Alcaraz et al. BMC Genomics 2010, 11:332 http://www.biomedcentral.com/1471-2164/11/332 groups of genes for processes such as sporulation and competence underlines the natural variability of the genus and emphasizes the need for further exploration of these differences. Methods Reciprocal Best Hits (RBH) We undertook an RBH approach as previously described [80,81]in order to identify all of the orthologous pairs among 20 complete genomes of the Bacillus. Predicted, translated gene models for each genome were used and required coverage of 70% of both genes with an e-value of 10-5 at an effective database size of 107. Core genome All pairwise RBH shared genes were compared and the common dataset of shared genes amongst all strains was defined as the core genome. We use the COG classifica- tion schema [41] to classify gene functions. The acro- nyms utilized in this study for the B. subtilis genes are the most widely used and we therefore used them for the entire core genome. Pan-genome Here, we define the pan-genome as the total set of genes within the 20 Bacillus genomes, including plasmid and extra-chromosomal elements (when available). A total set of 19,043 homologous families were comprised of a total of 155,747 genes, as identified by RBH, with a cut-off e- value of 10-5[80]. COG classification was conducted with each representative from the homologous families. Evolutionary Analysis To compute similarity between genomes using RBH pair- wise information, we took advantage of our RBH bit-score results using a Genomic Similarity Score (GSS) [37]that had a range from 0 to 1.A maximum score was obtained when two compared proteomes were identical and a GSS distance matrix was used to build a Neighbor-Joining tree. Alignments for 16S rRNA were done using MUSCLE [82] and the phylogenetic tree was created using PhyML [83]. The 20 universally conserved gene COG phylogeny was constructed as previously described[16,40]. Each translated gene of the core genome was aligned and concatenated using ClustalW-MPI [84]. The phylogenetic tree was con- structed using PhyML [83] with the JTT substitution model as has been done before with translated and conca- tenated sequences [40], given the alignment length (308,782 aa). The gamma distributed rates and 1,000 boot- straps were estimated from the dataset. Statistical analyses All statistical analyses were conducted on R (2.6.2) [85]. Heat maps were generated with the gplots library of R [86]. Page 14 of 17 Sporulation genes The genes were defined by the Kyoto Encyclopedia of Genes and Genomes KEGG BRITE Hierarchies [65]. RBH analysis, as previously described, was conducted with each KO gene annotated for B. subtilis in the BRITE hierarchy. RBH results were then parsed into presence/absence to map the conservancy of sporulation genes across the Bacillus. Bacillus sp. m3-13 isolation The strain was isolated from a desiccation lagoon in the Churince system located in Cuatro Cienegas in Coa- huila, Mexico (26°50.830’N, 102°09.335’W) by Rene Cer- ritos as described for B. coahuilensis [26]. Genome sequencing, assembly, and annotation Bacillus sp. m3-13 was sequenced using the 454 FLX system (454 Life Sciences) with a 20-fold coverage. Assembly was done with Newbler, Celera Assembler [87], and Phrap [88] resulting in 50 contigs and a total of 4,137,575 bp assembled. Gene prediction was done using Glimmer v3.0 [89] and GeneMark.hmm [90]. Automated annotation was performed with BASys [91] checked and proofed manually. The Whole Genome Shotgun (WGS) Bacillus sp. m3-13 project has been deposited at DDBJ/EMBL/GenBank under the project accession [ACPC00000000]. The ver- sion described in this paper (ACPC01000000) is the first version. Additional file 1: Core genome concatenate alignment in FASTA format. Additional file 2: Supplementary tables. Table S1. Current Bacillus sp. genome projects. Table S2. GI numbers, gene acronyms, and annotation for the 814 core genome genes. Table S3. Sporulation core genes. Abbreviations CGH: Comparative Genome Hybridization; COG: Cluster of Orthologous Groups; GSS: Genome Similarity Score; HGT: Horizontal Gene Transfer; KEGG: Kyoto Encyclopedia of Genes and Genomes; ML: Maximum likelihood; MLSA: Multi Locus Sequence Analysis; MLST: Multi Locus Sequence Typing; RBH: Reciprocal Blast Hits; SASP: Small Acid Soluble Protein; WGS: Whole Genome Shotgun. Acknowledgements This work was supported by a CONACyT-SEP grant 57507to VS, VS and LEE worked on this manuscript during their sabbatical at UCI with a DGAPA and an UCMExus grant respectively, Howard Hughes Medical Institute Grant 55005946 (to LHE), and a research grant from Cinvestav to GO. LDA was a recipient of a fellowship from CONACyT. We acknowledge Beatriz Jiménez and Gustavo Hernandez for sequencing assistance at Langebio-Cinvestav. We are grateful for the work of Rene Cerritos in the isolation of Bacillus sp. m3-13, Michael Travisano (U. Minnesota), and Alex Mira (CSISP, Spain) for their comments on the manuscript. Author details 1Departamento de Ingeniería Genética, Centro de Investigación y de Estudios Avanzados del I.P.N. Campus Guanajuato, AP 629 Irapuato,