Thirty-eight isolates of Lactobacillus gallinarum cultured from the crops of broiler chickens were screened for the presence of genes encoding S-layer proteins. All of the isolates had two S-protein genes, which were designated Lactobacillus gallinarum S-protein (lgs) genes. One gene in each isolate was either lgsA or lgsB. The Lactobacillus isolates were further characterized by pulsed-field gel electrophoresis of DNA digests, which grouped the isolates into 17 genotypes (strains). The second gene in each of eight representative strains was sequenced and shown to differ among strains (lgsC, lgsD, lgsE, lgsF, lgsG, lgsH, and lgsI). The genome of each strain thus encoded a common S-protein (encoded by either lgsA or lgsB) and a strain-specific S-protein. The extraction of cell surface proteins from cultures of the eight strains showed that each strain produced a single S-protein that was always encoded by the strain-specific lgs gene. Two of the strains were used to inoculate chickens maintained in a protected environment which were Lactobacillus-free prior to inoculation. DNAs and RNAs extracted from the digesta of the chickens were used for PCR and reverse transcription-PCR, respectively, to demonstrate the presence and transcription of lgs genes in vivo. In both cases, only the strain-specific gene was transcribed. Both of the strains adhered to the crop epithelium, consistent with published data predicting that S-proteins of lactobacilli are adhesins. The results of this study provide a basis for the investigation of gene duplication and sequence variation as mechanisms by which bacterial strains of the same species can share the same habitat.

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

The atp operon is highly conserved among eubacteria, and it has been considered a molecular marker as an alternative to the 16S rRNA gene. PCR primers were designed from the consensus sequences of the atpD gene to amplify partial atpD sequences from 12 Bifidobacterium species and nine Lactobacillus species. All PCR products were sequenced and aligned with other atpD sequences retrieved from public databases. Genes encoding the subunits of the F(1)F(0)-ATPase of Bifidobacterium lactis DSM 10140 (atpBEFHAGDC) were cloned and sequenced. The deduced amino acid sequences of these subunits showed significant homology with the sequences of other organisms. We identified specific sequence signatures for the genus Bifidobacterium and for the closely related taxa Bifidobacterium lactis and Bifidobacterium animalis and Lactobacillus gasseri and Lactobacillus johnsonii, which could provide an alternative to current methods for identification of lactic acid bacterial species. Northern blot analysis showed that there was a transcript at approximately 7.3 kb, which corresponded to the size of the atp operon, and a transcript at 4.5 kb, which corresponded to the atpC, atpD, atpG, and atpA genes. The transcription initiation sites of these two mRNAs were mapped by primer extension, and the results revealed no consensus promoter sequences. Phylogenetic analysis of the atpD genes demonstrated that the Lactobacillus atpD gene clustered with the genera Listeria, Lactococcus, Streptococcus, and Enterococcus and that the higher G+C content and highly biased codon usage with respect to the genome average support the hypothesis that there was probably horizontal gene transfer. The acid inducibility of the atp operon of B. lactis DSM 10140 was verified by slot blot hybridization by using RNA isolated from acid-treated cultures of B. lactis DSM 10140. The rapid increase in the level of atp operon transcripts upon exposure to low pH suggested that the ATPase complex of B. lactis DSM 10140 was regulated at the level of transcription and not at the enzyme assembly step.

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.

We analyzed the tuf gene, encoding elongation factor Tu, from 33 strains representing 17 Lactobacillus species and 8 Bifidobacterium species. The tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these tuf sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of tuf sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as Lactobacillus casei group members. These tuf gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of tuf genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one tuf gene in the genomes of lactobacilli and bifidobacteria, but the tuf gene was arranged differently in the genomes of these two taxa. Our results revealed that the tuf gene in bifidobacteria is flanked by the same gene constellation as the str operon, as originally reported for Escherichia coli. In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the tuf gene in four Lactobacillus species indicated the same four-gene unit and suggested a novel tuf operon specific for the genus Lactobacillus.

In the early 1980s, a facultatively anaerobic, non-motile, short rod, designated 202(T), was isolated from a chicken crop and identified as a homofermentative lactic acid bacterium. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain was affiliated with the genus Lactobacillus, clustering within the Lactobacillus acidophilus-delbrueckii group. In this analysis, strain 202(T) appeared to be most closely related to the type strains of Lactobacillus intestinalis and Lactobacillus amylolyticus, with gene sequence similarities of 96.1 and 96.2 %, respectively. Strain 202(T) was found to differ from these two species, however, when investigated by multilocus sequence analysis, and it also differed in terms of some of its metabolic properties. On the basis of these observations, strain 202(T) is considered to represent a novel species in the genus Lactobacillus, for which the name Lactobacillus gigeriorum sp. nov. is proposed; the type strain is 202(T) (= CRBIP 24.85(T) = DSM 23908(T)).

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.

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.

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.

Members of the Lactobacillus acidophilus complex are associated with functional foods and dietary supplements because of purported health benefits they impart to the consumer. Many characteristics of these microorganisms are reported to be strain specific. Therefore, proper strain typing is essential for safety assessment and product labeling, and also for monitoring strain integrity for industrial production purposes. Fifty-two strains of the L. acidophilus complex (L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, and L. johnsonii) were genotyped using two established methods and compared to a novel multilocus sequence typing (MLST) scheme. PCR restriction fragment length polymorphism (PCR-RFLP) analysis of the hsp60 gene with AluI and TaqI successfully clustered 51 of the 52 strains into the six species examined, but it lacked strain-level discrimination. Random amplified polymorphic DNA PCR (RAPD-PCR) targeting the M13 sequence resulted in highly discriminatory profiles but lacked reproducibility. In this study, an MLST scheme was developed using the conserved housekeeping genes fusA, gpmA, gyrA, gyrB, lepA, pyrG, and recA, which identified 40 sequence types that successfully clustered all of the strains into the six species. Analysis of the observed alleles suggests that nucleotide substitutions within five of the seven MLST loci have reached saturation, a finding that emphasizes the highly diverse nature of the L. acidophilus complex and our unconventional application of a typically intraspecies molecular typing tool. Our MLST results indicate that this method could be useful for characterization and strain discrimination of a multispecies complex, with the potential for taxonomic expansion to a broader collection of Lactobacillus species.

To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species-specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231-bp species-specific band was found only in L. delbrueckii. A SNaPshot mini-sequencing assay using recA as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species-specific polymerase chain reaction combined with SNaPshot mini-sequencing.