Analysis of Sporulation in Bacillus cereus Biovar anthracis Which Contains an Insertion in the Gene for the Sporulation Factor σK

Abstract

Bacillus cereus biovar anthracis (Bcbva) is an untypical pathogen causing a fatal anthrax-like disease in a variety of wildlife species in African rainforest areas. In contrast to Bacillus anthracis and most species of the B. cereus group, all strains of the Bcbva cluster contain a 22 kb insertion in the sigK gene which encodes the essential late sporulation sigma factor σK. This insertion is excised during sporulation in a site-specific recombination process resulting in an intact sigK gene and a circular molecule. The sporulation kinetics of two strains each of Bcbva and B. anthracis were compared by the expression analysis of eight sporulation-associated genes, including sigK, using reverse transcriptase quantitative real-time PCR. In addition, morphological sporulation stages were analyzed and quantified by electron microscopy. Our results indicated that the necessary excision of the insertion in Bcbva neither delayed nor inhibited its sporulation. In two spontaneous mutants of Bcbva, the excision of the sigK insertion and sporulation were impeded due to mutations in the spo0A and spoVG regulator genes, respectively. The spo0A frameshift mutation was overcome by intragenic suppression in a revertant which was able to sporulate normally, despite an M171S amino acid exchange in the global regulator Spo0A. A screening of the NCBI database identified further strains of the B. cereus group which possess unrelated insertions in the sigK gene, and two strains containing almost identical insertions at the same gene position. Some of the sigK insertions encode putative prophages, whereas the Bcbva insertion encoded a type I restriction–modification system. The function of these insertions and if they are possibly essential for sporulation remains to be assessed.