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PubMed2001

Detection of Lactobacillus, Pediococcus, Leuconostoc, and Weissella species in human feces by using group-specific PCR primers and denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.

Adult↗
PubMed2003

ISLpl1 is a functional IS30-related insertion element in Lactobacillus plantarum that is also found in other lactic acid bacteria.

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.

Bacterial Proteins↗
PubMed2003

Ecology of uncultivated Oscillospira species in the rumen of cattle, sheep, and reindeer as assessed by microscopy and molecular approaches.

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium Oscillospira spp. was studied. Oscillospira-specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the Oscillospira clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an Oscillospira-specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large Oscillospira morphotypes, the presence of Oscillospira spp. was still detected by Oscillospira-specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of Oscillospira species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.

Animal Feed↗
PubMed2003

Case study of the distribution of mucosa-associated Bifidobacterium species, Lactobacillus species, and other lactic acid bacteria in the human colon.

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.

Adult↗
PubMed2003

Purification and characterization of brochocin A and brochocin B(10-43), a functional fragment generated by heterologous expression in Carnobacterium piscicola.

Brochothrix campestris ATCC 43754 produces a heat-stable, two-component, nonlantibiotic, class IIb bacteriocin, brochocin C (BrcC), that is active against a broad range of gram-positive bacteria, including spores of Clostridium botulinum. An improved purification method was developed for BrcC, in which n-butanol and chloroform extraction are used. Mass spectral characterization of the two components, brochocin A (BrcA) and brochocin B (BrcB), showed that both components are excreted into the medium by B. campestris as mature peptides consisting of 59 and 43 amino acids, respectively. Separate expression clones of BrcA and BrcB were constructed previously in Carnobacterium piscicola LV17C, but the products were not chemically characterized. Purification by the new protocol showed that BrcA is expressed as the mature 59-amino-acid peptide but that BrcB is produced by C. piscicola as a fragment, BrcB(10-43), which is cleaved at an internal Gly-Gly site. This fragment is not antimicrobial by itself, but in combination with BrcA it displays the full activity of the BrcC complex. Circular dichroism measurements revealed a high beta-sheet content in the secondary structure of both BrcA and BrcB(10-43), as well as in a 1:1 BrcA-BrcB(10-43) mixture. Separate expression clones of brcA and brcB were also constructed in Escherichia coli, but these clones only produced multiple fragments of the desired peptides with little or no activity.

Amino Acid Sequence↗
PubMed2003

Characterization of a theta-type plasmid from Lactobacillus sakei: a potential basis for low-copy-number vectors in lactobacilli.

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.

Amino Acid Sequence↗
PubMed2004

Generation and utilization of polyclonal antibodies to a synthetic C-terminal amino acid fragment of divercin V41, a class IIa bacteriocin.

Polyclonal antibodies have been generated by immunization of rabbits with a chemically synthesized C-terminal part of divercin V41 (DvnCt) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and reactivity of the DvnCt-KLH-generated antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) using supernatant from cultures of 13 representative lactic acid bacterium strains, and specificity was confirmed by Western blot analysis. Anti-DvnCt-KLH antibodies were able to recognize not only divercin V41 but also enterocin P and piscicocin V1b, two other members of the class IIa bacteriocins. Production and activity of DvnV41 were evaluated by ELISA and activity tests during the growth of Carnobacterium divergens V41 in MRS medium containing or not containing Tween 80. Divercin V41, enterocin P, and piscicocin V1b were therefore purified by a single-step immunoaffinity chromatography method using a Sepharose matrix CNBr-activated directed binding of anti-DvnCt-KLH polyclonal antibodies.

Amino Acid Sequence↗
PubMed2004

Novel expression system for large-scale production and purification of recombinant class IIa bacteriocins and its application to piscicolin 126.

Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.

Amino Acid Sequence↗
PubMed1962

Fermentation of the Yugoslavian pickled cabbage.

In Yugoslavia, cabbage is preserved by fermentation using a variety of methods. Bacteriological study of these fermentations has shown that the relative influence of the various lactic acid producing bacteria varies somewhat with the method of preservation. The heterofermenters, Leuconostoc mesenteroides and Lactobacillus brevis, exert a greater effect when cabbage is shredded than when the whole heads are packed in salt brine. Whole heads of cabbage preserved by fermentation in salt brine are a good source of ascorbic acid and provide a flavorful food.

Brassica↗
PubMed1975

Influence of wine composition on the heat resistance of potential spoilage organisms.

Pasteurization studies were conducted on 29 yeast and five lactic acid bacteria. In general the yeasts were more heat resistant in wine than were the bacteria. The one exception was a strain of Lactobacillus fructivorans that gave an average D-value of 1.7 min at 60 C. Alcohol was the wine constituent that had the greatest effect on resistance; D-values for all test species were inversely related to the ethanol concentration. The response of organisms to other factors such as pH, sugar, and sulfur dioxide varied with the species.

Ethanol↗
PubMed1975

Detection of mannitol formation by bacteria.

A test is described by means of which formation of mannitol from fructose by lactic acid bacteria can be readily detected. The test is based on removal of interference of residual fructose by dehydration with hydrochloric acid followed by thin-layer chromatography.

Bacteriological Techniques↗
PubMed2001

Identification of the UDP-MurNAc-pentapeptide:L-alanine ligase for synthesis of branched peptidoglycan precursors in Enterococcus faecalis.

Many species of gram-positive bacteria produce branched peptidoglycan precursors resulting from the transfer of various L-amino acids or glycine from amino acyl-tRNA to the epsilon-amino group of L-lysine. The UDP-MurNAc-pentapeptide:L-alanine ligase and alanyl-tRNA synthetase genes from Enterococcus faecalis were identified, cloned, and overexpressed in Escherichia coli. The purified enzymes were necessary and sufficient for tRNA-dependent addition of L-alanine to UDP-MurNAc-pentapeptide in vitro. The ligase belonged to the Fem family of proteins, which were initially identified genetically as factors essential for methicillin resistance in Staphylococcus aureus.

Alanine↗
PubMed2002

Plasmid-encoded asp operon confers a proton motive metabolic cycle catalyzed by an aspartate-alanine exchange reaction.

Tetragenococcus halophila D10 catalyzes the decarboxylation of L-aspartate with nearly stoichiometric release of L-alanine and CO(2). This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an L-aspartate-beta-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter --> aspD --> aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known L-aspartate-beta-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of L-aspartate-beta-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

Alanine↗
PubMed2002

A conserved C-terminal region in Gp71 of the small isometric-head phage LL-H and ORF474 of the prolate-head phage JCL1032 is implicated in specificity of adsorption of phage to its host, Lactobacillus delbrueckii.

Thirty-five phage-resistant mutants of Lactobacillus delbrueckii subsp. lactis ATCC 15808 were selected. Thirty-three of these mutants were assigned to the Bes group, while the remaining two were grouped under the Ads designation. Bes group mutants adsorbed phage LL-H but did not allow efficient phage development. Preliminary evidence suggests that these strains exhibit a mutation that changes the DNA specificity of a restriction-modification system. The Ads group mutants did not adsorb the small isometric-head phage LL-H. The results suggest that there are at least three different types of phage receptors in L. delbrueckii: two that are specific for small isometric-head phages and one that is specific for prolate-head phage JCL1032. Five LL-H host-range mutants which could overcome the adsorption block (a-type mutants) were selected and investigated by sequencing the genes g71 and g17, which encode minor and major tail proteins, respectively. Each of the a-type mutants carried a nucleotide change at the 3' end of gene g71. No mutations were observed in gene g17. Comparison of the gene product of g71 of phage LL-H with its homolog in JCL1032 (ORF474) showed that these proteins had very similar C-terminal regions. No similarities were found at the N-terminal part of the proteins. We conclude that the C-terminal portion of the protein encoded by g71 of phage LL-H and its homolog in phage JCL1032 determines the adsorption specificities of these phages on L. delbrueckii.

Amino Acid Sequence↗
PubMed2005

CitI, a transcription factor involved in regulation of citrate metabolism in lactic acid bacteria.

A large variety of lactic acid bacteria (LAB) can utilize citrate under fermentative conditions. Although much information concerning the metabolic pathways leading to citrate utilization by LAB has been gathered, the mechanisms regulating these pathways are obscure. In Weissella paramesenteroides (formerly called Leuconostoc paramesenteroides), transcription of the citMDEFCGRP citrate operon and the upstream divergent gene citI is induced by the presence of citrate in the medium. Although genetic experiments have suggested that CitI is a transcriptional activator whose activity can be modulated in response to citrate availability, specific details of the interaction between CitI and DNA remained unknown. In this study, we show that CitI recognizes two A+T-rich operator sites located between citI and citM and that the DNA-binding affinity of CitI is increased by citrate. Subsequently, this citrate signal propagation leads to the activation of the cit operon through an enhanced recruitment of RNA polymerase to its promoters. Our results indicate that the control of CitI by the cellular pools of citrate provides a mechanism for sensing the availability of citrate and adjusting the expression of the cit operon accordingly. In addition, this is the first reported example of a transcription factor directly functioning as a citrate-activated switch allowing the cell to optimize the generation of metabolic energy.

Bacterial Proteins↗
PubMed2004

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

We developed a new molecular tool based on rpoB gene (encoding the beta subunit of RNA polymerase) sequencing to identify streptococci. We first sequenced the complete rpoB gene for Streptococcus anginosus, S. equinus, and Abiotrophia defectiva. Sequences were aligned with these of S. pyogenes, S. agalactiae, and S. pneumoniae available in GenBank. Using an in-house analysis program (SVARAP), we identified a 740-bp variable region surrounded by conserved, 20-bp zones and, by using these conserved zones as PCR primer targets, we amplified and sequenced this variable region in an additional 30 Streptococcus, Enterococcus, Gemella, Granulicatella, and Abiotrophia species. This region exhibited 71.2 to 99.3% interspecies homology. We therefore applied our identification system by PCR amplification and sequencing to a collection of 102 streptococci and 60 bacterial isolates belonging to other genera. Amplicons were obtained in streptococci and Bacillus cereus, and sequencing allowed us to make a correct identification of streptococci. Molecular signatures were determined for the discrimination of closely related species within the S. pneumoniae-S. oralis-S. mitis group and the S. agalactiae-S. difficile group. These signatures allowed us to design a S. pneumoniae-specific PCR and sequencing primer pair.

Base Sequence↗
PubMed2004

Antimicrobial susceptibilities of invasive pediatric Abiotrophia and Granulicatella isolates.

Abiotrophia and Granulicatella species have been associated with various infections. Antimicrobial susceptibility data for these nutritionally variant streptococcus-like organisms, especially for pediatric isolates, are very limited. Little is known about the genetic bases of their resistance mechanisms. We report the results of identification to bacterial species level, antimicrobial susceptibility testing, macrolide resistance testing, and detection of genes encoding that resistance for a collection of 15 pediatric clinical isolates from normally sterile sites. Our results indicate that the prevalence of beta-lactam and macrolide resistance is high and that both erm and mef are found in these isolates.

Base Sequence↗