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.

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)).

Women are at significant risk of heterosexually transmitted human immunodeficiency virus (HIV) infection, with the mucosal epithelium of the cervix and vagina serving as a major portal of entry. The cervicovaginal mucosa naturally harbors dynamic microflora composed predominantly of lactobacilli, which may be genetically modified to serve as a more efficient protective barrier against the heterosexual transmission of HIV. We selected a vaginal strain of Lactobacillus, L. jensenii 1153, for genetic modification to display surface-anchored anti-HIV proteins. Genomic sequencing analyses revealed that L. jensenii 1153 encodes several unique high-molecular-weight cell wall-anchored proteins with a C-terminal cell wall sorting LPQTG motif. In this report, we employed these proteins to express a surface-anchored two-domain CD4 (2D CD4) molecule in L. jensenii 1153. Our studies indicated that the C-terminal cell wall sorting signal LPQTG motif alone is insufficient to drive the surface expression of heterologous proteins, and the display of surface-anchored 2D CD4 molecules required native sequences of a defined length upstream of the unique C-terminal LPQTG cell wall sorting signal and the positively charged C terminus in a Lactobacillus-based expression system. The modified L. jensenii strain displayed 2D CD4 molecules that were uniformly distributed on bacterial surfaces. The surface-anchored 2D CD4 molecule was recognized by a conformation-dependent anti-CD4 antibody, suggesting that the expressed proteins adopted a native conformation. The establishment of this Lactobacillus-based surface expression system, with potential broad applicability, represents a major step toward developing an inexpensive yet durable approach to topical microbicides for the mitigation of heterosexual transmission of HIV and other mucosally transmitted viral pathogens.

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.

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.