterized. The protein sequence deduced from the isolated gene had a calculated molecular mass of 18.548 kDa and was therefore named HSP 18.55. The gene codes for a protein homologous to the previously characterized HSP 19.3 and HSP 18.5 and is co-transcribed with an upstream gene of unknown function. Analysis of the 5' flanking region of the hsp 18.55 gene revealed the presence of putative cis elements able to bind alternative sigma factor sigma(B). Based on its structure, the gene was classified as belonging to class II of the heat shock genes according to Bacillus subtilis nomenclature for shock-responsive genes. Expression of the newly identified small heat shock gene, analyzed by RT-PCR and northern blot analysis, was induced by a wide range of abiotic stresses including heat, cold and ethanol, suggesting that the small family of heat shock genes is probably involved in the general stress response in wine L. plantarum. Moreover, the expression of hsp 18.5, hsp 18.55 and hsp 19.3 genes, analyzed over a complete culture cycle, revealed that early growing cells contained substantial amounts of hsp 18.5, hsp 18.55 and hsp 19.3 mRNAs, which rapidly declined upon entry into stationary phase.
|
38. |
Miller KW,
Ray P,
Steinmetz T,
Hanekamp T,
Ray B,
( 2005 ) Gene organization and sequences of pediocin AcH/PA-1 production operons in Pediococcus and Lactobacillus plasmids. PMID : 15613003 : DOI : 10.1111/j.1472-765X.2004.01627.x Abstract >>
To determine the locations and sequences of pediocin AcH production genes in Pediococcus parvulus ATO77 from vegetables, Lactobacillus plantarum WHE92 from Muenster cheese, and a lactose-fermenting isolate Pediococcus pentosaceus S34 from buffalo milk. Plasmid curing, Southern blot hybridization, and DNA sequence analysis indicate that pediocin AcH production genes are encoded by highly similar operons in unique plasmids designated pATO77 from P. parvulus ATO77, pS34 from P. pentosaceus S34, and pWHE92 from Lact. plantarum WHE92. Structure, immunity and secretion system genes are linked together in the operons, and the promoter sequences are the same. The amino acid sequences of the encoded proteins are highly conserved between plasmids. Pediocin AcH production genes are located within a plasmid-borne operon cassette in all lactic acid bacterial strains examined to date. All four genes needed for production are present within a single plasmid in each strain. This is the first demonstration that the expression of a class IIa bacteriocin is directed by a common gene cassette that has been disseminated to unique plasmids in different genera of lactic acid bacteria. These plasmids should be useful for expressing pediocin AcH in Pediococcus and Lactobacillus strains used in food production.
|
39. |
de las Rivas B,
Marcobal A,
Muñoz R,
( 2004 ) Complete nucleotide sequence and structural organization of pPB1, a small Lactobacillus plantarum cryptic plasmid that originated by modular exchange. PMID : 15518876 : DOI : 10.1016/j.plasmid.2004.09.001 Abstract >>
A small cryptic plasmid designated pPB1 was isolated from Lactobacillus plantarum BIFI-38 and its complete 2899 bp nucleotide sequence was determined. Sequence analysis revealed four putative open reading frames. Based on sequence analysis two modules could be identified. First, the replication module consisted of a sequence coding for a replication protein (RepB) and its corresponding target site, and two putative repressor proteins (RepA and RepC). Sequence analysis indicated the possible synthesis of an antisense RNA that might regulate RepB production. A putative lagging-strand initiation site was also found, suggesting that pPB1 replicates via a rolling circle mechanism. The second module of pPB1 consisted of a sequence coding for a putative mobilization protein and its corresponding oriT site. Since the nucleotide sequence of the replication module showed 94.5% identity to the similar region on the Leuconostoc lactis plasmid pCI411, and the nucleotide sequence of the mobilization module had 97.5% identity to L. plantarum plasmid pLB4, it is concluded that pPB1 originated by modular exchange between two such plasmids by homologous recombination. Putative recombination sites where crossover might have taken place were also identified.
|
40. |
Spano G,
Capozzi V,
Vernile A,
Massa S,
( 2004 ) Cloning, molecular characterization and expression analysis of two small heat shock genes isolated from wine Lactobacillus plantarum. PMID : 15357727 : DOI : 10.1111/j.1365-2672.2004.02359.x Abstract >>
Understanding the molecular response to stress tolerance of wine Lactobacillus plantarum. Two genes codifying for heat shock proteins were cloned from wine L. plantarum. The coding regions of the two heat shock genes are 420 and 444 nucleotides long, and started with an ATG codon suggesting that they were translated. The protein sequences deduced from the isolated genes have a molecular mass of 18.483 and 19.282 kDa, respectively, and were therefore named hsp18.5 and hsp19.3. The expression of small heat shock genes was analysed by RT-PCR analysis. Moreover, the 5' and 3' noncoding regions were cloned and sequenced. The expression of the heat shock genes was strongly induced by heat, cold and ethanol stress. Analysis of the 5' and 3' flanking regions of hsp18.5 and hsp19.3 genes, revealed the presence of an inverted repeat sequence (TTAGCACTC-N(9)-GAGTGCTAA) homologue to the CIRCE elements found to the upstream regulatory region of heat shock operons, and an inverted sequence that could form a stem and loop structure that it is likely to function as a transcriptional terminator. Based on their structures, the genes were classified as belonging to Class I of heat shock genes according to the B. subtilis nomenclature of heat response genes. Small heat shock genes isolated from wine L. plantarum might have a role in preventing damage by cold stress.
|
41. |
Lorquet F,
Goffin P,
Muscariello L,
Baudry JB,
Ladero V,
Sacco M,
Kleerebezem M,
Hols P,
( 2004 ) Characterization and functional analysis of the poxB gene, which encodes pyruvate oxidase in Lactobacillus plantarum. PMID : 15175288 : DOI : 10.1128/JB.186.12.3749-3759.2004 PMC : PMC419957 Abstract >>
The pyruvate oxidase gene (poxB) from Lactobacillus plantarum Lp80 was cloned and characterized. Northern blot and primer extension analyses revealed that transcription of poxB is monocistronic and under the control of a vegetative promoter. poxB mRNA expression was strongly induced by aeration and was repressed by glucose. Moreover, Northern blotting performed at different stages of growth showed that poxB expression is maximal in the early stationary phase when glucose is exhausted. Primer extension and in vivo footprint analyses revealed that glucose repression of poxB is mediated by CcpA binding to the cre site identified in the promoter region. The functional role of the PoxB enzyme was studied by using gene overexpression and knockout in order to evaluate its implications for acetate production. Constitutive overproduction of PoxB in L. plantarum revealed the predominant role of pyruvate oxidase in the control of acetate production under aerobic conditions. The DeltapoxB mutant strain exhibited a moderate (20 to 25%) decrease in acetate production when it was grown on glucose as the carbon source, and residual pyruvate oxidase activity that was between 20 and 85% of the wild-type activity was observed with glucose limitation (0.2% glucose). In contrast, when the organism was grown on maltose, the poxB mutation resulted in a large (60 to 80%) decrease in acetate production. In agreement with the latter observation, the level of residual pyruvate oxidase activity with maltose limitation (0.2% maltose) was less than 10% of the wild-type level of activity.
|
42. |
Gury J,
Barthelmebs L,
Tran NP,
Diviès C,
Cavin JF,
( 2004 ) Cloning, deletion, and characterization of PadR, the transcriptional repressor of the phenolic acid decarboxylase-encoding padA gene of Lactobacillus plantarum. PMID : 15066807 : DOI : 10.1128/aem.70.4.2146-2153.2004 PMC : PMC383121 Abstract >>
Lactobacillus plantarum displays a substrate-inducible padA gene encoding a phenolic acid decarboxylase enzyme (PadA) that is considered a specific chemical stress response to the inducing substrate. The putative regulator of padA was located in the padA locus based on its 52% identity with PadR, the padA gene transcriptional regulator of Pediococcus pentosaceus (L. Barthelmebs, B. Lecomte, C. Divi?s, and J.-F. Cavin, J. Bacteriol. 182:6724-6731, 2000). Deletion of the L. plantarum padR gene clearly demonstrates that the protein it encodes is the transcriptional repressor of divergently oriented padA. The padR gene is cotranscribed with a downstream open reading frame (ORF1), the product of which may belong to a group of universal stress proteins (Usp). The padR deletion mutant overexpressed padA constitutively, and the padA promoter appears to be tightly regulated in this bacterium. Gel mobility shift assays using the padA gene promoter region and purified PadR expressed in Escherichia coli indicated that operator DNA binding by PadR was not eliminated by addition of p-coumarate. Gel mobility shift assays using partially purified extracts of native PadR protein from both phenolic acid-induced and noninduced L. plantarum cells demonstrate that inactivation of PadR by phenolic acids requires the integrity of L. plantarum and mediation by a specific protein absent in E. coli.
|
43. |
Maldonado A,
Jiménez-Díaz R,
Ruiz-Barba JL,
( 2004 ) Induction of plantaricin production in Lactobacillus plantarum NC8 after coculture with specific gram-positive bacteria is mediated by an autoinduction mechanism. PMID : 14973042 : DOI : 10.1128/jb.186.5.1556-1564.2004 PMC : PMC344433 Abstract >>
Plantaricin NC8 (PLNC8), a coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8, has recently been purified and genetically characterized. Analysis of an 8.1-kb NC8 DNA region downstream of the PLNC8 operon revealed the presence of at least four operons involved in bacteriocin production, showing high homology to the plantaricin cluster in L. plantarum C11. However, we found a three-component regulatory operon involving a quorum-sensing mechanism. Two of these components, the induction factor (PLNC8IF) and the histidine kinase, are novel, while the response regulator is identical to PlnD from C11. Homologous expression of plNC8IF in NC8 allowed constitutive bacteriocin production. Heterologous expression of this gene in Lactococcus lactis MG1363 produced supernatants which promoted bacteriocin production in NC8. Reverse transcription-PCR studies indicated that cocultivation of NC8 with inducing cells promoted transcription of the bacteriocin and regulatory operons in NC8. An identical result was obtained after addition of an external source of PLNC8IF. We propose that the presence of specific bacteria could act as an environmental signal that is able to switch on bacteriocin production in L. plantarum NC8 via a quorum-sensing mechanism mediated by PLNC8IF.
|
44. |
Abriouel H,
Herrmann A,
Stärke J,
Yousif NM,
Wijaya A,
Tauscher B,
Holzapfel W,
Franz CM,
( 2004 ) Cloning and heterologous expression of hematin-dependent catalase produced by Lactobacillus plantarum CNRZ 1228. PMID : 14711694 : DOI : 10.1128/aem.70.1.603-606.2004 PMC : PMC321233 Abstract >>
Lactobacillus plantarum CNRZ 1228 exhibited heme-dependent catalase activity under environmental conditions similar to those encountered during sausage fermentation. The 1,455-bp catalase gene (katL) was cloned and encoded a protein of 484 amino acids. Expression of katL in a heterologous host showed that katL encodes a functional catalase. PCR screening of selected strains of lactic acid bacteria for katL indicated the presence of similar genes in other strains of lactobacilli.
|
45. |
Spano G,
Chieppa G,
Beneduce L,
Massa S,
( 2004 ) Expression analysis of putative arcA, arcB and arcC genes partially cloned from Lactobacillus plantarum isolated from wine. PMID : 14678173 : Abstract >>
The aim of this paper was to study if homofermentative strains (Lacobacillus plantarum) capable of malolactic fermentation in wine can degrade arginine via the ADI pathway. Homofermentative lactic acid bacteria (LAB) isolated from a typical red wine were investigated for their ability to produce citrulline. Citrulline was formed suggesting that the arginine metabolism takes place via the arginine deiminase (ADI) pathway and not via the arginase/urease pathway. Ammonia was also detected with Nessler's reagent, and all the strains examined were able to produce ammonia. Identification of homofermentative LAB was performed using 16S ribosomal sequence analysis. The strains were further classified as belonging to L. plantarum species. Furthermore, the genes encoding for the three pathway enzymes (ADI, ornithine transcarbamylase, carbamate kinase) were partially cloned and gene expression was performed at two different pH values (3.6 and 4.5). The results suggest that citrulline production in wine, could be performed by homofermentative LAB. Homofermentative malolactic bacteria (L. plantarum) may degrade arginine through the ADI pathway.
|
46. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 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.
|
47. |
Gueneau de Novoa P,
Williams KP,
( 2004 ) The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts. PMID : 14681369 : DOI : 10.1093/nar/gkh102 PMC : PMC308836 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.
|
48. |
Miller BJ,
Franz CM,
Cho GS,
du Toit M,
( 2011 ) Expression of the malolactic enzyme gene (mle) from Lactobacillus plantarum under winemaking conditions. PMID : 21404095 : DOI : 10.1007/s00284-011-9914-4 Abstract >>
Malolactic fermentation (MLF) plays an important role in the production of wine, especially red wines, resulting in microbial stability, deacidification, as well as contributing to the aroma profile. MLF can be influenced by a number of factors. In this study, the influence of pH and ethanol on expression of the structural malolactic enzyme gene (mle) from Lactobacillus plantarum was investigated in a synthetic wine media, as well as in wine using quantitative PCR. Expression of mle was shown to be inducible by the presence of malic acid, with increased expression in the middle of MLF. Expression of mle was also shown to be increased at low pH values and decreased in the presence of ethanol. This indicates the role of MLF in acid tolerance and the negative impact of ethanol on the completion of MLF. The results therefore provide further evidence that L. plantarum should be applied as co-inoculation for MLF where alcohol will initially not have a negative impact on the malic acid degradation.
|
49. |
Madoroba E,
Steenkamp ET,
Theron J,
Scheirlinck I,
Cloete TE,
Huys G,
( 2011 ) Diversity and dynamics of bacterial populations during spontaneous sorghum fermentations used to produce ting, a South African food. PMID : 21300507 : DOI : 10.1016/j.syapm.2010.11.016 Abstract >>
Ting is a spontaneously fermented sorghum food that is popular for its sour taste and unique flavour. Insight of the microbial diversity and population dynamics during sorghum fermentations is an essential component of the development of starter cultures for commercial production of ting. In this study, bacterial populations associated with spontaneous sorghum fermentations were examined using a culture-independent strategy based on denaturing gradient gel electrophoresis and sequence analysis of V3-16S rRNA gene amplicons, and a culture-dependent strategy using conventional isolation based on culturing followed by 16S rRNA and/or pheS gene sequence analysis. The entire fermentation process was monitored over a 54 h period and two phases were observed with respect to pH evolution and microbial succession. The first phase of the process (0-6h) was characterized by relatively high pH conditions and the presence of Enterococcus mundtii, albeit that this species was only detected with the culture-dependent approach. The second phase of the fermentation process (12-54 h) was characterized by increased acidity and the predominance of a broader range of lactic acid bacteria, including Lactococcus lactis, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Weissella cibaria, Enterococcus faecalis, and a close relative of Lactobacillus curvatus, as well as some members of the Enterobacteriaceae family. The Lb. curvatus-like species was only detected with PCR-DGGE, while the majority of the other species was only detected using the culture-dependent approach. These findings highlighted the fact that a combination of both approaches was essential in revealing the microbial diversity and dynamics during spontaneous sorghum fermentations.
|
50. |
Venugopal H,
Edwards PJ,
Schwalbe M,
Claridge JK,
Libich DS,
Stepper J,
Loo T,
Patchett ML,
Norris GE,
Pascal SM,
( 2011 ) Structural, dynamic, and chemical characterization of a novel S-glycosylated bacteriocin. PMID : 21395300 : DOI : 10.1021/bi200217u Abstract >>
Bacteriocins are bacterial peptides with specific activity against competing species. They hold great potential as natural preservatives and for their probiotic effects. We show here nuclear magnetic resonance-based evidence that glycocin F, a 43-amino acid bacteriocin from Lactobacillus plantarum, contains two �]-linked N-acetylglucosamine moieties, attached via side chain linkages to a serine via oxygen, and to a cysteine via sulfur. The latter linkage is novel and has helped to establish a new type of post-translational modification, the S-linked sugar. The peptide conformation consists primarily of two �\-helices held together by a pair of nested disulfide bonds. The serine-linked sugar is positioned on a short loop sequentially connecting the two helices, while the cysteine-linked sugar presents at the end of a long disordered C-terminal tail. The differing chemical and conformational stabilities of the two N-actetylglucosamine moieties provide clues about the possible mode of action of this bacteriostatic peptide.
|
51. |
Stepper J,
Shastri S,
Loo TS,
Preston JC,
Novak P,
Man P,
Moore CH,
Havlí?ek V,
Patchett ML,
Norris GE,
( 2011 ) Cysteine S-glycosylation, a new post-translational modification found in glycopeptide bacteriocins. PMID : 21251913 : DOI : 10.1016/j.febslet.2011.01.023 Abstract >>
O-Glycosylation is a ubiquitous eukaryotic post-translational modification, whereas early reports of S-linked glycopeptides have never been verified. Prokaryotes also glycosylate proteins, but there are no confirmed examples of sidechain glycosylation in ribosomal antimicrobial polypeptides collectively known as bacteriocins. Here we show that glycocin F, a bacteriocin secreted by Lactobacillus plantarum KW30, is modified by an N-acetylglucosamine �]-O-linked to Ser18, and an N-acetylhexosamine S-linked to C-terminal Cys43. The O-linked N-acetylglucosamine is essential for bacteriostatic activity, and the C-terminus is required for full potency (IC(50) 2 nM). Genomic context analysis identified diverse putative glycopeptide bacteriocins in Firmicutes. One of these, the reputed lantibiotic sublancin, was shown to contain a hexose S-linked to Cys22.
|
52. |
Lucena BT,
dos Santos BM,
Moreira JL,
Moreira AP,
Nunes AC,
Azevedo V,
Miyoshi A,
Thompson FL,
de Morais MA,
( 2010 ) Diversity of lactic acid bacteria of the bioethanol process. PMID : 21092306 : DOI : 10.1186/1471-2180-10-298 PMC : PMC2999616 Abstract >>
Bacteria may compete with yeast for nutrients during bioethanol production process, potentially causing economic losses. This is the first study aiming at the quantification and identification of Lactic Acid Bacteria (LAB) present in the bioethanol industrial processes in different distilleries of Brazil. A total of 489 LAB isolates were obtained from four distilleries in 2007 and 2008. The abundance of LAB in the fermentation tanks varied between 6.0 �� 105 and 8.9 �� 108 CFUs/mL. Crude sugar cane juice contained 7.4 �� 107 to 6.0 �� 108 LAB CFUs. Most of the LAB isolates belonged to the genus Lactobacillus according to rRNA operon enzyme restriction profiles. A variety of Lactobacillus species occurred throughout the bioethanol process, but the most frequently found species towards the end of the harvest season were L. fermentum and L. vini. The different rep-PCR patterns indicate the co-occurrence of distinct populations of the species L. fermentum and L. vini, suggesting a great intraspecific diversity. Representative isolates of both species had the ability to grow in medium containing up to 10% ethanol, suggesting selection of ethanol tolerant bacteria throughout the process. This study served as a first survey of the LAB diversity in the bioethanol process in Brazil. The abundance and diversity of LAB suggest that they have a significant impact in the bioethanol process.
|
53. |
Pan Q,
Zhang L,
Li J,
Chen T,
Chen W,
Wang G,
Yin J,
( 2011 ) Characterization of pLP18, a novel cryptic plasmid of Lactobacillus plantarum PC518 isolated from Chinese pickle. PMID : 21255609 : DOI : 10.1016/j.plasmid.2011.01.001 Abstract >>
A cryptic plasmid of Lactobacillus plantarum PC518 isolated from Chinese pickle, designated pLP18, was sequenced and characterized. It is a 1806-bp circular molecule with a G+C content of 37.5%. Sequence analysis of pLP18 revealed three putative open reading frames (ORFs), in which ORF1 contained conserved motifs of pMV158-family Rep proteins and showed 60% similarity with the Rep protein of pPSC22, a member of rolling-circle replication (RCR) pMV158 family. The double strand origin (dso) of pMV158 family and the single strand origin A (ssoA) located upstream of the rep gene. The putative cop and rnaII genes were predicted to be regulatory genes controlling copy number of pLP18. The results of Southern hybridization suggested that pLP18 replicate via the RCR mechanism. Furthermore, the relative copy number of pLP18 was estimated to be about 24 copies per chromosome equivalent by quantitative PCR.
|
54. |
Zhou H,
Hao Y,
Xie Y,
Yin S,
Zhai Z,
Han B,
( 2010 ) Characterization of a rolling-circle replication plasmid pXY3 from Lactobacillus plantarum XY3. PMID : 20353802 : DOI : 10.1016/j.plasmid.2010.03.003 Abstract >>
The complete nucleotide sequence of cryptic plasmid pXY3 isolated from Lactobacillus plantarum strain XY3 has been determined. It consisted of a 2968-bp circular molecule with a G+C content of 39%. Sequence analysis of pXY3 revealed three putative open reading frames (ORFs). Based on sequence similarity, the Rep protein shared 89% and 88% identity with Rep proteins of pLF24 and pWCFS102, respectively, which belonged to the rolling-circle replication (RCR) pMV158 family. A ssoT-like single-strand origin (sso) and a typical pMV158 family double-strand origin (dso) located upstream of the rep gene. Southern blot analysis indicated pXY3 replicate via a rolling-circle (RC) mechanism. Furthermore, the relative copy number of pXY3 was estimated to be about 97 copies per chromosome equivalent by real-time PCR.
|
55. |
Brod FC,
Vernal J,
Bertoldo JB,
Terenzi H,
Arisi AC,
( 2010 ) Cloning, expression, purification, and characterization of a novel esterase from Lactobacillus plantarum. PMID : 20033357 : DOI : 10.1007/s12033-009-9232-2 Abstract >>
Lactobacillus plantarum is an important lactic acid bacterium, usually found as natural inhabitant of food, such as fermented vegetables and meat products. However, little information about lactic acid bacteria, especially concerning L. plantarum, as a source of useful enzymes has been reported. The aim of this study was to clone, express in Escherichia coli, purify, and characterize an esterase from L. plantarum ATCC 8014. The esterase gene (1014 bp) was amplified and cloned in pET14b expression vector to express a His(6)-tagged protein in E. coli. Recombinant L. plantarum esterase was purified by Ni-NTA resin, presenting an apparent molecular mass of about 38 kDa. It presented highest activity at pH 6.0 and 40 degrees C. Also, it presented preference for p-nitrophenyl butyrate, but hydrolyzed more efficiently p-nitrophenyl acetate. Besides, this study shows, for the first time, CD data about secondary structure of an esterase from L. plantarum.
|
56. |
Hata T,
Tanaka R,
Ohmomo S,
( 2010 ) Isolation and characterization of plantaricin ASM1: a new bacteriocin produced by Lactobacillus plantarum A-1. PMID : 19939484 : DOI : 10.1016/j.ijfoodmicro.2009.10.021 Abstract >>
Bacteriocins produced by lactic acid bacteria showing stability even in neutral and weak alkaline pH were screened, and a new bacteriocin produced by Lactobacillus plantarum A-1, plantaricin ASM1 (PASM1) was purified and characterized. This bacteriocin which is heat-stable but digested by trypsin inhibits the growth of lactic acid bacterial species, such as Lactobacillus, Leuconostoc, and Enterococcus. PASM1 showed stability in a wide pH range compared to nisin A. The bacteriocin was purified using cation exchange, hydrophobic interaction, and reverse-phase high-performance liquid chromatography. The activity of the purified bacteriocin was obtained as one fraction. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the fraction showed a mass of 5045.7Da. Combining the data obtained from amino acid and DNA sequencing, the primary sequence of PASM1 was determined. The sequence of the corresponding gene revealed that the peptide is ribosomally synthesized as a 64 amino acid precursor containing a 21 amino acid N-terminal extension of the double-glycine type. The mature peptide consists of 43 amino acids, which could contain two intramolecular disulfide bonds in the structure. Three putative open reading frames were located upstream of the PLNA1 gene. These genes may encode the thioredoxin family proteins and a response regulator both of which have been suggested to regulate expression of the PASM1 gene and the processing of its leader peptide. PASM1 has no reported homologue bacteriocins. Stability in a wide pH range and heat indicates its potential for application in food preservation.
|
57. |
Rogne P,
Haugen C,
Fimland G,
Nissen-Meyer J,
Kristiansen PE,
( 2009 ) Three-dimensional structure of the two-peptide bacteriocin plantaricin JK. PMID : 19538999 : DOI : 10.1016/j.peptides.2009.06.010 Abstract >>
The three-dimensional structures of the two peptides, PlnJ and PlnK, that constitutes the two-peptide bacteriocin plantaricin JK have been solved in water/TFE and water/DPC-micellar solutions using nuclear magnetic resonance (NMR) spectroscopy. PlnJ, a 25 residue peptide, has an N-terminal amphiphilic alpha-helix between Trp-3 and Tyr-15. The 32 residues long PlnK forms a central amphiphilic alpha-helix between Gly-9 and Leu-24. Measurements of the effect on anti-microbial activity of single glycine replacements in PlnJ and PlnK show that Gly-13 and Gly-17 in both peptides are very sensitive, giving more than a 100-fold reduction in activity when large residues replace glycine. In variants where other glycine residues, Gly-20 in PlnJ and Gly-7, Gly-9, Gly-24 and Gly-25 in PlnK, were replaced, the activity was reduced less than 10-fold. It is proposed that the detrimental effect on activity when exchanging Gly-13 and Gly-17 in PlnJ and PlnK is a result of reduced ability of the two peptides to interact through the GxxxG-motifs constituting Gly-13 and Gly-17.
|
58. |
Chen YS,
Miyashita M,
Suzuki K,
Sato H,
Hsu JS,
Yanagida F,
( 2010 ) Lactobacillus pobuzihii sp. nov., isolated from pobuzihi (fermented cummingcordia). PMID : 19783610 : DOI : 10.1099/ijs.0.016873-0 Abstract >>
Twenty-one homofermentative lactic acid bacteria were isolated from fermented cummingcordia (pobuzihi), a traditional food in Taiwan. The isolates had identical 16S rRNA gene sequences that were distinct from those of other lactobacilli, and their closest neighbours in the 16S rRNA gene sequence phylogenetic tree were strains of Lactobacillus acidipiscis. Levels of DNA-DNA relatedness between representative pobuzihi isolates and strains of L. acidipiscis were 17% and below. Furthermore, the new isolates could be differentiated clearly from L. acidipiscis NBRC 102163T and NBRC 102164 in terms of acid production from L-arabinose, rhamnose, mannitol, lactose and 5-ketogluconate. It was concluded that the new isolates represent a single novel species of the genus Lactobacillus, for which the name Lactobacillus pobuzihii sp. nov. is proposed. The type strain is E100301T (=RIFY 6501T =NBRC 103219T =KCTC 13174T).
|
59. |
Avila M,
Jaquet M,
Moine D,
Requena T,
Peláez C,
Arigoni F,
Jankovic I,
( 2009 ) Physiological and biochemical characterization of the two alpha-L-rhamnosidases of Lactobacillus plantarum NCC245. PMID : 19423635 : DOI : 10.1099/mic.0.027789-0 Abstract >>
This work is believed to be the first report on the physiological and biochemical characterization of alpha-l-rhamnosidases in lactic acid bacteria. A total of 216 strains representing 37 species and eight genera of food-grade bacteria were screened for alpha-l-rhamnosidase activity. The majority of positive bacteria (25 out of 35) were Lactobacillus plantarum strains, and activity of the L. plantarum strain NCC245 was examined in more detail. The analysis of alpha-l-rhamnosidase activity under different growth conditions revealed dual regulation of the enzyme activity, involving carbon catabolite repression and induction: the enzyme activity was downregulated by glucose and upregulated by l-rhamnose. The expression of the two alpha-l-rhamnosidase genes rhaB1 and rhaB2 and two predicted permease genes rhaP1 and rhaP2, identified in a probable operon rhaP2B2P1B1, was repressed by glucose and induced by l-rhamnose, showing regulation at the transcriptional level. The two alpha-l-rhamnosidase genes were overexpressed and purified from Escherichia coli. RhaB1 activity was maximal at 50 degrees C and at neutral pH and RhaB2 maximal activity was detected at 60 degrees C and at pH 5, with high residual activity at 70 degrees C. Both enzymes showed a preference for the alpha-1,6 linkage of l-rhamnose to beta-d-glucose, hesperidin and rutin being their best substrates, but, surprisingly, no activity was detected towards the alpha-1,2 linkage in naringin under the tested conditions. In conclusion, we identified and characterized the strain L. plantarum NCC245 and its two alpha-l-rhamnosidase enzymes, which might be applied for improvement of bioavailability of health-beneficial polyphenols, such as hesperidin, in humans.
|
60. |
Tanganurat W,
Quinquis B,
Leelawatcharamas V,
Bolotin A,
( 2009 ) Genotypic and phenotypic characterization of Lactobacillus plantarum strains isolated from Thai fermented fruits and vegetables. PMID : 19219901 : DOI : 10.1002/jobm.200800185 Abstract >>
Ten Lactobacillus strains originally isolated from Thai fruits and vegetables fermentation were characterized by various phenotypic and genotypic methods. The phenotypic analysis using the method of carbohydrate fermentation patterns (API50CHL) revealed that the isolates belonged to the L. plantarum species. This was further confirmed by 16S rRNA gene sequencing. Multilocus sequence typing (MLST) revealed a strongly clonal population structure and a low genotypic diversity in this collection. However, the analyzed L. plantarum population demonstrated a higher level of diversification after API50CHL that reflects the role of available carbohydrate sources in bacterial evolution. Our results support the postulate that a combination of conventional biochemical and genotyping methods allows a thorough characterization and identification of isolates. We propose that genotypic characterization could be complemented by biochemical characterization to discriminate L. plantarum strains.
|
61. |
Araque I,
Gil J,
Carreté R,
Bordons A,
Reguant C,
( 2009 ) Detection of arc genes related with the ethyl carbamate precursors in wine lactic acid bacteria. PMID : 19219988 : DOI : 10.1021/jf803421w Abstract >>
Trace amounts of the carcinogen ethyl carbamate can appear in wine by the reaction of ethanol with compounds such as citrulline and carbamyl phosphate, which are produced from arginine degradation by some wine lactic acid bacteria (LAB). In this work, the presence of arc genes for the arginine-deiminase pathway was studied in several strains of different species of LAB. Their ability to degrade arginine was also studied. To detect the presence of arc genes, degenerate primers were designed from the alignment of protein sequences in already sequenced LAB. The usefulness of these degenerate primers has been proven by sequencing some of the amplified PCR fragments and searching for homologies with published sequences of the same species and related ones. Correlation was found between the presence of genes and the ability to degrade arginine. Degrading strains included all heterofermentative lactobacilli, Oenococcus oeni , Pediococcus pentosaceus , and some strains of Leuconostoc mesenteroides and Lactobacillus plantarum .
|
62. |
Kim JH,
Sunako M,
Ono H,
Murooka Y,
Fukusaki E,
Yamashita M,
( 2008 ) Characterization of gene encoding amylopullulanase from plant-originated lactic acid bacterium, Lactobacillus plantarum L137. PMID : 19111640 : DOI : 10.1263/jbb.106.449 Abstract >>
A starch-hydrolyzing lactic acid bacterium, Lactobacillus plantarum L137, was isolated from traditional fermented food made from fish and rice in the Philippines. A gene (apuA) encoding an amylolytic enzyme from Lactobacillus plantarum L137 was cloned, and its nucleotide sequence was determined. The apuA gene consisted of an open reading frame of 6171 bp encoding a protein of 2056 amino acids, the molecular mass of which was calculated to be 215,625 Da. The catalytic domains of amylase and pullulanase were located in the same region within the middle of the N-terminal region. The deduced amino acid sequence revealed four highly conserved regions that are common among amylolytic enzymes. In the N-terminal region, a six-amino-acid sequence (Asp-Ala/Thr-Ala-Asn-Ser-Thr) is repeated 39 times, and a three-amino-acid sequence (Gln-Pro-Thr) is repeated 50 times in the C-terminal region. The apuA gene was subcloned in L. plantarum NCL21, which is a plasmid-cured derivative of the wild-type L137 strain and has no amylopullulanase activity, and the gene was overexpressed under the control of its own promoter. The ApuA enzyme from this recombinant L. plantarum NCL21 harboring apuA gene was purified. The enzyme has both alpha-amylase and pullulanase activities. The N-terminal sequence of the purified enzyme showed that the signal peptide was cleaved at Ala(36) and the molecular mass of the mature extracellular enzyme is 211,537 Da. The major reaction products from soluble starch were maltotriose (G3) and maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. From these results, we concluded that ApuA is an amylolytic enzyme belonging to the amylopullulanase family.
|
63. |
Iwamoto K,
Tsuruta H,
Nishitaini Y,
Osawa R,
( 2008 ) Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917(T). PMID : 18653299 : DOI : 10.1016/j.syapm.2008.05.004 Abstract >>
The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.
|
64. |
Yin S,
Hao Y,
Zhai Z,
Li R,
Huang Y,
Tian H,
Luo Y,
( 2008 ) Characterization of a cryptic plasmid pM4 from Lactobacillus plantarum M4. PMID : 18557944 : DOI : 10.1111/j.1574-6968.2008.01229.x Abstract >>
A cryptic plasmid from Lactobacillus plantarum M4 isolated from fresh milk, designated as pM4, was sequenced and characterized. It was 3320 bp in length with a G+C content of 38.73 mol%. The plasmid pM4 was predicted to encode three putative ORFs, in which ORF1 shared 99% and 98% homology, respectively, with the Rep proteins of reported plasmids pWCFS101 and pF8801, members of the rolling circle replication (RCR) pC194 family. Sequence analysis revealed a typical pC194 family double strand origin (dso) and a putative single strand origin (sso) located upstream of the rep gene. Mung bean nuclease analysis and Southern hybridization confirmed the presence of single-stranded DNA (ssDNA) intermediates, suggesting that pM4 belongs to the RCR pC194 family. Accumulation of ssDNA in rifampicin-treated strains implied that the host-encoded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA. Furthermore, the relative copy number of pM4 was estimated to be about 25 in each cell by real-time PCR. The new RCR plasmid would be valuable in constructing cloning vectors for application in the food industry.
|
65. |
Li R,
Zhai Z,
Yin S,
Huang Y,
Wang Q,
Luo Y,
Hao Y,
( 2009 ) Characterization of a rolling-circle replication plasmid pLR1 from Lactobacillus plantarum LR1. PMID : 18839247 : DOI : 10.1007/s00284-008-9280-z Abstract >>
A cryptic plasmid from Lactobacillus plantarum LR1, designated pLR1, was sequenced and characterized. It consisted of a 2066-bp circular molecule with a G + C content of 52.7%. The plasmid pLR1 was predicted to contain five putative ORFs, in which ORF1 shared 93% and 92% identity with Rep proteins of pLP1 and pC30il, members of rolling-circle replication (RCR) pC194 family. Detection of single-stranded DNA (ssDNA) intermediates by Southern hybridization and mung bean nuclease treatment confirmed that pLR1 replicated via the RCR mechanism. Accumulation of ssDNA in rifampicin-treated strains implied that the host-coded RNA polymerase was involved in the conversion of ssDNA to double-stranded DNA (dsDNA). Furthermore, the copy number of pLR1 was estimated to be 36 in each cell by real-time polymerase chain reaction.
|
66. |
Fimland N,
Rogne P,
Fimland G,
Nissen-Meyer J,
Kristiansen PE,
( 2008 ) Three-dimensional structure of the two peptides that constitute the two-peptide bacteriocin plantaricin EF. PMID : 18555030 : DOI : 10.1016/j.bbapap.2008.05.003 Abstract >>
The three-dimensional structures of the two peptides plantaricin E (plnE; 33 residues) and plantaricin F (plnF; 34 residues) constituting the two-peptide bacteriocin plantaricin EF (plnEF) have been determined by nuclear magnetic resonance (NMR) spectroscopy in the presence of DPC micelles. PlnE has an N-terminal alpha-helix (residues 10-21), and a C-terminal alpha-helix-like structure (residues 25-31). PlnF has a long central alpha-helix (residues 7-32) with a kink of 38+/-7 degrees at Pro20. There is some flexibility in the helix in the kink region. Both helices in plnE are amphiphilic, while the helix in plnF is polar in its N-terminal half and amphiphilic in its C-terminal half. The alpha-helical content obtained by NMR spectroscopy is in agreement with CD studies. PlnE has two GxxxG motifs which are putative helix-helix interaction motifs, one at residues 5 to 9 and one at residues 20 to 24, while plnF has one such motif at residues 30 to 34. The peptides are flexible in these GxxxG regions. It is suggested that the two peptides lie parallel in a staggered fashion relative to each other and interact through helix-helix interactions involving the GxxxG motifs.
|
67. |
Navarro L,
Rojo-Bezares B,
Sáenz Y,
Díez L,
Zarazaga M,
Ruiz-Larrea F,
Torres C,
( 2008 ) Comparative study of the pln locus of the quorum-sensing regulated bacteriocin-producing L. plantarum J51 strain. PMID : 18819721 : DOI : 10.1016/j.ijfoodmicro.2008.08.004 Abstract >>
Lactobacillus plantarum J51 strain was isolated from a Rioja red wine and it showed bacteriocin activity against a wide range of lactic acid bacteria of oenological importance. These characteristics conferred L. plantarum J51 a high interest both in wine microbiology and in the study of bacteriocin production. In this work the bacteriocin production regulated under the "quorum-sensing" mechanism is observed and the pln locus of the bacteriocin-producing L. plantarum J51 is fully characterized. A 20,667 bp fragment was completely sequenced (GenBank accession number DQ340868), and showed five operons (plNC8betaalphac, plnLR-like, plnABCD, plnEFI, plnGHSTUVW) and a new region containing a putative operon with three new orfs that could encode a putative two-peptide bacteriocin.
|
68. |
Scheirlinck I,
Van der Meulen R,
Van Schoor A,
Vancanneyt M,
De Vuyst L,
Vandamme P,
Huys G,
( 2008 ) Taxonomic structure and stability of the bacterial community in belgian sourdough ecosystems as assessed by culture and population fingerprinting. PMID : 18310426 : DOI : 10.1128/AEM.02771-07 PMC : PMC2293155 Abstract >>
A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.
|
69. |
Rojo-Bezares B,
Sáenz Y,
Navarro L,
Jiménez-Díaz R,
Zarazaga M,
Ruiz-Larrea F,
Torres C,
( 2008 ) Characterization of a new organization of the plantaricin locus in the inducible bacteriocin-producing Lactobacillus plantarum J23 of grape must origin. PMID : 18193201 : DOI : 10.1007/s00203-007-0342-6 Abstract >>
Lactobacillus plantarum J23 was previously characterized as a bacteriocin-producer-strain when it was cocultured with other lactic acid bacteria. In this work, the genetic organization of the pln locus in the J23 strain was studied and compared with those of previously described L. plantarum C11, WCFS1 and NC8 strains. A new organization of the plantaricin locus was detected in the J23 strain. The sequenced fragment (20,266 bp) comprised plnJLR, plnMNOP, plnEFI, plnGHSTUVWXY, and plNC8IF-plNC8HK-plnD operons, as well as a new region that includes three new orfs (GenBank accession number DQ323671). When the J23 pln gene sequences were compared with those included in the GenBank database, the identity of the putative encoded proteins was in the range 67.1-100%. The regulatory system and the repertoire of putative bacteriocins of the J23 pln locus presented important differences with respect to the ones of C11, WCFS1 and NC8, such as the absence of plnK and the presence of a larger plnJ gene than the previously described for the other L. plantarum strains. The pln locus in L. plantarum strains seems to be a mosaic-like structure with different modules and reorganizations that presents highly conserved regions related to transport and bacteriocin maturation and variable regions related to regulation and bacteriocin production.
|
70. |
Chouayekh H,
Bejar W,
Rhimi M,
Jelleli K,
Mseddi M,
Bejar S,
( 2007 ) Characterization of an l-arabinose isomerase from the Lactobacillus plantarum NC8 strain showing pronounced stability at acidic pH. PMID : 18031349 : DOI : 10.1111/j.1574-6968.2007.00961.x Abstract >>
Gene araA encoding the l-arabinose isomerase (l-AI) from Lactobacillus plantarum NC8 was cloned and expressed in Escherichia coli. It encodes a polypeptide of 474 residues having 55% identities with l-AIs from Bacillus stearothermophilus US100 and Thermus sp. IM6501. The active form of the purified recombinant l-AI NC8 enzyme is a hexamer composed of six identical 55-kDa subunits. The purified enzyme was optimally active at 60 degrees C and pH 7.5. It required divalent cations such as Co(2+) and Mn(2+) for maximal activity and thermostability. The l-AI NC8 was exceptionally active and stable at acidic pH. Indeed, it exhibited 68% of its maximal activity at pH 5.5 and retained 89% of activity after a 24-h incubation at pH 5. The apparent K(m) values of the enzyme for l-arabinose and d-galactose were 43.4 and 69.7 mM, respectively, and its catalytic efficiency was c. 10-fold higher for the physiological substrate l-arabinose (15.5 mM(-1) min(-1)) than d-galactose (1.6 mM(-1) min(-1)). The bioconversion yield of d-galactose to d-tagatose by the purified l-AI NC8 after 6 h at 60 degrees C was 30%.
|
71. |
Naser SM,
Dawyndt P,
Hoste B,
Gevers D,
Vandemeulebroecke K,
Cleenwerck I,
Vancanneyt M,
Swings J,
( 2007 ) Identification of lactobacilli by pheS and rpoA gene sequence analyses. PMID : 18048724 : DOI : 10.1099/ijs.0.64711-0 Abstract >>
The aim of this study was to evaluate the use of the phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) partial gene sequences for species identification of members of the genus Lactobacillus. Two hundred and one strains representing the 98 species and 17 subspecies were examined. The pheS gene sequence analysis provided an interspecies gap, which in most cases exceeded 10 % divergence, and an intraspecies variation of up to 3 %. The rpoA gene sequences revealed a somewhat lower resolution, with an interspecies gap normally exceeding 5 % and an intraspecies variation of up to 2 %. The combined use of pheS and rpoA gene sequences offers a reliable identification system for nearly all species of the genus Lactobacillus. The pheS and rpoA gene sequences provide a powerful tool for the detection of potential novel Lactobacillus species and synonymous taxa. In conclusion, the pheS and rpoA gene sequences can be used as alternative genomic markers to 16S rRNA gene sequences and have a higher discriminatory power for reliable identification of species of the genus Lactobacillus.
|
72. |
Blaiotta G,
Fusco V,
Ercolini D,
Aponte M,
Pepe O,
Villani F,
( 2008 ) Lactobacillus strain diversity based on partial hsp60 gene sequences and design of PCR-restriction fragment length polymorphism assays for species identification and differentiation. PMID : 17993558 : DOI : 10.1128/AEM.01711-07 PMC : PMC2223197 Abstract >>
A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.
|
73. |
Chen YY,
Liang NY,
Curtis JM,
Gänzle MG,
( 2016 ) Characterization of Linoleate 10-Hydratase of Lactobacillus plantarum and Novel Antifungal Metabolites. PMID : 27757104 : DOI : 10.3389/fmicb.2016.01561 PMC : PMC5047880 Abstract >>
Lactobacilli convert linoleic acid to the antifungal compound 10-hydroxy-12-octadecenoic acid (10-HOE) by linoleate 10-hydratase (10-LAH). However, the effect of this conversion on cellular membrane physiology and properties of the cell surface have not been demonstrated. Moreover, Lactobacillus plantarum produces 13-hydroxy-9-octadecenoic acid (13-HOE) in addition to 10-HOE, but the antifungal activity of 13-HOE was unknown. Phylogenetic analyses conducted in this study did not differentiate between 10-LAH and linoleate 13-hydratase (13-LAH). Thus, linoleate hydratases (LAHs) must be characterized through their differences in their activities of linoleate conversion. Four genes encoding putative LAHs from lactobacilli were cloned, heterologous expressed, purified and identified as FAD-dependent 10-LAH. The unsaturated fatty acid substrates stimulated the growth of lactobacilli. We also investigated the role of 10-LAH in ethanol tolerance, membrane fluidity and hydrophobicity of cell surfaces in lactobacilli by disruption of lah. Compared with the L. plantarum lah deficient strain, 10-LAH in wild-type strain did not exert effect on cell survival and membrane fluidity under ethanol stress, but influenced the cell surface hydrophobicity. Moreover, deletion of 10-LAH in L. plantarum facilitated purification of 13-HOE and demonstration of its antifungal activity against Penicillium roqueforti and Aspergillus niger.
|
74. |
Xu H,
Liu W,
Zhang W,
Yu J,
Song Y,
Menhe B,
Zhang H,
Sun Z,
( 2015 ) Use of multilocus sequence typing to infer genetic diversity and population structure of Lactobacillus plantarum isolates from different sources. PMID : 26511725 : DOI : 10.1186/s12866-015-0584-4 PMC : PMC4625847 Abstract >>
Lactobacillus plantarum is a lactic acid bacterium (LAB) of considerable industrial interest since it has an important role in the production of fermented food. In the present study, the genetic diversity and population structure within 186 L. plantarum isolates was determined based on a novel MLST scheme employing eight housekeeping genes. These isolates had originated from different sources and geographic regions: 179 isolates were from our own culture collection and originated from China and Mongolia and seven isolates were type or reference isolates from other collections. The results showed that 179 isolates and seven reference isolates could be assigned to 73 different sequence types (STs), forming ten clonal complexes (CCs) and 23 singletons. There were 158 polymorphic sites detected in total, and the nucleotide diversity per site varied from 0.00401 in clpX to 0.03220 in groEL. The minimum spanning tree analyses suggested that the evolution of L. plantarum isolates have little relationship with ecological sources have similar nucleotide diversity. Phylogenetic trees and structure indicated that there were six lineages in the L. plantarum isolates used in our study. Split-decomposition and ClonalFrame analysis indicated that recombination had occurred throughout the population of L. plantarum, but it occurred at a low frequency in these eight loci. We deduced that L. plantarum isolates from the same ecological niches have similar genetic diversity and population structure. The MLST scheme presented in this study provides abundant sequence data for L. plantarum and enabled global comparisons of isolates associated with various environmental origins to be made. This will further advance our understanding of the microbial ecology of this industrially important LAB.
|
75. |
Bhushan B,
Tomar SK,
Mandal S,
( 2016 ) Phenotypic and genotypic screening of human-originated lactobacilli for vitamin B12 production potential: process validation by micro-assay and UFLC. PMID : 27234139 : DOI : 10.1007/s00253-016-7639-9 Abstract >>
Vitamin B12 (B12) production is a strain specific, rare and hidden functional attribute of lactobacilli and a cogent protocol for selection of such isolates from the herd of lactobacilli is required. The present study included isolation of lactobacilli from human samples (milk and fecal), screening them by a polyphasic (three-phase) methodology for probable B12 production potential and validating the screening protocol by exploring selected strains for in vitro vitamin production (two-phase fermentation) and quantification [micro-assay and ultra fast liquid chromatography (UFLC)]. Fifty-nine Lactobacillus strains were recovered from tested biological samples. Contrary to screening inapplicabilities of first [growth potential (GP) in B12-free medium] and second phases (GP in B12-free and cobalt chloride-supplemented conditions), third phase (cbiK gene detection on genomic DNA) alone was revealed as a validated strategy for selection of two probable B12-producing lactobacilli. Microbiological assay confirmed production and bioavailability of produced vitamin, while UFLC testing validated the results by precisely quantifying the cyanocobalamin (industrially produced bio-available form of B12) in cell extracts of both possible B12 producers [BHM10 (10.91 �� 1.55 �gg/l) and BCF20 (23.90 �� 1.73 �gg/l)] and positive standard [Lactobacillus reuteri DSM20016 (20.03 �� 4.17 �gg/l)]. Moreover, this study generates a novel report for genomic detection, partial amplification and sequencing of cbiK gene in Lactobacillus plantarum species (both BHM10 and BCF20). In conclusion, contrary to first two phases, cbiK gene detection strategy successfully selects B12-producing strains from a group of human-originated lactobacilli and can be used in the future for similar screening studies.
|
76. |
Bates EE,
Gilbert HJ,
( 1989 ) Characterization of a cryptic plasmid from Lactobacillus plantarum. PMID : 2695401 : DOI : 10.1016/0378-1119(89)90491-5 Abstract >>
The complete nucleotide sequence of pLB4, a cryptic plasmid isolated from Lactobacillus plantarum NCDO1088 has been determined. Three open reading frames, which encode proteins of 42, 25 and 6 kDa, have been identified. In vitro transcription/translation of pLB4-derived DNA restriction fragments confirm the existence of all three polypeptides, which show homology to replication proteins and site-specific recombinases from other Gram+ plasmids. Three major regions of dyad symmetry with delta G of -28.8, -15.0 and -17.0 kcal were observed. One of these regions contains a sequence which shows perfect homology to the nick site of the Gram+ replicons, pE194, pLS1 and pADB201. In addition, a 21-bp sequence located upstream from the site-specific recombinase shows 80% homology to the recombination sites of pE194 and pT181.
|
77. |
Rumjuankiat K,
Perez RH,
Pilasombut K,
Keawsompong S,
Zendo T,
Sonomoto K,
Nitisinprasert S,
( 2015 ) Purification and characterization of a novel plantaricin, KL-1Y, from Lactobacillus plantarum KL-1. PMID : 25862353 : DOI : 10.1007/s11274-015-1851-0 Abstract >>
Three bacteriocins from Lactobacillus plantarum KL-1 were successfully purified using ammonium sulfate precipitation, cation-exchange chromatography and reverse-phase HPLC. The bacteriocin peptides KL-1X, -1Y and -1Z had molecular masses of 3053.82, 3498.16 and 3533.16 Da, respectively. All three peptides were stable at pH 2-12 and 25 �XC and at high temperatures of 80 and 100 �XC for 30 min and 121 �XC for 15 min. However, they differed in their susceptibility to proteolytic enzymes and their inhibition spectra. KL-1Y showed broad inhibitory activities against Gram-positive and Gram-negative bacteria, including Salmonella enterica serovar Enteritidis DMST 17368, Pseudomonas aeruginosa ATCC 15442, P. aeruginosa ATCC 9027, Escherichia coli O157:H7 and E. coli ATCC 8739. KL-1X and -1Z inhibited only Gram-positive bacteria. KL-1X, KL-1Y and KL-1Z exhibited synergistic activity. The successful amino acid sequencing of KL-1Y had a hydrophobicity of approximately 30 % and no cysteine residues suggested its novelty, and it was designated "plantaricin KL-1Y". Plantaricin KL-1Y exhibited bactericidal activity against Bacillus cereus JCM 2152(T). Compared to nisin, KL-1Y displayed broad inhibitory activities of 200, 800, 1600, 800, 400 and 400 AU/mL against the growth of Bacillus coagulans JCM 2257(T), B. cereus JCM 2152(T), Listeria innocua ATCC 33090(T), Staphylococcus aureus TISTR 118, E. coli O157:H7 and E. coli ATCC 8739, respectively, whereas nisin had similar activities against only B. coagulans JCM 2257(T) and B. cereus JCM 2152(T). Therefore, the novel plantaricin KL-1Y is a promising antimicrobial substance for food safety uses in the future.
|
78. |
Hou F,
Miyakawa T,
Kitamura N,
Takeuchi M,
Park SB,
Kishino S,
Ogawa J,
Tanokura M,
( 2015 ) Structure and reaction mechanism of a novel enone reductase. PMID : 25702712 : DOI : 10.1111/febs.13239 Abstract >>
Recently, a novel gut-bacterial fatty acid metabolism, saturation of polyunsaturated fatty acid, that modifies fatty acid composition of the host and is expected to improve our health by altering lipid metabolism related to the onset of metabolic syndrome, was discovered in Lactobacillus plantarum AKU 1009a. Enzymes constituting the pathway catalyze sequential reactions of free fatty acids without CoA or acyl carrier protein. Among these enzymes, CLA-ER was identified as an enone reductase that can saturate the C=C bond in the 10-oxo-trans-11-octadecenoic acid (KetoB) to produce 10-oxo-octadecanoic acid (KetoC). This enzyme is the sole member of the NADH oxidase/flavin reductase family that has been identified to exert an enone reduction activity. Here, we report both the structure of holo CLA-ER with cofactor FMN and the KetoC-bound structure, which elucidate the structural basis of enone group recognition of free fatty acids and provide the unique catalytic mechanism as an enone reductase in the NADH oxidase/flavin reductase family. A 'cap' structure of CLA-ER underwent a large conformational change upon KetoC binding. The resulting binding site adopts a sandglass shape and is positively charged at one side, which is suitable to recognize a fatty acid molecule with enone group. Based on the crystal structures and enzymatic activities of several mutants, we identified C51, F126 and Y101 as the critical residues for the reaction and proposed an alternative electron transfer pathway of CLA-ER. These findings expand our understanding of the complexity of fatty acid metabolism. The atomic coordinates have been deposited in the Protein Data Bank (PDB), www.pdb.org (PDB ID 4QLX, 4QLY).
|
79. |
Tajabadi N,
Baradaran A,
Ebrahimpour A,
Rahim RA,
Bakar FA,
Manap MY,
Mohammed AS,
Saari N,
( 2015 ) Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production. PMID : 25757029 : DOI : 10.1111/1751-7915.12254 PMC : PMC4476817 Abstract >>
Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36�XC, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products.
|
80. |
Zhang B,
Zuo F,
Yu R,
Zeng Z,
Ma H,
Chen S,
( 2015 ) Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells. PMID : 26370773 : DOI : 10.1038/srep14109 PMC : PMC4572922 Abstract >>
Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest.
|
81. |
Sun Z,
Harris HM,
McCann A,
Guo C,
Argimón S,
Zhang W,
Yang X,
Jeffery IB,
Cooney JC,
Kagawa TF,
Liu W,
Song Y,
Salvetti E,
Wrobel A,
Rasinkangas P,
Parkhill J,
Rea MC,
O'Sullivan O,
Ritari J,
Douillard FP,
Paul Ross R,
Yang R,
Briner AE,
Felis GE,
de Vos WM,
Barrangou R,
Klaenhammer TR,
Caufield PW,
Cui Y,
Zhang H,
O'Toole PW,
( 2015 ) Expanding the biotechnology potential of lactobacilli through comparative genomics of 213 strains and associated genera. PMID : 26415554 : DOI : 10.1038/ncomms9322 PMC : PMC4667430 Abstract >>
Lactobacilli are a diverse group of species that occupy diverse nutrient-rich niches associated with humans, animals, plants and food. They are used widely in biotechnology and food preservation, and are being explored as therapeutics. Exploiting lactobacilli has been complicated by metabolic diversity, unclear species identity and uncertain relationships between them and other commercially important lactic acid bacteria. The capacity for biotransformations catalysed by lactobacilli is an untapped biotechnology resource. Here we report the genome sequences of 213 Lactobacillus strains and associated genera, and their encoded genetic catalogue for modifying carbohydrates and proteins. In addition, we describe broad and diverse presence of novel CRISPR-Cas immune systems in lactobacilli that may be exploited for genome editing. We rationalize the phylogenomic distribution of host interaction factors and bacteriocins that affect their natural and industrial environments, and mechanisms to withstand stress during technological processes. We present a robust phylogenomic framework of existing species and for classifying new species.
|
82. |
Skaugen M,
( 1989 ) The complete nucleotide sequence of a small cryptic plasmid from Lactobacillus plantarum. PMID : 2623083 : Abstract >>
The complete nucleotide sequence of a cryptic plasmid isolated from a Lactobacillus plantarum strain has been determined. The plasmid, designated pC30i1, has a molecular size of 2140 bp and a GC content of 37%. The sequence contains one major open reading frame (ORF R) of 951 bp, encoding a basic polypeptide of 317 amino acids, and a molecular weight of 36,956. ORF R shows extensive sequence similarity with genes coding for replication-associated proteins in a group of gram-positive plasmids known to replicate via single-stranded intermediates (ssDNA plasmids), and a stretch of 9 amino acids in the translation of ORF R closely matches a conserved region in these proteins, as well as the active site of the phi X174 Rep protein. Sequences similar to the ssDNA plasmid origins of replication are also present in the pC30i1 sequence, strengthening the hypothesis that pC30i1 belongs to the ssDNA plasmid family. The other main feature of the pC30i1 sequence is a noncoding region consisting of 14 direct, imperfect repeats of a 17-bp sequence, which may have an incompatibility function.
|
83. |
Tai HF,
Foo HL,
Abdul Rahim R,
Loh TC,
Abdullah MP,
Yoshinobu K,
( 2015 ) Molecular characterisation of new organisation of plnEF and plw loci of bacteriocin genes harbour concomitantly in Lactobacillus plantarum I-UL4. PMID : 26077560 : DOI : 10.1186/s12934-015-0280-y PMC : PMC4467070 Abstract >>
Bacteriocin-producing Lactic acid bacteria (LAB) have vast applications in human and animal health, as well as in food industry. The structural, immunity, regulatory, export and modification genes are required for effective bacteriocin biosynthesis. Variations in gene sequence, composition and organisation will affect the antimicrobial spectrum of bacteriocin greatly. Lactobacillus plantarum I-UL4 is a novel multiple bacteriocin producer that harbours both plw and plnEF structural genes simultaneous which has not been reported elsewhere. Therefore, molecular characterisation of bacteriocin genes that harboured in L. plantarum I-UL4 was conducted in this study. Under optimised conditions, 8 genes (brnQ1, napA1, plnL, plnD, plnEF, plnI, plnG and plnH) of plnEF locus and 2 genes (plw and plwG) of plw locus were amplified successfully from genomic DNA extracted from L. plantarum I-UL4 using specific primers designed from 24 pln genes selected randomly from reported plw, plS, pln423 and plnEF loci. DNA sequence analysis of the flanking region of the amplified genes revealed the presence of two pln loci, UL4-plw and UL4-plnEF loci, which were chromosomally encoded as shown by Southern hybridisation. UL4-plw locus that contained three ORFs were arranged in one operon and possessed remarkable amino acid sequence of LMG2379-plw locus, suggesting it was highly conserved. Interestingly, the UL4-plnEF locus appeared to be a composite pln locus of JDM1-plnEF and J51-plnEF locus in terms of genetic composition and organisation, whereby twenty complete and one partial open reading frames (ORFs) were aligned and organised successfully into five operons. Furthermore, a mutation was detected in plnF structural gene which has contributed to a longer bacteriocin peptide. Plantaricin EF and plantaricin W encoded by plnEF and plnW loci are classified as class I bacteriocin and class II bacteriocin molecules respectively. The concurrent presence of two pln loci encoding bacteriocins from two different classes has contributed greatly to the broad inhibitory spectrum of L. plantarum I-UL4. The new genetic composition and organisation of plnEF locus and concurrent presence of plnEF and plnW loci indicated that L. plantarum I-UL4 is a novel multiple bacteriocin producer that possesses vast potentials in various industries.
|
84. |
Li L,
Yu YG,
Fei YT,
Liu DM,
Luo TH,
Chen G,
Wu H,
( 2014 ) Molecular characterization of Lactobacillus plantarum DMDL 9010, a strain with efficient nitrite degradation capacity. PMID : 25423449 : DOI : 10.1371/journal.pone.0113792 PMC : PMC4244150 Abstract >>
Nitrites commonly found in food, especially in fermented vegetables, are potential carcinogens. Therefore, limiting nitrites in food is critically important for food safety. A Lactobacillus strain (Lactobacillus sp. DMDL 9010) was previously isolated from fermented vegetables by our group, and is not yet fully characterized. A number of phenotypical and genotypical approaches were employed to characterize Lactobacillus sp. DMDL 9010. Its nitrite degradation capacity was compared with four other Lactobacillus strains, including Lactobacillus casei subsp. rhamnosus 719, Lactobacillus delbrueckii subsp. bulgaricu 1.83, Streptococcus thermophilus 1.204, and lactobacillus plantarum 8140, on MRS medium. Compared to these four Lactobacillus strains, Lactobacillus sp. DMDL 9010 had a significantly higher nitrite degradation capacity (P<0.001). Based on 16S rDNA sequencing and sequence comparison, Lactobacillus sp. DMDL 9010 was identified as either Lactobacillus plantarum or Lactobacillus pentosus. To further identify this strain, the flanking regions (922 bp and 806 bp upstream and downstream, respectively) of the L-lactate dehydrogenase 1 (L-ldh1) gene were amplified and sequenced. Lactobacillus sp. DMDL 9010 had 98.92 and 76.98% sequence identity in the upstream region with L. plantarum WCFS1 and L. pentosus IG1, respectively, suggesting that Lactobacillu sp. DMDL 9010 is an L. plantarum strain. It was therefore named L. plantarum DMDL 9010. Our study provides a platform for genetic engineering of L. plantarum DMDL 9010, in order to further improve its nitrite degradation capacity.
|
85. |
Murphree CA,
Heist EP,
Moe LA,
( 2014 ) Antibiotic resistance among cultured bacterial isolates from bioethanol fermentation facilities across the United States. 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.
|
86. |
Yang B,
Chen H,
Gu Z,
Tian F,
Ross RP,
Stanton C,
Chen YQ,
Chen W,
Zhang H,
( 2014 ) Synthesis of conjugated linoleic acid by the linoleate isomerase complex in food-derived lactobacilli. PMID : 24750362 : DOI : 10.1111/jam.12524 PMC : PMC4306591 Abstract >>
To assess strains of lactobacilli for their capacity to produce functional fatty acid-conjugated linoleic acid. To assess the linoleate isomerase for CLA production in the most efficient CLA producer. In this study, strains of food-derived lactobacilli were cultured in media with linoleic acid and CLA production was assessed. Most of the selected strains produced CLA at different levels, with Lactobacillus plantarum ZS2058 being the most efficient CLA producer converting over 50% of linoleic acid to c9, t11-CLA and t9, t11-CLA. Some intermediates 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid were determined via GC-MS. The genes coding the multicomponent linoleate isomerase containing myosin-cross-reactive antigen, short-chain dehydrogenase/oxidoreductase and acetoacetate decarboxylase for CLA production in Lact. plantarum ZS2058 were cloned and expressed in Escherichia coli. With the mixture of recombinant E. coli, c9, t11-CLA and three kinds of intermediates were produced from linoleic acid, which were in line with those in the lactobacilli. The ability for CLA production by lactobacilli exhibited variation. Lactobacillus plantarum and Lact. bulgaricus were the most efficient producers in the selected strains. Lact. plantarum ZS2058 converted linoleic acid to CLAs with 10-hydroxy-cis-12-octadecenoic acid, 10-oxo-cis-12-octadecenoic acid and 10-oxo-trans-11-octadecenoic acid as intermediates. The multiple-step reactions for CLA production catalysed by multicomponent linoleate isomerase in Lact. plantarum ZS2058 were confirmed successfully. Multicomponent linoleate isomerase provides important results for the illustration of the mechanism for CLA production in lactic acid bacteria. Food-derived lactobacilli with CLA production ability offers novel opportunities for functional foods development.
|
87. |
Heiss S,
Grabherr R,
Heinl S,
( 2015 ) Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH. PMID : 26038184 : DOI : 10.1016/j.plasmid.2015.05.004 Abstract >>
Lactobacillus plantarum CD033, a strain isolated from grass silage in Austria, harbors a 7.9 kb plasmid designated pCD033. Sequence analysis identified 14 open reading frames and 8 of these were supposed to be putative coding sequences. Gene annotation revealed no putative essential genes being plasmid encoded, but a plasmid addiction system based on a PemI/PemK-like toxin-antitoxin system, able to stabilize plasmid maintenance. Absence of a replication initiation protein, a double strand origin as well as a single strand origin on plasmid pCD033 suggests replication via a new type of theta mechanism, whereby plasmid replication is potentially initiated and regulated by non-coding RNA. Detailed examination of segregational stability of plasmid vectors consisting of pCD033-fragments, combined with a selection marker, resulted in definition of a stably maintained minimal replicon. A gene encoding a RepB/OrfX-like protein was found to be not essential for plasmid replication. Alignment of the amino acid sequence of this protein with related proteins unveiled a highly conserved amino acid motif (LLDQQQ). L. plantarum CD033 was cured of pCD033 resulting in the novel plasmid free strain L. plantarum 3NSH. Plasmid curing demonstrated that no essential features are provided by pCD033 under laboratory conditions.
|
88. |
Bouia A,
Bringel F,
Frey L,
Kammerer B,
Belarbi A,
Guyonvarch A,
Hubert JC,
( 1989 ) Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904. PMID : 2517345 : Abstract >>
To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage phi X174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5' region has been identified and is functional in Bacillus subtilis.
|
89. |
Gu XC,
Luo XG,
Wang CX,
Ma DY,
Wang Y,
He YY,
Li W,
Zhou H,
Zhang TC,
( 2014 ) Cloning and analysis of bile salt hydrolase genes from Lactobacillus plantarum CGMCC No. 8198. PMID : 24375235 : DOI : 10.1007/s10529-013-1434-9 Abstract >>
Genes coding for bile salt hydrolase of Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from silage, were identified, analyzed and cloned. L. plantarum strongly resisted the inhibitory effects of bile salts and also decreased serum cholesterol levels by 20% in mice with hypercholesterolemia. Using RT-PCR analysis, bsh2, bsh3 and bsh4 were upregulated by bile salts in a dose-dependent manner. All three bsh genes had high similarity with those of other Lactobacillus strains. All three recombinant BSHs had high activities for the hydrolysis of glycodeoxycholic acids and taurodeoxycholic acids.
|
90. |
Zhang X,
Zhang S,
Shi Y,
Shen F,
Wang H,
( 2014 ) A new high phenyl lactic acid-yielding Lactobacillus plantarum IMAU10124 and a comparative analysis of lactate dehydrogenase gene. PMID : 24861375 : DOI : 10.1111/1574-6968.12483 Abstract >>
Phenyl lactic acid (PLA) has been widely reported as a new natural antimicrobial compound. In this study, 120 Lactobacillus plantarum strains were demonstrated to produce PLA using high-performance liquid chromatography. Lactobacillus plantarum IMAU10124 was screened with a PLA yield of 0.229 g L(-1) . Compared with all previous reports, this is the highest PLA-producing lactic acid bacteria (LAB) when grown in MRS broth without any optimizing conditions. When 3.0 g L(-1) phenyl pyruvic acid (PPA) was added to the medium as substrate, PLA production reached 2.90 g L(-1) , with the highest 96.05% conversion rate. A lowest PLA-yielding L. plantarum IMAU40105 (0.043 g L(-1)) was also screened. It was shown that the conversion from PPA to PLA by lactic dehydrogenase (LDH) is the key factor in the improvement of PLA production by LAB. Comparing the LDH gene of two strains, four amino acid mutation sites were found in this study in the LDH of L. plantarum IMAU10124.
|
91. |
Zuo F,
Yu R,
Feng X,
Khaskheli GB,
Chen L,
Ma H,
Chen S,
( 2014 ) Combination of heterogeneous catalase and superoxide dismutase protects Bifidobacterium longum strain NCC2705 from oxidative stress. PMID : 24903816 : DOI : 10.1007/s00253-014-5851-z Abstract >>
Bifidobacteria are generally sensitive to oxidative stress caused by reactive oxygen species (ROS). To improve oxidative-stress tolerance, the superoxide dismutase (SOD) gene from Streptococcus thermophilus (StSodA) and the heme-dependent catalase (KAT) gene from Lactobacillus plantarum (LpKatL) were heterologously expressed in Bifidobacterium longum strain NCC2705. Three types of strain NCC2705 transformants were obtained: with transgenic SOD expression, with transgenic KAT expression, and with coexpression of the two genes. Intracellular expression of the genes and their functional role in oxidative-stress resistance were evaluated. In response to oxidative stress, B. longum NCC2705/pDP401-LpKatL (expressing LpKatL) and NCC2705/pDP-Kat-Sod (coexpressing LpKatL and StSodA) rapidly degraded exogenous H2O2 and the peroxides generated as a byproduct of aerobic cultivation, preventing oxidative damage to DNA and RNA. Individual expression of StSodA or LpKatL both improved B. longum NCC2705 cell viability. Survival rate of strain NCC2705 was further improved by combining SOD and KAT expression. The two enzymes played complementary roles in ROS-scavenging pathways, and coexpression led to a synergistic beneficial effect under conditions of intensified oxidative stress. Our results illustrate that heterogeneous expression of heme-dependent KAT and Mn(2+)-dependent SOD is functional in the B. longum oxidative-stress response, and synergistic protection is achieved when their expressions are combined.
|
92. |
Matoba Y,
Tanaka N,
Noda M,
Higashikawa F,
Kumagai T,
Sugiyama M,
( 2013 ) Crystallographic and mutational analyses of tannase from Lactobacillus plantarum. PMID : 23836494 : DOI : 10.1002/prot.24355 Abstract >>
Tannin acylhydrolase (EC 3.1.1.20) referred commonly as tannase catalyzes the hydrolysis of the galloyl ester bond of tannins to release gallic acid. Although the enzyme is useful for various industries, the tertiary structure is not yet determined. In this study, we determined the crystal structure of tannase produced by Lactobacillus plantarum. The tannase structure belongs to a member of �\/�]-hydrolase superfamily with an additional "lid" domain. A glycerol molecule derived from cryoprotectant solution was accommodated into the tannase active site. The binding manner of glycerol to tannase seems to be similar to that of the galloyl moiety in the substrate.
|
93. |
Bernardo D,
Sánchez B,
Al-Hassi HO,
Mann ER,
Urdaci MC,
Knight SC,
Margolles A,
( 2012 ) Microbiota/host crosstalk biomarkers: regulatory response of human intestinal dendritic cells exposed to Lactobacillus extracellular encrypted peptide. PMID : 22606249 : DOI : 10.1371/journal.pone.0036262 PMC : PMC3351486 Abstract >>
The human gastrointestinal tract is exposed to a huge variety of microorganisms, either commensal or pathogenic; at this site, a balance between immunity and immune tolerance is required. Intestinal dendritic cells (DCs) control the mechanisms of immune response/tolerance in the gut. In this paper we have identified a peptide (STp) secreted by Lactobacillus plantarum, characterized by the abundance of serine and threonine residues within its sequence. STp is encoded in one of the main extracellular proteins produced by such species, which includes some probiotic strains, and lacks cleavage sites for the major intestinal proteases. When studied in vitro, STp expanded the ongoing production of regulatory IL-10 in human intestinal DCs from healthy controls. STp-primed DC induced an immunoregulatory cytokine profile and skin-homing profile on stimulated T-cells. Our data suggest that some of the molecular dialogue between intestinal bacteria and DCs may be mediated by immunomodulatory peptides, encoded in larger extracellular proteins, secreted by commensal bacteria. These peptides may be used for the development of nutraceutical products for patients with IBD. In addition, this kind of peptides seem to be absent in the gut of inflammatory bowel disease patients, suggesting a potential role as biomarker of gut homeostasis.
|
94. |
Xi X,
Fan J,
Hou Y,
Gu J,
Shen W,
Li Z,
Cui Z,
( 2013 ) Characterization of three cryptic plasmids from Lactobacillus plantarum G63 that was isolated from Chinese pickle. PMID : 23899664 : DOI : 10.1016/j.plasmid.2013.07.004 Abstract >>
Three plasmids from Lactobacillus plantarum G63, pG6301, pG6302 and pG6303, were sequenced and have molecular sizes of 3516-bp, 9112-bp and 10047-bp, respectively. We determined the replicons of these plasmids. The pG6301 plasmid carried a replication gene that functioned by the rolling-circle replication mechanism. The Rep protein of pG6302 shared extremely low similarity with a reported plasmid, pLME300, which replicated by a bi-directional mechanism. Both the Rep protein and OriV analyses indicated a similar replication mechanism in pG6302. Conversely, we found neither the Rep protein nor OriV when we analyzed the whole sequence of pG6303. This finding may illustrate a novel replication mechanism. Additionally, the transposon, a mobilization element, was analyzed and compared to a similar insertion sequence (IS) element. A predicted lysozyme gene, pG6303 guhA, was heterologously expressed, but no activity was detected. The pG6302 and pG6303 plasmids contain new replicons and may be useful vector candidates for future molecular manipulation of L. plantarum.
|
95. |
Pal G,
Srivastava S,
( 2014 ) Cloning and heterologous expression of plnE, -F, -J and -K genes derived from soil metagenome and purification of active plantaricin peptides. PMID : 23884205 : DOI : 10.1007/s00253-013-5097-1 Abstract >>
Plantaricin gene-specific primers were used to obtain plnE, -F, -J and -K structural gene amplicons from soil metagenome. These amplicons were cloned and expressed in pET32a (+) vector in Escherichia coli BL21 (DE3). PlnE, -F, -J and -K peptides were expressed as His-tagged-fusion proteins and were separated by Ni(2+) -chelating affinity chromatography. The peptides were released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. The cleaved peptides were further analysed for antimicrobial activity and found to be active against Listeria innocua NRRL B33314, Micrococcus luteus MTCC 106 and lactic acid bacteria, such as Enterococcus casseliflavus NRRL B3502, Lactococcus lactis lactis NRRL 1821, Lactobacillus curvatus NRRL B4562 and Lactobacillus plantarum NRRL B4496. E. coli has been successfully exploited as a host for heterologous expression with a significant yield of fused and cleaved peptides in the range of 8-12 and 1-1.5 mg/l of the culture, respectively. Heterologous expression, therefore, can be used to overcome the constraints of low yield often reported from a native strain.
|
96. |
Hevia A,
Martínez N,
Ladero V,
Alvarez MA,
Margolles A,
Sánchez B,
( 2013 ) An extracellular Serine/Threonine-rich protein from Lactobacillus plantarum NCIMB 8826 is a novel aggregation-promoting factor with affinity to mucin. PMID : 23892754 : DOI : 10.1128/AEM.01657-13 PMC : PMC3811371 Abstract >>
Autoaggregation in lactic acid bacteria is directly related to the production of certain extracellular proteins, notably, aggregation-promoting factors (APFs). Production of aggregation-promoting factors confers beneficial traits to probiotic-producing strains, contributing to their fitness for the intestinal environment. Furthermore, coaggregation with pathogens has been proposed to be a beneficial mechanism in probiotic lactic acid bacteria. This mechanism would limit attachment of the pathogen to the gut mucosa, favoring its removal by the human immune system. In the present paper, we have characterized a novel aggregation-promoting factor in Lactobacillus plantarum. A mutant with a knockout of the D1 gene showed loss of its autoaggregative phenotype and a decreased ability to bind to mucin, indicating an adhesion role of this protein. In addition, heterologous production of the D1 protein or an internal fragment of the protein, characterized by its abundance in serine/threonine, strongly induced autoaggregation in Lactococcus lactis. This result strongly suggested that this internal fragment is responsible for the bioactivity of D1 as an APF. To our knowledge, this is the first report on a gene coding for an aggregation-promoting factor in Lb. plantarum.
|
97. |
Jalilsood T,
Baradaran A,
Ling FH,
Mustafa S,
Yusof K,
Rahim RA,
( 2014 ) Characterization of pR18, a novel rolling-circle replication plasmid from Lactobacillus plantarum. PMID : 24785193 : DOI : 10.1016/j.plasmid.2014.04.004 Abstract >>
Lactobacillus plantarum PA18, a strain originally isolated from the leaves of Pandanus amaryllifolius, contains a pR18 plasmid. The pR18 plasmid is a 3211bp circular molecule with a G+C content of 35.8%. Nucleotide sequence analysis revealed two putative open reading frames, ORF1 and ORF2, in which ORF2 was predicted (317 amino acids) to be a replication protein and shared 99% similarity with the Rep proteins of pLR1, pLD1, pC30il, and pLP2000, which belong to the RCR pC194/pUB110 family. Sequence analysis also indicated that ORF1 was predicted to encode linA, an enzyme that enzymatically inactivates lincomycin. The result of Southern hybridization and mung bean nuclease treatment confirmed that pR18 replicated via the RCR mechanism. Phylogenetic tree analysis of pR18 plasmid proteins suggested that horizontal transfer of antibiotic resistance determinants without genes encoding mobilization has not only occurred between Bacillus and Lactobacillus but also between unrelated bacteria. Understanding this type of transfer could possibly play a key role in facilitating the study of the origin and evolution of lactobacillus plasmids. Quantitative PCR showed that the relative copy number of pR18 was approximately 39 copies per chromosome equivalent.
|
98. |
Kishino S,
Takeuchi M,
Park SB,
Hirata A,
Kitamura N,
Kunisawa J,
Kiyono H,
Iwamoto R,
Isobe Y,
Arita M,
Arai H,
Ueda K,
Shima J,
Takahashi S,
Yokozeki K,
Shimizu S,
Ogawa J,
( 2013 ) Polyunsaturated fatty acid saturation by gut lactic acid bacteria affecting host lipid composition. PMID : 24127592 : DOI : 10.1073/pnas.1312937110 PMC : PMC3816446 Abstract >>
In the representative gut bacterium Lactobacillus plantarum, we identified genes encoding the enzymes involved in a saturation metabolism of polyunsaturated fatty acids and revealed in detail the metabolic pathway that generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates. Furthermore, we observed these intermediates, especially hydroxy fatty acids, in host organs. Levels of hydroxy fatty acids were much higher in specific pathogen-free mice than in germ-free mice, indicating that these fatty acids are generated through polyunsaturated fatty acids metabolism of gastrointestinal microorganisms. These findings suggested that lipid metabolism by gastrointestinal microbes affects the health of the host by modifying fatty acid composition.
|
99. |
( 1997 ) Molecular characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum: gene cloning, transcriptional analysis, overexpression in Escherichia coli, purification, and characterization. PMID : 9143125 : PMC : PMC168485 Abstract >>
By using degenerate primers designed from the first 19 N-terminal amino acids of Lactobacillus plantarum p-coumaric acid decarboxylase (PDC), a 56-bp fragment was amplified from L. plantarum in PCRs and used as a probe for screening an L. plantarum genomic bank. Of the 2,880 clones in the genomic bank, one was isolated by colony hybridization and contained a 519-bp open reading frame (pdc gene) followed by a putative terminator structure. The pdc gene is expressed on a monocistronic transcriptional unit, which is transcribed from promoter sequences homologous to Lactococcus promoter sequences. No mRNA from pdc and no PDC activity were detected in uninduced cell extracts, indicating that the expression is transcriptionally regulated by p-coumaric acid, which corresponds to an activation factor up to 6,000. The pdc gene was overexpressed constitutively in Escherichia coli, and the recombinant enzyme was purified and characterized.
|
100. |
( 1997 ) The alanine racemase gene is essential for growth of Lactobacillus plantarum. PMID : 9171436 : DOI : 10.1128/jb.179.11.3804-3807.1997 PMC : PMC179184 Abstract >>
The Lactobacillus plantarum alr gene encoding alanine racemase was cloned by complementation of an Escherichia coli Alr- DadX- double mutant strain. Knockout of the alr gene abolished all measurable alanine racemase activity, and the mutant was shown to be strictly dependent on D-alanine for growth.
|
101. |
( 1996 ) Genetic analysis of the replication region of the Lactobacillus plasmid vector pPSC22. PMID : 9157488 : Abstract >>
The sequence and genetic organization of the 1,600-bp replication region of the Lactobacillus vector pPSC22, a plasmid derived from a 7-kb cryptic plasmid of L. plantarum used for the cloning of heterologous genes in several lactobacilli, were determined. Sequence analysis revealed the presence of a plus origin of replication containing the two functional elements nic and bind, required for initiation of the leading strands typical of the rolling circle (RC)-replicating plasmids belonging to the pLS1 family. Two open reading frames (copA and repA) were located within the Lactobacillus portion of pPSC22. The repA gene product, a 234-amino acid protein, showed homologies with the Rep protein of the streptococcal plasmid pLS1 and contained the three conserved domains detected in most Rep proteins of RC-replicating plasmids and ss-coliphages. The genetic organization of the replication region of pPSC22 shared relevant homologies with the lactococcal plasmids pWVO1 and pFX2.
|
102. |
( 1997 ) Molecular characterization of the alpha-amylase genes of Lactobacillus plantarum A6 and Lactobacillus amylovorus reveals an unusual 3' end structure with direct tandem repeats and suggests a common evolutionary origin. PMID : 9370276 : DOI : 10.1016/s0378-1119(97)00309-0 Abstract >>
The alpha-amylase gene (amyA) of Lactobacillus plantarum A6 was isolated from the genome by polymerase chain reaction with degenerated oligonucleotides, synthesized according to the tryptic peptide amino acid sequences of the purified enzyme. Nucleic acid sequence analysis revealed one open reading frame of 2739 bp encoding a 913 amino acid protein. The amylase appears to be divided into two equal parts. The N-terminal part has the typical characteristics of the well-known alpha-amylase family (65% identity with the alpha-amylase of Bacillus subtilis and 97% identity with the partial sequence available for the alpha-amylase of Lactobacillus amylovorus). The C-terminal part displays a fairly unusual structure. It consists of four direct tandem repeated sequences of 104 amino acids sharing 100% similarity. The complete nucleotide sequence of the alpha-amylase gene of L. amylovorus was also determined. An open reading frame of 2862 bp encoding a 954 amino acid protein was identified. Perfect homology between the two amyA genes was observed in the N-terminal region. The C-terminal part of L. amylovorus alpha-amylase also included tandem repeat units but striking differences were observed: (i) the addition of one repeat unit; (ii) a shorter, 91 amino acid repetition unit. These structural homologies suggest that both genes have a common ancestor and may have evolved independently by duplication with subsequent recombination and mutation.
|
103. |
( 1995 ) A bacteriocin-like peptide induces bacteriocin synthesis in Lactobacillus plantarum C11. PMID : 8817486 : Abstract >>
In this study, we show that bacteriocin production in Lactobacillus plantarum C11 is an inducible process triggered by a secreted protein factor produced by the bacteriocin producer itself. The induction factor was identified to be plantaricin A, a bacteriocin-like peptide whose gene (plnA) is located in the same operon as a two-component regulatory system (plnBCD). When L. plantarum C11 cultures were depleted for plantaricin A, either by growing individual colonies on agar plates or by starting a new culture with a highly diluted inoculum, no bacteriocin was produced during the following growth. When chemically synthesized plantaricin A or purified bacterially produced plantaricin A was added to non-producing cultures, bacteriocin production was induced. Only 1 ng ml-1 plantaricin A is sufficient to induce the bacteriocin production in non-producing L. plantarum C11, and bacteriocin activity appears in the growth medium approximately 150 min after induction. Northern analyses, using a plnA-specific probe, demonstrated that plantaricin A is able to induce its own synthesis by transcription of the plnABCD operon, and this is observed approximately 15 min after adding plantaricin A. Furthermore, heterologous expression of the plnABCD operon in a Lactobacillus sake strain showed that the conditioned growth medium contained the active induction factor. Neither synthetic nor expressed plantaricin A from the heterologous system possesses any bacteriocin activity, suggesting that plantaricin A is primarily an induction factor and not a bacteriocin as claimed earlier.
|
104. |
( 1996 ) Molecular cloning of manganese catalase from Lactobacillus plantarum. PMID : 8939876 : DOI : 10.1074/jbc.271.47.29521 Abstract >>
A genomic clone encoding manganese-containing catalase has been isolated from lactic acid bacterium Lactobacillus plantarum, sequenced, and expressed in Escherichia coli cells with an inducible expression system. The primary structure of the enzyme deduced from the nucleotide sequence, that comprises 266 amino acid residues, showed no significant homology with that of any other proteins registered on the available data bases. No peptide motifs conserved among active sites of proteins including manganese-containing enzymes were found. The E. coli cells carrying an expression construct, in which the 5'-noncoding region of the manganese catalase gene was replaced with the lac promoter, highly induced a protein reacting with the antiserum to manganese catalase. The prediction of secondary structure from the deduced primary structure suggested that the L. plantarum manganese catalase, that is classified as a novel protein on the basis of its primary structure, has a main structural motif formed by four near parallel helices between which is the catalytic site manganese.
|
105. |
( 1996 ) Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11. PMID : 8755874 : DOI : 10.1128/jb.178.15.4472-4483.1996 PMC : PMC178213 Abstract >>
Lactobacillus plantarum C11 secretes a small cationic peptide, plantaricin A, that serves as induction signal for bacteriocin production as well as transcription of plnABCD. The plnABCD operon encodes the plantaricin A precursor (PlnA) itself and determinants (PlnBCD) for a signal transducing pathway. By Northern (RNA) and sequencing analyses, four new plantaricin A-induced operons were identified. All were highly activated in concert with plnABCD upon bacteriocin induction. Two of these operons (termed plnEFI and plnJKLR) each encompass a gene pair (plnEF and plnJK, respectively) encoding two small cationic bacteriocin-like peptides with double-glycine-type leaders. The open reading frames (ORFs) encoding the bacteriocin-like peptides are followed by ORFs (plnI and -L, respectively) encoding cationic hydrophobic proteins resembling bacteriocin immunity proteins. On the third operon (termed plnMNOP), a similar bacteriocin-like ORF (plnN) and a putative immunity ORF (either plnM or -P) were identified as well. These findings suggest that two bacteriocins of two-peptide type (mature PlnEF and PlnJK) and a bacteriocin of one-peptide type (mature PlnN) could be responsible for the observed bacteriocin activity. The last operon (termed plnGHSTUV) contains two ORFs (plnGH) apparently encoding an ABC transporter and its accessory protein, respectively, known to be involved in processing and export of peptides with precursor double-glycine-type leaders. Promoter structure was established. A conserved regulatory-like box encompassing two direct repeats was identified in the promoter regions of all five plantaricin A-induced operons. These repeats may serve as regulatory elements for gene expression.
|
106. |
( 1994 ) Use of homologous expression-secretion signals and vector-free stable chromosomal integration in engineering of Lactobacillus plantarum for alpha-amylase and levanase expression. PMID : 8017927 : PMC : PMC201496 Abstract >>
The genuine alpha-amylase gene from Bacillus licheniformis (amyL) is not expressed in Lactobacillus plantarum, but replacement of the amyL promoter by a strong L. plantarum promoter leads to efficient expression of the gene and secretion of more than 90% of the alpha-amylase into the culture supernatant. A series of L. plantarum genetic cassettes (transcription and translation with or without secretion) were cloned by translation fusion of random DNA fragments to the silent amyL coding frame in the pGIP212 probe vector (P. Hols, A. Baulard, D. Garmyn, B. Delplace, S. Hogan, and J. Delcour, Gene 118:21-30, 1992). Five different cassettes were sequenced and found to harbor genetic signals similar to those of other gram-positive bacteria. The functions of the cloned cassettes and the cassettes isolated previously from Enterococcus faecalis were compared in E. faecalis and L. plantarum, respectively. All signals were well recognized in L. plantarum, but cassettes isolated from L. plantarum led to a low level of amylase production in E. faecalis, suggesting that the L. plantarum signals are more species specific. Six transcriptional or translational fusions were constructed to express the Bacillus subtilis levanase gene (sacC) in L. plantarum. All of these constructions were capable of inducing levanase production and secretion in the culture supernatant, and, furthermore, L. plantarum strains harboring the most efficient fusions could grow in MRS medium containing inulin as the major carbon source. Finally, a two-step chromosomal integration procedure was used to achieve efficient stabilization of an amylase construction without any residual resistance marker or vector sequence.
|
107. |
( 1994 ) The gene encoding plantaricin A, a bacteriocin from Lactobacillus plantarum C11, is located on the same transcription unit as an agr-like regulatory system. PMID : 8117074 : PMC : PMC201284 Abstract >>
Purification and amino acid sequencing of plantaricin A, a bacteriocin from Lactobacillus plantarum C11, revealed that maximum bacteriocin activity is associated with the complementary action of two almost-identical peptides, alpha and beta (J. Nissen-Meyer, A. G. Larsen, K. Sletten, M. Daeschel, and I. F. Nes, J. Gen. Microbiol. 139:1973-1978, 1993). A 5-kb chromosomal HindIII restriction fragment containing the structural gene of plantaricin A was cloned and sequenced. Only one gene encoding plantaricin A was found. The gene, termed plnA, encodes a 48-amino-acid precursor peptide, of which the 22 and 23 C-terminal amino acids correspond to the purified peptides. Northern (RNA) blot analysis demonstrated that a probe complementary to the coding strand of the plantaricin A gene hybridized to a 3.3-kb mRNA transcript. Further analysis of the 3.3-kb transcript demonstrated that it contains three additional open reading frames (plnB, plnC and plnD) downstream of plnA. The DNA sequences of plnB, plnC, and plnD revealed that their products closely resemble members of bacterial two-component signal transduction systems. The strongest homology was found to the accessory gene regulatory (agr) system, which controls expression of exoproteins during post-exponential growth in Staphylococcus aureus. The finding that plnABCD are transcribed from a common promoter suggests that the biological role played by the bacteriocin is somehow related to the regulatory function of the two-component system located on the same operon.
|
108. |
( 1993 ) Molecular analysis of the rolling-circle replicating plasmid pA1 of Lactobacillus plantarum A112. PMID : 8439153 : PMC : PMC202090 Abstract >>
Lactobacillus plantarum A112 has four different plasmids. Plus-origin-specific probes were used to determine that the smallest, cryptic plasmid, pA1 (2,820 bp), showed homology to the pE194 plasmid family. This subclass of plasmids uses the rolling-circle mode of replication. Subsequent analysis of plasmid pA1 demonstrated that it generates single-stranded DNA intermediates, and sequence analysis revealed that it contains three putative open reading frames (ORFs): ORF1, ORF2, and ORF3, which could encode proteins designated RepA (47 amino acids [aa]) and RepB (196 aa) and a protein of 103 aa, respectively. Two of these proteins, RepA (5.6 kDa) and RepB (26 kDa), were identified in in vitro transcription translation assays. The RepA protein contains a characteristic alpha-helix-turn-alpha-helix motif typical of DNA-binding proteins that act as DNA-binding repressors. The RepB protein shows a significant similarity with replication initiation proteins of the pE194 family of plasmids that use the rolling-circle mode of replication. Plasmid pA1 is able to replicate in Escherichia coli and Lactobacillus lactis subsp. lactis as well as in other L. plantarum strains.
|
109. |
( 1994 ) Cloning and expression of the plasmid encoded beta-D-galactosidase gene from a Lactobacillus plantarum strain of dairy origin. PMID : 7958766 : DOI : 10.1111/j.1574-6968.1994.tb07157.x Abstract >>
The beta-galactosidase (beta-Gal) gene from Lactobacillus plantarum C3.8 was cloned and expressed in Lactococcus lactis and Escherichia coli. Hybridization experiments indicated that the gene is located on a plasmid and is present in other strains of Lactobacillus plantarum. Its sequence is very similar to a Leuconostoc lactis beta-Gal gene. Expression of the gene, both in Lactobacillus plantarum and in Lactococcus lactis, was four-fold higher in cells growth in lactose compared to those grown in glucose. The presence of the beta-Gal gene in Lactococcus lactis allowed this bacterium to be efficient in clotting milk.
|
110. |
Elisha BG,
Courvalin P,
( 1995 ) Analysis of genes encoding D-alanine:D-alanine ligase-related enzymes in Leuconostoc mesenteroides and Lactobacillus spp. PMID : 7828933 : DOI : 10.1016/0378-1119(94)00692-l Abstract >>
Degenerate oligodeoxyribonucleotides complementary to sequences encoding conserved amino acid (aa) motifs in D-alanine:D-alanine ligases (Ddl) were used to amplify approx. 600-bp fragments from glycopeptide-resistant strains of Leuconostoc mesenteroides (Lm), Lactobacillus plantarum, La. salivarius and La. confusus, and from a susceptible strain of La. leichmannii. Comparison of the deduced aa sequences of the PCR products revealed that the Ddl-related enzymes of resistant Lm and Lactobacillus spp. are more akin to each other (47-63% aa identity) than to that of susceptible La. leichmannii (33-37% aa identity), indicating that the Ddl-related enzymes in these intrinsically resistant species of Gram+ bacteria exhibit structural differences with those in susceptible species. The Ddl-related enzymes, VanA and VanB, implicated in acquired resistance to glycopeptides in enterococci, were not closely related to their counterparts in Lm and Lactobacillus spp., as they displayed only 26-32% aa identity.
|
111. |
Vaiman D,
Mercier D,
Moazami-Goudarzi K,
Eggen A,
Ciampolini R,
Lépingle A,
Velmala R,
Kaukinen J,
Varvio SL,
Martin P,
( 1994 ) A set of 99 cattle microsatellites: characterization, synteny mapping, and polymorphism. PMID : 7545949 : Abstract >>
Cattle microsatellite clones (136) were isolated from cosmid (10) and plasmid (126) libraries and sequenced. The dinucleotide repeats were studied in each of these sequences and compared with dinucleotide repeats found in other vertebrate species where information was available. The distribution in cattle was similar to that described for other mammals, such as rat, mouse, pig, or human. A major difference resides in the number of sequences present in the bovine genome, which seemed at best one-third as large as in other species. Oligonucleotide primers (117 pairs) were synthesized, and a PCR product of expected size was obtained for 88 microsatellite sequences (75%). Synteny or chromosome assignment was searched for each locus with PCR amplification on a panel of 36 hamster/bovine somatic cell hybrids. Of our bovine microsatellites, eighty-six could be assigned to synteny groups of chromosomes. In addition, 10 other microsatellites--HEL 5, 6, 9, 11, 12, 13 (Kaukinen and Varvio 1993), HEL 4, 7, 14, 15--as well as the microsatellite found in the kappa-casein gene (Fries et al. 1990) were mapped on the hybrids. Microsatellite polymorphism was checked on at least 30 unrelated animals of different breeds. Almost all the autosomal and X Chr microsatellites displayed polymorphism, with the number of alleles varying between two and 44. We assume that these microsatellites could be very helpful in the construction of a primary public linkage map of the bovine genome, with an aim of finding markers for Economic Trait Loci (ETL) in cattle.
|
112. |
Ekblad B,
Kristiansen PE,
( 2019 ) NMR structures and mutational analysis of the two peptides constituting the bacteriocin plantaricin S. PMID : 30787405 : DOI : 10.1038/s41598-019-38518-6 PMC : PMC6382864 Abstract >>
The structure of the individual peptides of the two-peptide bacteriocin plantaricin S, an antimicrobial peptide produced by a Lactobacillus plantarum strain, has been determined in DPC micelles. The two peptides of plantaricin S, Pls-�\ and Pls-�], form an �\-helix from and including residue 8 to 24 with a less structured region around residue 16-19 and an amphiphilic �\-helix from and including residue 7 to 23, respectively. Activity assays on single amino acid-substituted GxxxG and GxxxG-like motifs show that substituting the Ser and Gly residues in the G9xxxG13 motif in Pls-�\ and the S17xxxG21 motif in Pls-�] reduced or drastically reduced the antimicrobial activity. The two-peptide bacteriocin muricidin contains GxxxG-like motifs at similar positions and displays 40-50% amino acid identity with plantaricin S. Activity assays of combinations of the peptides that constitute the bacteriocins plantaricin S and muricidin show that some combinations are highly active. Furthermore, sequence alignments show that the motifs important for plantaricin S activity align with identical motifs in muricidin. Based on sequence comparison and activity assays, a membrane-inserted model of plantaricin S in which the two peptides are oriented antiparallel relative to each other and where the GxxxG and GxxxG-like motifs important for activity come close in space, is proposed.
|
113. |
Song Y,
He Q,
Zhang J,
Qiao J,
Xu H,
Zhong Z,
Zhang W,
Sun Z,
Yang R,
Cui Y,
Zhang H,
( 2018 ) Genomic Variations in Probiotic Lactobacillus plantarum P-8 in the Human and Rat Gut. PMID : 29867805 : DOI : 10.3389/fmicb.2018.00893 PMC : PMC5951974 Abstract >>
The effects of probiotics on host gastrointestinal health have become an area of particular interest in the field of probiotic research. However, the impact of the host intestinal environment on genomic changes in probiotic organisms remains largely unknown. To investigate, Lactobacillus plantarum P-8, a well-studied probiotic bacterium, was consumed by healthy human volunteers and rats. Then, the persistence and genomic stability of P-8 in the host gut were surveyed. qPCR results revealed that after the consumption of one dose, P-8 could be detected in the host gastrointestinal tract for 4-5 weeks. By contrast, after 4 successive weeks of consumption, P-8 could be detected for up to 17 weeks after consumption ceased. In total, 92 P-8 derived strains were isolated from fecal samples and their genomes were sequenced and analyzed. Comparative genomic analysis detected 19 SNPs, which showed the characteristics of neutral evolution in the core genome. In nearly half of samples (n = 39, 42%), the loss of one to three plasmids was observed. The frequent loss of plasmids indicated reductive evolution in the accessory genome under selection pressure within the gastrointestinal tract. We also observed a 609-bp 23S rRNA homologous fragment that may have been acquired from other species after intake. Our findings offer insight into the complex reactions of probiotics to the gut environment during survival in the host. The in vivo genomic dynamics of L. plantarum P-8 observed in this study will aid the commercial development of probiotics with more stable characteristics.
|
114. |
( 2013 ) Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins. PMID : 23395591 : DOI : 10.1016/j.micres.2013.01.003 Abstract >>
An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated.
|
115. |
( 2013 ) Crystal structure of tannase from Lactobacillus plantarum. PMID : 23648840 : DOI : 10.1016/j.jmb.2013.04.032 Abstract >>
Tannins are water-soluble polyphenolic compounds in plants. Hydrolyzable tannins are derivatives of gallic acid (3,4,5-trihydroxybenzoic acid) or its meta-depsidic forms that are esterified to polyol, catechin, or triterpenoid units. Tannases are a family of esterases that catalyze the hydrolysis of the galloyl ester bond in hydrolyzable tannins to release gallic acid. The enzymes have found wide applications in food, feed, beverage, pharmaceutical, and chemical industries since their discovery more than a century ago, although little is known about them at the molecular level, including the details of the catalytic and substrate binding sites. Here, we report the first three-dimensional structure of a tannase from Lactobacillus plantarum. The enzyme displays an �\/�] structure, featured by a large cap domain inserted into the classical serine hydrolase fold. A catalytic triad was identified in the structure, which is composed of Ser163, His451, and Asp419. During the binding of gallic acid, the carboxyl group of the molecule forges hydrogen-bonding interactions with the catalytic triad of the enzyme while the three hydroxyl groups make contacts with Asp421, Lys343, and Glu357 to form another hydrogen-bonding network. Mutagenesis studies demonstrated that these residues are indispensable for the activity of the enzyme. Structural studies of the enzyme in complex with a number of substrates indicated that the interactions at the galloyl binding site are the determinant force for the binding of substrates. The single galloyl binding site is responsible for the esterase and depsidase activities of the enzyme.
|
116. |
( 2013 ) Patagonian red wines: selection of Lactobacillus plantarum isolates as potential starter cultures for malolactic fermentation. PMID : 23546829 : DOI : 10.1007/s11274-013-1337-x Abstract >>
The aim of this study was to evaluate fifty-three Lactobacillus plantarum isolates obtained from a Patagonian red wine, molecularly identified and typified using RAPD analysis, in order to select starter cultures for malolactic fermentation (MLF). The results obtained suggest a considerable genetic diversity, taking into account that all L. plantarum isolates were obtained from one cellar and one vintage. Based on the capacity to tolerate a concentration of 14 % ethanol in MRS broth for 2 days, eight isolates were selected for the subsequent analysis. The incidence of various wine stress factors (ethanol, acid pH, lysozyme and sulfur dioxide) on isolates growth was studied. Besides, glucosidase and tannase activities were evaluated, and the presence of genes involved in the synthesis of biogenic amines was examined by PCR. A previously characterized indigenous Oenococcus oeni strain was included with comparative purposes. Differences in technologically relevant characteristics were observed among the eight L. plantarum selected isolates, revealing an isolate-dependent behavior. Detectable glucosidase and tannase activities were found in all isolates. The presence of genes encoding histidine and tyrosine descarboxylases and putrescine carbamoyltransferase was not detected. The ability of L. plantarum isolates to grow and consume L-malic acid in simulated laboratory-scale vinifications revealed that two of them could be considered as possible MLF starter cultures for Patagonian red wines. These isolates will be subjected to further analysis, for a final winery technological characterization.
|
117. |
( 2013 ) Characterization of pMRI 5.2, a rolling-circle-type plasmid from Lactobacillus plantarum BFE 5092 which harbours two different replication initiation genes. PMID : 23220639 : DOI : 10.1016/j.plasmid.2012.11.005 Abstract >>
Plasmid pMRI 5.2 from Lactobacillus plantarum BFE 5092 was sequenced and analysed. The sequence consists of 5206bp with a mol% G+C content of 35.8%. Nine putative open reading frames were identified. A typical pC194 family double strand origin (dso) and a putative single strand origin (sso) were predicted upstream of a rep gene. This rep gene encoded a replication protein of 314 amino acids exhibiting 98% amino acid sequence identity to the Rep protein of plasmid pLAB1000 from Lactobacillus hilgardii. A mob gene encoding a mobilization protein was also identified and this protein showed high amino acid similarity to Mob proteins from various L. plantarum plasmids. Downstream of the mob gene, a second putative replication region was identified that is similar to the pMV158 family of plasmids. It contains a dso as well as a putative sso, and encodes the 52 amino acid repressor-like protein RepA, the replication initiation protein RepB of 215 amino acids, and the 48 amino acid RepC that is similar to ORFD of the lactococcal plasmid pWVO1. RT-PCR and qRT-PCR expression analyses of the rep and repB genes showed that the repB gene was expressed at a higher level. To confirm that the plasmid replicated by the rolling-circle-type mechanism, the presence of a characteristic single strand intermediate DNA was shown to be produced during replication. Plasmid copy number was ca. 30 per equivalent chromosome copy number based on qRT-PCR analyses. The plasmid also encodes four additional putative proteins of unknown function. The unusual feature of a rolling-circle plasmid having two different plasmid-encoded replication initiation proteins from different replicon families suggests that the genes for these may have originated from different plasmids.
|
118. |
( 2012 ) Tyrosine-containing peptides are precursors of tyramine produced by Lactobacillus plantarum strain IR BL0076 isolated from wine. PMID : 22963406 : DOI : 10.1186/1471-2180-12-199 PMC : PMC3492074 Abstract >>
Biogenic amines are molecules with allergenic properties. They are found in fermented products and are synthesized by lactic acid bacteria through the decarboxylation of amino acids present in the food matrix. The concentration of biogenic amines in fermented foodstuffs is influenced by many environmental factors, and in particular, biogenic amine accumulation depends on the quantity of available precursors. Enological practices which lead to an enrichment in nitrogen compounds therefore favor biogenic amine production in wine. Free amino acids are the only known precursors for the synthesis of biogenic amines, and no direct link has previously been demonstrated between the use of peptides by lactic acid bacteria and biogenic amine synthesis. Here we demonstrate for the first time that a Lactobacillus plantarum strain isolated from a red wine can produce the biogenic amine tyramine from peptides containing tyrosine. In our conditions, most of the tyramine was produced during the late exponential growth phase, coinciding with the expression of the tyrDC and tyrP genes. The DNA sequences of tyrDC and tyrP in this strain share 98% identity with those in Lactobacillus brevis consistent with horizontal gene transfer from L. brevis to L. plantarum. Peptides amino acids are precursors of biogenic amines for Lactobacillus plantarum strain IR BL0076.
|
119. |
( 1998 ) Expression of the bglH gene of Lactobacillus plantarum is controlled by carbon catabolite repression. PMID : 9642194 : PMC : PMC107296 Abstract >>
A newly identified bglH gene coding for a phospho-beta-glucosidase of Lactobacillus plantarum was isolated and expressed in Escherichia coli. The sequence analysis of the cloned DNA fragment showed an open reading frame encoding a 480-amino-acid protein with a calculated molecular mass of 53 kDa. The bglH gene was shown to be expressed on a monocistronic transcriptional unit. Its transcription was repressed 10-fold in L. plantarum cells grown on glucose compared to the beta-glucoside salicin as a sole carbon source. A catabolite-responsive element (CRE) spanning from -3 to +11 with respect to the transcriptional start point was found, and its functionality was assessed by mutational analysis. In vitro and in vivo DNA binding experiments suggested the occurrence of a DNA-protein complex at the CRE site, which would mediate glucose repression of bglH expression.
|
120. |
( 1998 ) Molecular analysis of the locus responsible for production of plantaricin S, a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10. PMID : 9572965 : PMC : PMC106244 Abstract >>
A 4.5-kb region of chromosomal DNA carrying the locus responsible for the production of plantaricin S, a two-peptide bacteriocin produced by Lactobacillus plantarum LPCO10 (R. Jim?nez-D?az, J. L. Ruiz-Barba, D. P. Cathcart, H. Holo, I. F. Nes, K. H. Sletten, and P. J. Warner, Appl. Environ. Microbiol. 61:4459-4463, 1995), has been cloned, and the nucleotide sequence has been elucidated. Two genes, designated plsA and plsB and encoding peptides alpha and beta, respectively, of plantaricin S, plus an open reading frame (ORF), ORF2, were found to be organized in an operon. Northern blot analysis showed that these genes are cotranscribed, giving a ca. 0.7-kb mRNA, whose transcription start point was determined by primer extension. Nucleotide sequences of plsA and plsB revealed that both genes are translated as bacteriocin precursors which include N-terminal leader sequences of the double-glycine type. The role of ORF2 is unknown at the moment, although it might be expected to encode an immunity protein of the type described for other bacteriocin operons. In addition, several other potential ORFs have been found, including some which may be responsible for the regulation of bacteriocin production. Two of them, ORF8 and ORF14, show strong homology with histidine protein kinase and response regulator genes, respectively, which have been found to be involved in the regulation of the production of other bacteriocins from lactic acid bacteria. A third ORF, ORF5, shows homology with gene agrB from Staphylococcus aureus, which is involved in the mechanism of regulation of the virulence phenotype in this species. Thus, an agr-like regulatory system for the production of plantaricin S is postulated.
|
331, Shih-Pin Rd., Hsinchu 30062, Taiwan
Phone: +886-3-5223191
E-mail: bcrcweb@firdi.org.tw
web maintainance: +886-3-5223191 ext 593
Copyright © 2018.BCRC All rights reserved.The duplication or use of information and data such as texts or images or any linkage the website at the "bcrc.firdi.org.tw" is only permitted with the indication of the source or with prior approval by the BCRC(Bioresource Collection and Research Center).