Sarmiento-Rubiano et al. BMC Genomics 2010, 11:504 http://www.biomedcentral.com/1471-2164/11/504 Page 13 of 16 Figure 6 Genome conservation on L. johnsonii NCC533 microarrays. Scatter plot diagram of hybridization profiles for L. taiwanensis BL263 (y axis) versus L. gasseri ATCC 33323T (x axis). The axis values represent the hybridization signal ratios expressed in a log2 scale. For each gene, data points were colour-coded according to the hybridization profile of a third strain, L johnsonii ATCC 33200T. Gene conservation colour was obtained from the signal ratio for each gene in log2 scale. It ranged from black (presence in ATCC 32200T of a homologous gene with respect to the reference strain NCC533) to white (absence of a gene). For instance, a white circle in the top left corner of the plot represents a gene present in BL263 and absent in both ATCC 33323T and ATCC 32200T. experiments (Table 4). Taken together, these results indicate that the five L. taiwanensis isolates constitute distinct strains. The microarray-based CGH has been used to charac- terize bacterial intra-species genetic diversity at the whole-genome level [41-46]. It has also been used for genome comparisons between species of the same genus or closely related genus that differ in environmental ori- gin or virulence potential [47-49]. CGH analyses by microarrays have also been used to discriminate between the species of the L. acidophilus complex [26]. Here, microarray-based CGH technology has been applied to characterize a novel species by determining the gene by gene boundaries with its nearest phylogenetic neighbour. The rapid accumulation in the last years of complete genome bacterial sequences (at present there are 721 complete or in progress deposited genomes in the phy- lum Firmicutes, 57 of them of the genus Lactobacillus, at the National Center for Biotechnology Information), and the general trend towards an increase in genome sequen- cing projects will offer the opportunity of applying the DNA microarray-based CGH as an alternative to the classical whole genome DNA macroarray hybridizations for bacterial species determination. At a more affordable price than full genome sequencing, the CGH analysis renders many more information parameters than whole DNA re-association. Although the CGH microarrays pre- sented the limitation of be unable to detect novel genes, it offers the re-association ratio for each ORF, the plot of the signal to ratio frequency, and the mapping of the conserved genes on the reference genome, thus providing a different ground for intra and inter-species compari- sons. This work has shown that the inter-species profile comparison of the novel species L. taiwanensis clearly demonstrated its separate status respect to L. johnsonii. The values obtained when comparing the relatedness of L. taiwanensis BL263 and L. johnsonii NCC533 using DNA-DNA hybridization with macro or microarrays may look dissimilar: whole DNA re-association values were 25% for the macroarray method, but in microarray CGH analysis 51% of the L. johnsonii NCC533 genes were conserved in L. taiwanensis. In this latter experi- ment, the hybridization ratios distribution of the con- served genes in L. taiwanensis is centred on -1 (Fig. 5A), which corresponds to a 50% reduced signal inten- sity. Therefore, 51% of conserved genes showing about 50% of signal intensity approximates 25% of global intensity, as observed by DNA-DNA hybridization. A number of features on L. johnsonii NCC533 genome [13] that are conserved on L. taiwanensis BL263 may contribute to the adaptation of this bacterium to its eco- logical niche. Interestingly, our combined CGH results with two L. johnsonii strains showed that L. taiwanensis BL263 is closer to the gut isolate L. johnsonii BL261 than to the blood isolate L. johnsonii ATCC 33200T. The L. johnsonii NCC533 genome encodes 16 putative phosphoenolpyruvate: sugar phosphotransferase systems (PTSs) and several of them, including the ones anno- tated for fructose, glucose and cellobiose transport, are specifically induced in the gastrointestinal tract (GIT) [14]. CGH results showed that four PTSs, hypothetically involved in transport and metabolism of fructose, cello- biose, trehalose and sucrose are conserved in L. taiwa- nensis BL263. These predictions are supported by the API 50CH results showed for L. taiwanensis BL263 (Table 3). A putative maltose/maltodextrin utilization gene cluster, which includes a gene coding for a putative neopullulanase/maltogenic a-amylase, is conserved in L. taiwanensis BL263, suggesting that this strain may use starch-degraded products. Other enzymes that may con- tribute to the survival/persistence of L. taiwanensis BL263 in the GIT are bile salt hydrolases (BSH). These enzymes have been almost exclusively found in bacterial species associated with the GIT and its role has been largely discussed [see review [50]. L. taiwanensis BL263 exhibited taurocholic and taurodeoxycholic acid decon- jugation activity (L. A. Sarmiento-Rubiano and M. J. Yebra, unpublished results). This is in agreement with