Sarmiento-Rubiano et al. BMC Genomics 2010, 11:504 http://www.biomedcentral.com/1471-2164/11/504 Results 16S rRNA encoding gene and multilocus sequence analyses positioned the novel Lactobacillus isolates within the Lactobacillus acidophilus group Sequences of the 16S rRNA gene (approximately 1,440 bp) of the rat isolates Lactobacillus sp. BL263, BL301, BL302, BL303 and BL304 were determined. Sequences of BL263, BL301, BL302 and BL303 were identical (data not shown) and shared a 99.9% sequence identity with BL304 strain. A preliminary analysis showed that these sequences were very similar to sequences of L. gasseri and L. johnsonii. Subsequently, a phylogenetic tree was obtained by using the tools available in RDP [32]. On the basis of this reconstruction, sequences of species close to L. gasseri and L. johnsonii were selected for a detailed phylogenetic reconstruction by maximum likeli- hood. During the preparation of this manuscript, the novel L. taiwanensis species isolated from silage was described [39]. The phylogenetic reconstruction based on 16S rRNA gene sequences suggested that these strains belong to L. taiwanensis species and that they are closely related to L. johnsonii and L. gasseri (Fig. 1). The identity between the 16S rRNA gene of L. taiwa- nensis and L. johnsonii and L. gasseri strains ranged from 99.2% to 99.7% and 98.5% to 99.5%, respectively. This is higher than the generally accepted >97% identity threshold for the species definition, but this problem is known for long in the acidophilus group [24]. The analysis of the recA, pheS, pyrG and tuf gene sequences from strains BL263, BL301, BL302, BL303, BL304 and L. taiwanensis DSM 21401T also led to phy- logenetic trees that clearly separated a cluster encom- passing L. taiwanensis and the rat intestinal isolates from all the L. johnsonii and L. gasseri strains as indi- cated by the bootstrap values (Fig. 2 and Fig. 3). There- fore, we concluded that our isolates belong to L. taiwanensis. These phylogenetic reconstructions were in general agreement with that obtained with the 16S rRNA gene sequences described above. Although highly conserved, those genes showed a higher degree of varia- bility between related bacteria than the 16S rRNA gene. Therefore, the multilocus sequence analysis provided higher discriminating power which is more suited for phylogenetic and taxonomic reconstruction within the closely related species that constitute the L. acidophilus group. Phenotypic and protein profile analyses did not resolve species The carbohydrate fermentation patterns, and the growth profiles of 26 lactobacilli (including the novel isolates and members of the L. acidophilus group) at different temperatures and salt concentrations were used to Page 5 of 16 construct a dendrogram (Table 3 and Fig. 4). Similarly, a comparative analysis of whole-cell protein patterns was performed with bacterial protein extracts of the novel isolates and other members of the L. acidophilus group (Fig. 4). Neither the phenotypic dendrogram, nor the cluster analysis of the SDS-PAGE protein profiles showed a good agreement with the phylogenetic rela- tionships inferred from genetic markers. These phenoty- pic and protein profiles analyses agree with previous taxonomic analyses [40] that showed a lack of correla- tion between phylogenetic positioning and physiological/ biochemical properties in lactobacilli. Notwithstanding, L. taiwanensis strains grouped together in a subcluster of the phenotypic analysis with an internal similarity above 30% (Fig. 4). DNA-DNA hybridization experiments confirmed the separate status of Lactobacillus taiwanensis strains at the species level DNA similarity values were determined by DNA-DNA hybridization with 26 Lactobacillus strains. DNA of the 26 strains was immobilized in membrane blots and the genomic DNA of six Lactobacillus type strains and L. taiwanensis BL263 strain was used as probe (Table 4). Relative hybridization values obtained from intra-species hybridization assays with L. intestinalis, L. acidophilus or L. crispatus strains always rendered values above 82%, while inter-species DNA re-associa- tion values between those species were low (1% to 18%). Results obtained with L. johnsonii, L. gasseri and L. taiwanensis were less clear-cut. L. johnsonii and L. gasseri intra-species hybridization values were greater than 71%, but L. johnsonii BL261 and L. gasseri BL223 only showed values of 57% and 62% with their respective type strains. Nevertheless, those values still fall within relaxed species delineation boundaries (50- 70% DNA reassociation) [20]. L. taiwanensis showed intra-specific hybridization values in the range of 73% to 95%. Inter-species hybridizations between L. johnso- nii and L. gasseri strains rendered values up to 46%, and always higher than with the other species included in this assay, supporting that they are closely related but distinct species. L. taiwanensis strains also showed high reassociation rates with L. johnsonii and L. gasseri, their inter-species hybridization values ranging from 21-40% and from 10-26%, respectively. Therefore, these results also demonstrated the separate status of L. taiwanensis strains at the species level, and undoubtedly placed them within the L. acidophilus complex. Inside this group they showed closer DNA homology to L. johnsonii and L. gasseri than to L. cris- patus and L. acidophilus in agreement with the pre- vious phylogenetic analyses.