Evolutionary history and functional characterization of three large genes involved in sporulation in Bacillus cereus group bacteria

Abstract

The Bacillus cereus group of bacteria is a group of closely related species that are of medical and economic relevance, including B. anthracis, B. cereus, and B. thuringiensis. Bacteria from the Bacillus cereus group encode three large, highly conserved genes of unknown function (named crdA, crdB, and crdC) that are composed of 16 to 35 copies of a repeated domain of 132 amino acids at the protein level. Bioinformatic analysis revealed that there is a phylogenetic bias in the genomic distribution of these genes and that strains harboring all three large genes mainly belong to cluster III of the B. cereus group phylogenetic tree. The evolutionary history of the three large genes implicates gain, loss, duplication, internal deletion, and lateral transfer. Furthermore, we show that the transcription of previously identified antisense open reading frames in crdB is simultaneously regulated with its host gene throughout the life cycle in vitro, with the highest expression being at the onset of sporulation. In B. anthracis, different combinations of double- and triple-knockout mutants of the three large genes displayed slower and less efficient sporulation processes than the parental strain. Altogether, the functional studies suggest an involvement of these three large genes in the sporulation process.

Fig. 1.

Chromosomal organization and gene structure of crdA, crdB, and crdC. The sense genes crdA (A) (chromosomal location is shown with ×2 zoom), crdB (B), and crdC (C) are shown as blue arrows in the 5′ to 3′ direction. The sequences encoding the repeated domains (132 aa) are illustrated as black-outlined boxes. In crdA, discontinuity was introduced (12 domains were left out); crdA (BT9727_1474) is flanked by BT9727_1471 (hypothetical protein) and BT9727_1475 (acetyltransferase), crdB (BT9727_3414) is flanked by BT9727_3413 (hypothetical protein) and BT9727_3416 (cytochrome c defective protein), and crdC is flanked by BT9727_3304 (acetyltransferase) and BT9727_3306 (short-chain dehydrogenase). The locations of the antisense ORFs X and Y in crdB are represented as red and green arrows, respectively. A size bar for orientation is included.

Fig. 2.

Unrooted whole-genome phylogenetic tree of 85 sequenced B. cereus group strains. The names of the strains are color coded according to the presence of orthologs of the three large genes crdA, crdB, and crdC. In addition, for each isolate the presence of supplementary genes that carry one or several homologous copies of the common 132-aa domain is indicated by colored filled circles. The seven major clusters of the B. cereus group population are indicated by Roman numerals (I to VII). Although none of the 85 genomes are assigned to cluster II, the branching point of this cluster is indicated for reference with Table 1 (cluster II would emerge in the vicinity of the node circled with a dashed line, by extrapolation from the B. cereus group supertree available at the HyperCAT database [http://mlstoslo.uio.no]). The 19 B. anthracis isolates form a clonal complex and are represented here as a single lineage. The tree is based on the concatenation of the sequences conserved among the chromosomes of all strains (a total of 1,452,373 bp, with gaps removed). All nodes in the tree have a bootstrap support of >99%, on the basis of 1,000 replicates.

Fig. 3.

Venn diagram of the distribution of crdA, crdB, and crdC in 85 sequenced B. cereus group strains.

Fig. 4.

Transcriptional regulation of crdB and its antisense ORFs in MGM during the complete life cycle of B. thuringiensis subsp. konkukian 97-27. The figure shows the transcriptional regulation of crdB (A) and its antisense transcript ORF Y (B) in MGM on the basis of real-time qPCR experiments after 3 h, 5 h, 8 h, and 24 h in culture, covering the bacterium's complete life cycle. Transcriptional regulation is reported as log₂ fold change and related to the initial mRNA level at 3 h, which was set to 1. (C) Log₂ relative ratio between the sense and antisense transcripts for each time point. Error bars indicate standard deviations.