Alcaraz et al. BMC Genomics 2010, 11:332 http://www.biomedcentral.com/1471-2164/11/332 licheniformis-pumilus, B. anthracis-thuringiensis-cereus, and a novel group, Bacillus sp. NRRLB-14911-coahui- lensis-m3-13, The strain Geobacillus kaustophilusis within the major groups in its own leaf but deep into the Bacillus, whereas Oceanobacillus iheyensis is basally located outside the major groups. A sister group is formed by B. halodurans-clausii falling on the edge of the main Bacillus groups. Another distinctive feature of the core genome phylogeny, as compared with the tradi- tional 16S rRNA and the universally conserved COG phylogenies, is its robustness that is reflected by the generally high bootstrap replica values. Still, this tree is not fully resolved, as shown by the position of the G. kauustophilus leaf. We further measured the distance between species with a Genomic Similarity Score (GSS) [37]. This mea- surement is based on the sum of bit-scores of shared orthologs, detected as RBH, and normalized against the sum of bit-scores of the compared genes against them- selves (self-bit-scores). It has a range from 0 to 1 with a maximum reached when two compared proteomes are identical. There is an average of 2,539 ± 561 shared genes between different Bacillus species and an average GSS of 0.5637 ± 0.0039. A distance matrix of the GSS scores of the shared orthologs for the 20 Bacillus groups was plotted in a Neighbor-Joining tree to evaluate the resolution of a pairwise, shared orthologs index as an evolutionary distance tool (Figure 2B). All of the inner groups shown in the core genome phylogeny appear in this clustering, although inner sister groups are clustered showing the ambiguity of the deep nodes. GSS can therefore be used as a complementary approach, as an index to clarify relationships among organisms using whole pairwise shared orthologs. GSS takes into account the maximum comparable pairwise genome shared, in contrast to the regular phylogenetic reconstruction, which can only compare a common dataset of homolo- gous genes. A 16S rRNA Maximum Likelihood phylogeny of the selected strains is shown in Figure 2C. Major groups, such as the B. cereus and B. subtilis’ groups, are main- tained with low bootstrap support (< 50), while the aquatic Bacillus group is paraphyletic within this phylo- geny. The use of 20 conserved concatenated Cluster of Orthologous Groups (COGs) as described in [38-40] for Bacillus (5,299 amino acid alignment length) to perform an ML phylogeny (Figure 2D) resulted in a tree that shows also a paraphyletic aquatic Bacillus group and lower bootstrap support when comparing inner groups like B. cereus group to the core genome phylogeny. We noted that the 16S phylogeny places G. kaustophilus as internal clade within the Bacillus genus, but with con- siderable substitution rates shown in the large branch length, while B. clausii and O. iheyensis are placed in Page 5 of 17 the same clade though with very low bootstrap support; in contrast, the conserved universal COGs (uCOGs) phylogeny places O. iheyensis close to a branch formed by B. halodurans and B. clausii. Neither the 16S rRNA nor the uCOGs resolve the internal clades with as much support as the core genome phylogeny. Functional composition of the core genome of sequenced Bacillus To understand the functional roles of the genes that constitute the core and pan-genome, we took advantage of the COGs functional classification [41]. This is a clas- sification system based on orthologous relations among genes. We used the COGs to map the core genome, pan-genome, and each of the four groups defined in the core phylogeny (Figure 3): i) B. anthracis-cereus-thurin- giensis, ii) B. subtilis-licheniformis-pumilus, iii) B. clau- sii-halodurans, iv) B. coahuilensis-m313-nrrlb14911, v) G. kaustophilus-O. iheyensis. These last two do not form a phylogenetically related group, however, for the func- tional analysis we chose to group them given the simila- rities in the environments from which these strains were recovered. Using this strategy, we grouped Bacillus representatives that had shared evolutionary and ecolo- gical features and then searched for over/under repre- sented gene functions within each group to underscore relevant gene functions for each evolutionary group. We observed significant differences in the COGs categories of core-pan-genome-groups (Chi square = 753.72; d.f. = 126; p-value < 2.2e-16). Figure 3 shows a heat plot map of the ratio of normalized genes to total gene content for each COG category. Several expected features arose from this analysis, such as the predominance of genes in COG category J (translation and ribosomal structure genes; core = 0.11/average = 0.06) within the core. This was expected as most of the conserved universal COGs are contained within this category [40]. Their conserva- tion across taxa and functional restrains are precisely why they are chosen as gold standards for phylogenetics in addition to 16S rRNA genes. Other over-represented categories, although only within the core, are C (energy production and conversion; core = 0.07/average = 0.06) and L (replication, recombination, and repair; core = 0.07/average = 0.05). Highly represented categories included COGs E (amino acid transport and metabo- lism; core = 0.09/average = 0.1), R (function unknown; core = 0.11/average = 0.12), and S (poorly characterized genes; core = 0.06/average = 0.09) that were not only in the core but also in the pan-genome and in all of the Bacillus groups. Interestingly, several core genes of the COG R and S are conserved across the entire Bacillus with some clearly being involved in the sporulation pro- cess, including spmA, spmB, yaaT, spoIVFB, spoVB, spoVR, and others (See Additional file 2: Table S2).