Alcaraz et al. BMC Genomics 2010, 11:332 http://www.biomedcentral.com/1471-2164/11/332 pan-genome, and in particular the functional categori- zation of the accessory genomes and their relationship or lack thereof with the environment, may provide insight into these important biological questions. There are several ways to classify this group according to biochemistry, lifestyles, and/or growth on different substrates. One classification of the Bacillus splits them into three major classes [1]: pathogenic, environmental, and those used for industrial purposes. The pathogenic class is represented by B. anthracis, B. cereus, and B. thuringiensis. Environmental Bacilli are quite diverse and include B. subtilis, B. pumilus, B. halodurans, and B. coa- huilensis. The strain B. licheniformis is a well known representative of an industrial strain [1]. This classifica- tion is useful for introducing the metabolic diversity of the genus, but it provides no guidance on a phylogenetic classification of the Bacillus for research purposes. In addition, this type of classification does not consider any aquatic Bacillus. There is need for a classification method that could take advantage of the nearly 130 genome pro- jects of the genus. With more than 108 complete and draft genome sequences available to date, Bacillus is one of the most represented genera in the genomic databases. Although there are close to 1,000 complete prokaryo- tic genome sequences to date, the group is highly biased toward pathogenic isolates [4,5]. Among 85 Bacillus genomes, 61% are devoted to the cereus-anthracis-thur- ingiensis group (See Additional file 2: Table S1). Several researchers have used this overrepresentation as an advantage to perform comparative genomic studies aimed at defining the population structure and finding genetic markers for pathovar identification [6-9]. Despite the oversampling of pathogens, genomes of Bacillus iso- lated from a wide range of environments are available, including hydrothermal vents [10], tidal flats [11], soil [12], alkaline environments [13,14], shallow marine water [15], and a shallow water column from an oligo- trophic environment [16]. The presence of Bacillus in these different environments reflects the broad meta- bolic capabilities of a widely distributed genus. In a report of the intra-species diversity of Streptococ- cus agalactiae [17], the “pan-genome” concept was defined as the sum of the core genome (comprising genes present in all analyzed strains) and the “accessory” genome (comprising all strain-specific genes) [18,19]. This concept has been expanded for comparisons at other taxonomic levels, such as family [20,21] and for defining the universal ancestor hypothetical core [22]. Most traditional markers for species identification, such as 16S rRNA genes, Comparative Genome Hybridization (CGH), and the classical measures of phenotypic similar- ity, mask the real genetic diversity since they rely mainly on core-genome genes [17,23,24]. Another approach to unveil the microbial diversity used mostly by population Page 2 of 17 geneticists and by clinical microbiologists are the Multi Locus Sequence Typing (MLST) and Multi Locus Sequence Analysis (MLSA) methods [25] relying on the analysis of internal fragments of housekeeping genes (usually 7 genes), which are useful for understanding populations dynamics, recombination, and pathogen diagnosis. Phylogenetic relationships can be obtained through more extensive genomic sampling, such as the one afforded by the genome sequences. Analyzing the whole set of conserved genes across a taxonomical level, such as the core genomes, will shed light about evolu- tionary and functional relationships among the related species. Several methods based on pairwise ortholog comparison and synteny strategies have been developed to assess the composition of core genomes [17,20,23]. In this study, we were interested in understanding the cohesion of the Bacillus genus at the genomic level by using the core and pan-genomes as the working units and taking advantage of the large dataset available. We have recently described the complete genome of Bacillus coahuilensis [16,26], which possesses one of the smallest genomes (3.35 Mb; 38% GC) reported for a free-living bacteria in the group, and have identified genes that allow this bacterium to survive in an aquatic oligo- trophic environment. We now report the genome sequence of another isolate, Bacillus sp. m3-13 with a genome size of 4.13 Mb from the same environment as B. coahuilensis. We compared these genomes to ask whether the common environment has selected for simi- lar features in the two genomes. These coincidences would be observed in their gene constitutions as cohe- sion of similar classes of metabolic genes. To obtain insight into the group’s biology we describe the relatedness within the Bacillus using whole genome information to reconstruct their evolutionary history tak- ing advantage of the dataset available from the complete and draft genomes of 20 Bacillus isolated from a wide range of environments. We compared the use of different conserved genes as well as pairwise shared genes to address local phylogenies and measure quantitatively the relatedness between species using the core genome. Ana- lysis of the functional categories of the core and pan-gen- ome revealed a clear separation between different groups and reflected the niche of the Bacillus strains. Finally, clustering of conserved/absent genes for competence and sporulation genes, distinctive processes of the Bacillus genus, showed that genetic mechanisms for sporulation are far more diverse across Bacillus than expected from studies of B. subtilis alone (Figure 1). Results Bacillus sp. m3-13 genome summary Bacillus sp. m3-13 genome was sequenced using a 454 FLX system (454 Life Sciences) with a 20-fold coverage.