Alcaraz et al. BMC Genomics 2010, 11:332 http://www.biomedcentral.com/1471-2164/11/332 aspartate phosphatases are either not conserved or exhi- bit changes that make them difficult to recognize by sequence similarity. The only coat gene present in the core is cotE, which is involved in the outer layer of the spore coat [68]. Germination genes are also found within the core. GerD is involved in the early germina- tion response to amino acids such as L-alanine and L-asparagine [69], whereas GerM has been hypothe- sized to bind to peptidoglycan [70]. The spore matura- tion proteins SpmA and SpmB are also part of the sporulation core and are involved in spore dehydration. These proteins provide spore resistance to moist heat, as was shown in Clostridium perfringens [71,72]. Dipi- colinic acid has been recognized as a core molecule that gives the spore radiation resistance [51]. The two units of dipicolinate synthase, spoVFA and spoVFB, are also present in the sporulation core. SspI appears to be the only universal SASP. An interesting observation of group B is the seemingly generalized gene loss in the aquatic/halophile Bacillus. This finding is not seen in the B. subtilis and B. anthra- cis-cereus-thuringiensis groups. Diverse sporulation genes, germination genes, coat and small acid proteins are the predominant categories absent from this group. Variation of conservancy decreases with several SASPs, such as SspB, SspD, SspH, and SspF. This suggests that despite these proteins being very abundant in the spore, they are diverse and exhibit redundancy in places where they are bound to DNA (3-6% of the total spore protein) [51]. Therefore, the absence of some genes may be com- pensated by the presence of others. The phosphatases RapF and RapI are involved in the phosphorelay and are poorly conserved across all Bacil- lus. The low conservation of phosphatase-related pro- teins is more prominent in groups C and D (RapA, B, C, D, E, G, and K;and PhrA, C, E, F, and G), suggesting that phosphorelay cascades may almost be strain-speci- fic. A similar situation is observed for several coat pro- teins that first appear in group B (CotB, D, F, H, JA, JB, JC, SA, and Z) and for germination proteins (GerE, PA, PC, PD, and PE). The cereus and subtilis group are similar in the A and B gene categories, but lack almost all genes described in categories C and D. Our results are consistent with pre- vious studies [6-9,66] on comparative genomics within the B. cereus group that show that the main differences among the groups reside in HGT mobilized elements [31,73] and not in the core genome. Our findings suggest that the sporulation sensor kinases, coat proteins, and SASPs of the various Bacillus species have evolved to be responsive to signals specific for particular environments. Even two very close strains, such as B. licheniformis DSM13 and ATCC 14580, differ Page 12 of 17 in the preservation of some genes, including sspJ and sspL. Discussion The increasing number of sequenced microbial genomes provides an ideal opportunity to re-evaluate approaches in understanding phylogenetic and functional differences among bacteria. Much of the understanding of microbial biodiversity has been studied by comparison of rRNA sequences. However, this approach has clear limitations, such as arbitrary cut-off values for sequence identity and the inability to resolve relationships between closely related groups [74]. For very close relatives, MLST and similar approaches can be used successfully to describe intra-diversity and resolve discrete clusters [75]. Both rRNA and MLST approaches use genes from the core genome [17], and in our analysis, we expanded the gene set for use in phylogenetic reconstruction in order to greatly increase our ability to resolve clusters. Testing inter-species phylogenetic cohesion of a group, such as the Bacillus, and taking advantage of 814 concatenated core genes, allowed us to obtain a robust phylogenetic reconstruction of the inner clusters that failed when comparing the same species with rRNA or universally conserved genes. Data obtained using metrics of taxa distance, such as GSS, for whole genome pairwise com- parisons that made use of the entire shared genetic information agreed with the cluster resolution. Of note, we described the aquatic Bacillus as a new group. We predict that this group will quickly gain importance given the numerous examples of aquatic representatives that have been identified through 16S rRNA gene sequencing in multiple environmental samplings. Today, given the constant improvement in cost/benefit of massive sequencing technologies, it is possible to think in whole genome shotgun (WGS) approaches to try to answer global internal group diversity. Although it is not yet the cheapest/feasible option for the regular laboratory, we suggest that the rapidly growing micro- bial genome database can be used to regularly and auto- matically build core genomes at intra-species, genus, family, order, and other taxonomic levels. This approach will aid in defining the functions of the genes behind different taxonomic ranks and provide the whole research community with specific genetic markers to perform detailed ecological and evolutionary analysis. Similar to the RBH approach, researchers can benefit from WGS projects in progress to define core genomes, since the number of unfinished genomes (1,777) almost doubles the amount of complete genomes (892). In this study, we used data from the sequenced genome of Bacillus sp. m3-13 with 22-fold coverage, the previously sequenced genome of B. coahuilensis [16], and the WGS