Circular representation of the B. megaterium chromosome. Circles (numbered 1 to 13, from outer to inner circle): circles 1 and 2, predicted open reading frames encoded on the QM B1551 plus (circle 1, blue) and minus (circle 2, red) strands; circle 3, predominant genes in conserved Bacillus region, showing tRNAs (brown), rRNA clusters (black), and sporulation and germination (dark green); circle 4, GC skew; circle 5, chromosomal regions of interest, including gas vesicle operons I and II (gvp; magenta), vitamin B₁₂ operons I and II (B12; brown), flagellar operons (fla; aqua), RecA genes (rec; black), arsenate resistance operons (ars; green), β-galactosidase genes (lac; orange), ethanolamine utiliazation (eut; brown), histidine biosynthesis (his; gray), and competence operons CEFG (com; purple); circles 6 and 7, comparative analysis of the QM B1551 chromosome identities (circle 6) and proteome (circle 7) to DSM319; circles 8 and 9, nonrandom distribution of genomic islands and islets in QM B1551 (circle 8) and DSM319 (circle 9); circles 10 and 11, chromosomal genes with QM B1551 plasmid-borne orthologs in QM B1551 (circle 10) and DSM319 (circle 11); circle 12, chi-square values to show GC deviations; circle 13, region of conserved synteny (red) neighboring the ori identified by the comparative study of genome architectures in related Bacilli (see Fig. S4 in the supplemental material), with the remainder of the genome colored green.

Circular representation of the B. megaterium plasmids pBM500, pBM600, and pBM700. Circles 1 and 2, predicted open reading frames encoded on the QM B1551 plus (circle 1, blue) and minus (circle 2, red) strands with the replication gene dnaA (green); circle 3 (circle 4 in pBM700 representation), GC skew; circle 4 (circle 5 in pBM700 representation), plasmid genes with chromosomal orthologs (green) in strains QM B1551 and DSM319. The two innermost circles are the chi square deviations from the average trinucleotide distribution and the results of a comparative analysis of each plasmid with the other seven indigenous plasmids of QM B1551, showing nucleotide identities (Fig. 1). (A) Regions and genes of interest in pBM700. Circle 3, tRNA-Cys and mobile genetic elements (black); circle 6, regulatory functions, including transcription regulators (red), two-component sensor histidine kinase systems (TCS; brown), adaptive response (light blue), and sporulation (orange); circle 7, metabolic features, including carbohydrate metabolism (brown) and two peptidases (gold); circle 8, several enzymes of unknown specificity (blue) and for antibiotic resistance (yellow). (B) Regions and genes of interest in pBM600. The origin amplicon is shown in black, with the identified replication gene in green. Circle 5, mobile genetic elements (red). The IS3 transposase is adjacent to an aggrecan core (proteoglycan) protein that is further distinguished by a deviating GC content, which suggests horizontal acquisition; circle 6, carbohydrate metabolism (brown) and sporulation genes (orange). (C) Regions and genes of interest in pBM500. Circle 5, regulatory functions, including transcription regulators (red) and three sigma factors (light blue); circle 6, metabolism features, including carbohydrate metabolism (brown) and fatty acid biosynthesis (magenta); circle 7, sporulation (orange) and erm(B) macrolide resistance (yellow); circle 8, regions of deviating GC content, membrane protein (blue), collagen-like protein (gold), and cytochrome P450 (green).

Genome synteny of B. megaterium. The genomes of strains QM B1551 and DSM319 are mostly colinear, but the distribution of isolate-specific genes is not random: gene insertions are rare in a 2-Mbp region around the origin of replication (ori). Each protein of the reference genome plotted on the x axis was used as the query sequence with BLASTP against the y axis subject genome. The N-terminal coordinates of the best hit proteins were plotted. The colors represent the level of similarity of the match, expressed by the BLAST score ratio (48).

Functional classification of the isolate-specific gene inventory of B. megaterium strains QM B1551 and DSM319 compared to the DSM319 chromosome. Unique genes in strains QM B1551 (A) and DSM319 (B) are indicated. (C) Functional classes of total chromosomal genes of DSM319.

Genome features of B. megaterium QM B1551. (A) Gas vesicle-forming proteins. The B. megaterium QM B1551 chromosome features two unique gas vesicle protein-forming loci (gvp) not found in any of the related species or genera. DSM319 has only operon II. The scale (in bp) indicates the genomic location of the gas vesicle loci, which are composed of 8 and 14 genes (gvpBRFGLSKJ and gvpAPQBRNFGLSKJTU). A possible araC regulator is also present. The loci are syntenic and share 8 homologous genes, with the notable exception of gvpN. The absence of colocalizing mobile genetic loci and average GC content suggest a duplication event in strain QM B1551 rather than acquisition via horizontal gene transfer. Homologous genes have been given identical colors and shaded in gray. (B) Vitamin B₁₂ biosynthesis. Both B. megaterium strains encode three phylogenetically unrelated loci that are associated with vitamin B₁₂ biosynthesis; the two multigene loci are shown here. The scale (in bp) indicates the genomic location of these loci in strain QM B1551. Regulatory genes are colored magenta. (C) Vitamin B₁₂-dependent loci. Several enzymes with dependencies on vitamin B₁₂ have been identified in the B. megaterium genome, such as ethanolamine ammonia-lyase (EutBC) and methylmalonyl-CoA mutase (MutAB). The genes eutCBA are also found at Mb 2.52 to 2.53 in both strains. (D) Flagellar biosynthesis. The two flagellar biosynthesis clusters, fla/che and fla/com/deg in QM B1551, are shown, with the upstream regulatory genes colored magenta. The fla/com/deg cluster contains flagellar, competence, and the extracellular enzyme regulatory genes degU and degS.

Growth of B. megaterium strains DSM319 (blue) and QM B1551 (red) on different nutrients. Strain-specific gene differences deduced from the analysis of the isolate-specific genes were validated by growth on different nutrients: (A) glucarate, (B) galactitol, (C) pullulan, (D) sorbitol. The optical density was recorded over a time course of up to 13 h. The experimental data were fitted to logistic or exponential growth curves.

Oxygen-independent biosynthesis of adenosylcobalamin. B. megaterium is able to synthesize vitamin B₁₂ in the absence of oxygen due to the presence of the signature gene cbiX, which results in insertion of cobalt at an early stage, in contrast to the oxygen-dependent pathway.

Distinct genomic locations for different functional classes of genes in B. megaterium QM B1551. The number of genes classified by function that were encoded within the conserved syntenic region flanking the ori (red) was compared to the number present in the remainder of the chromosome (green), according to Fig. 1, and to the total chromosomal number (gray). Those functions concentrated in the conserved region that were compared to the remainder of the functions are lettered in red.

Pan-genome analysis and genomic plasticity in the Bacillus group of organisms. (A) Core genes. For each reported number of genomes (n), the circles represent the number of genes in common in different randomly chosen combinations of Bacillus species, with a sampling size of 1,000. Diamonds show the median values for each distribution. The curve represents the exponential regression of the least squares fit of the function F_core(n) = κ_c exp[−n/τ_c] + tg_c(θ), based on the medians of the distribution. The extrapolated core genome size is shown as a horizontal dashed red line. (B) Gene discovery. By using the same sampling method as for panel A, the number of new genes found was plotted for increasing values of n. A power law regression for new genes discovered was fitted to the means of new gene counts (diamonds) for each value of n. The curve is the least squares fit of the exponential decay equation F_new(n) = κ_n exp[−n/τ_n] + tg_n(θ), based on the means of the distribution. The value of tg_n(θ) shown in this figure represents the number of new genes asymptotically predicted for further genome sequencing. (C) The Bacillus pan-genome. The total numbers of genes found according to the pan-genome analyses are shown for increasing values of the number (n) of Bacillus genomes sequenced, using medians and an exponential fit. Red diamonds indicate the means of the distributions. The dashed line represents the asymptotic prediction of the total number of genes expected to be found in the Bacillus pan-genome.