Sarmiento-Rubiano et al. BMC Genomics 2010, 11:504 http://www.biomedcentral.com/1471-2164/11/504 Page 7 of 16 Figure 2 Phylogenetic tree showing relationships among recA (A) and pheS (B) gene sequences of species in the Lactobacillus acidophilus group, including Lactobacillus taiwanensis, and species representing different lineages within the genus Lactobacillus. The trees were created using a maximum-likelihood approach and numbers at branch points are bootstrap values (based on 500 samplings expressed in percentages). Only bootstrap values over 75% are shown. Bar indicates sequence divergence. The trees have been arbitrarily rooted. In parenthesis are shown the GenBank accession numbers. Complete Genome Hybridization between L. taiwanensis BL263 and L. johnsonii NCC533 using DNA microarrays Firstly, the DNA microarray constructed with oligonu- cleotides representing all the coding regions from L. johnsonii strain NCC533 was tested in a self-hybridiza- tion experiment to validate the design of the microarrays. This step was necessary since the microarrays previously used for inter-species CGH in the L. acidophilus group [26] were amplicon-based and not oligonucleotide-based. Thus, L. johnsonii NCC533 strain was used as both the reference and the test strain, and as expected the distri- bution of the log2 ratios showed a normal distribution around zero (data not shown). The performance of the oligonucleotide microarray was further evaluated by hybridization with DNA from L. johnsonii ATCC 33200 type strain and L. johnsonii BL261. In the DNA-DNA hybridization assays, this latter strain fell into the relaxed definition of species. The distribution of the log2 ratios of the CGH results are shown in Fig. 5A. Approximately 83% of the ORFs present in NCC533 strain produced a log2 ratio of -3.5 or greater with these two strains, sug- gesting a majority of very similar DNA sequences. The rest of ORFs showed log2 values under -3.5, reflecting high divergence or absent genes in the test strains. The two L. johnsonii strains profiles were typical of intra-spe- cies comparisons. In contrast, L. taiwanensis and L. gasseri strains showed a significant deviation from zero for nearly all the genes, suggesting global sequence diver- gence between L. johnsonii and both tested strains (Fig. 5A). These strains gave a similar profile, characteristic of inter-species comparisons. However, L. taiwanensis have more probes (51%) with ratios of -3.5 or greater than L. gasseri (47%), showing slightly better gene conservation. This is in agreement with the results of DNA-DNA hybridization experiments. These results confirmed the separate status of L. taiwanensis from L. johnsonii at the level of species, and they showed that L. taiwanensis is closer to L. johnsonii than L. gasseri is to L. johnsonii. CGH results were mapped on the L. johnsonii NCC533 genome (Fig. 5B). The genetic differences observed in the intra-species analysis with the L. johnso- nii ATCC 33200 type strain closely match with the pre- vious analysis by amplicon-based microarrays [26], further validating the oligonucleotide-based microarrays used in this study. While L. taiwanensis BL263 shared fewer genes with the reference L. johnsonii NCC533 than the other L. johnsonii strains, it showed 78 genes which are not always present in L. johnsonii strains. They are mainly organized in 6 gene clusters: the sec locus, a hypothetical fimbrial protein, the bacteriocin lactacin F biosynthesis operon, the lactose utilization cluster, the sugar nucleotide dTDP-rhamnose synthesis