Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group

Abstract

BackgroundThe Bacillus cereus sensu lato group consists of six species (B. anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis, and B. weihenstephanensis). While classical microbial taxonomy proposed these organisms as distinct species, newer molecular phylogenies and comparative genome sequencing suggests that these organisms should be classified as a single species (thus, we will refer to these organisms collectively as the Bc species-group). How do we account for the underlying similarity of these phenotypically diverse microbes? It has been established for some time that the most rapidly evolving and evolutionarily flexible portions of the bacterial genome are regulatory sequences and transcriptional networks. Other studies have suggested that the sigma factor gene family of these organisms has diverged and expanded significantly relative to their ancestors; sigma factors are those portions of the bacterial transcriptional apparatus that control RNA polymerase recognition for promoter selection. Thus, examining sigma factor divergence in these organisms would concurrently examine both regulatory sequences and transcriptional networks important for divergence. We began this examination by comparison to the sigma factor gene set of B. subtilis.ResultsPhylogenetic analysis of the Bc species-group utilizing 157 single-copy genes of the family Bacillaceae suggests that several taxonomic revisions of the genus Bacillus should be considered. Within the Bc species-group there is little indication that the currently recognized species form related sub-groupings, suggesting that they are members of the same species. The sigma factor gene family encoded by the Bc species-group appears to be the result of a dynamic gene-duplication and gene-loss process that in previous analyses underestimated the true heterogeneity of the sigma factor content in the Bc species-group.ConclusionsExpansion of the sigma factor gene family appears to have preferentially occurred within the extracytoplasmic function (ECF) sigma factor genes, while the primary alternative (PA) sigma factor genes are, in general, highly conserved with those found in B. subtilis. Divergence of the sigma-controlled transcriptional regulons among various members of the Bc species-group likely has a major role in explaining the diversity of phenotypic characteristics seen in members of the Bc species-group.

Iron Regulates Expression of Bacillus cereus Hemolysin II via Global Regulator Fur

“…Target promoter DNA corresponding to the hlyII promoter region was PCR amplified using promoter-specific oligonucleotides hlyIIp-for and hlyIIp-rev ( Table 1). The fragments were 32 P labeled with T4 DNA kinase and purified using the Qiagen gel extraction kit. Binding reaction mixtures (20 l) contained 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 20 ng/l chicken erythrocyte DNA, 7.5% glycerol, 1 or 2 nM DNA, Fur-His 6 (various concentrations), and 0.1 mg/ml xylene cyanol.…”

“…The reaction mixture was incubated for 10 min at 37°C. Transcription complexes were used in footprinting or were supplemented with 500 M GTP, 50 M ATP and CTP, and [␣- 32 P]UTP to analyze in vitro transcription products. Transcription reactions were allowed to proceed for 10 min at 37°C before termination by addition of formamide buffer.…”

“…Data were fit to the Hill equation in SigmaPlot. For footprinting analysis, hlyII DNA fragment was 32 P end labeled at the nontemplate strand. Samples containing hlyII promoter complexes were footprinted with DNase I (33).…”

“…Three important species that belong to the B. cereus group are entomopathogenic Bacillus thuringiensis, deadly Bacillus anthracis, and B. cereus sensu stricto, the last of which has widely varying properties that determine its role both as a harmless, spore-forming soil microorganism and as a causative agent of food poisoning and endophthalmitis (19,38). These three species are closely related genetically and are now classified as a single species (22,32). B. cereus is considered to be an emerging pathogen (8), warranting a detailed investigation of the mechanisms and regulation of B. cereus toxin production.…”

Complete genome sequence of the nematicidal Bacillus thuringiensis MYBT18247

“…Because of the highly conserved 16S rRNAgenes, the classical 16S phylogeny of Bcsl strains is inconclusive. Thus, a combination of 16S and a seven gene multi-locus sequence typing scheme have been used to establish taxonomic relationships within species of the Bcslgroup [35,36]. Comparative genomics of the cry-gene loci has revealed remarkable proximity to elements of genome plasticity such as plasmids, transposons, insertion elements and prophages [2,[37][38][39].…”