Ole Andreas Økstad

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.

The positions of DNA regions close to the chromosome replication origin and terminus in growing cells of Escherichia coli have been visualized simultaneously, using new widely applicable reagents. Furthermore, the positions of these regions with respect to a replication factory-associated protein have been analysed. Time-lapse analysis has allowed the fate of origins, termini and the FtsZ ring to be followed in a lineage-specific manner during the formation of microcolonies. These experiments reveal new aspects of the E. coli cell cycle and demonstrate that the replication terminus region is frequently located asymmetrically, on the new pole side of mid-cell. This asymmetry could provide a mechanism by which the chromosome segregation protein FtsK, located at the division septum, can act directionally to ensure that the septal region is free of DNA before the completion of cell division.

Members of the Bacillus cereus group (B. anthracis, B. cereus, B. mycoides and B. thuringiensis) are well-known pathogens of mammals (B. anthracis and B. cereus) and insects (B. thuringiensis). The specific diseases they cause depend on their capacity to produce specific virulence factors, such as the lethal toxin of B. anthracis and the Cry toxins of B. thuringiensis. However, these Bacillus spp. also produce a variety of proteins, such as phospholipases C, which are known to act as virulence factors in various pathogenic bacteria. Few genes encoding these virulence factors have been characterized in pathogenic Bacillus spp. and little is known about the regulation of their expression. We had previously reported that in B. thuringiensis expression of the phosphatidylinositol-specific phospholipase C gene is regulated by the transcriptional activator PlcR. Here we report the identification of several extracellular virulence factor genes by the virtue of their PlcR-regulated expression. These PlcR-regulated genes encode degradative enzymes, cell-surface proteins and enterotoxins. The PlcR-regulated genes are widely dispersed on the chromosome and therefore do not constitute a pathogenic island. Analysis of the promoter region of the PlcR-regulated genes revealed the presence of a highly conserved palindromic region (TATGNAN4TNCATA), which is presumably the specific recognition target for PlcR activation. We found that the plcR gene is also present in and probably restricted to all the members of the B. cereus group. However, although the polypeptide encoded by the B. cereus PlcR gene is functionally equivalent to the B. thuringiensis regulator, the polypeptide encoded by the B. anthracis gene is truncated and not active as a transcriptional activator. PlcR is the first example described of a pleiotropic regulator involved in the control of extracellular virulence factor expression in pathogenic Bacillus spp. These results have implications for the taxonomic relationships among members of the B. cereus group, the virulence properties of these bacteria and the safety of B. thuringiensis-based biopesticides.

PlcR is a Bacillus cereus transcriptional regulator, which activates gene expression by binding to a nucleotidic sequence called the 'PlcR box'. To build a list of all genes included in the PlcR regulon, a consensus sequence was identified by directed mutagenesis. The reference strain ATCC14579 sequenced genome was searched for occurrences of this consensus sequence to produce a virtual regulon. PlcR control of these genes was confirmed by comparing gene expression in the reference strain and its isogenic Delta-plcR strain using DNA microarrays, lacZ fusions and proteomics methods. The resulting list included 45 genes controlled by 28 PlcR boxes. Forty of the PlcR controlled proteins were exported, of which 22 were secreted in the extracellular medium and 18 were bound or attached to cell wall structures (membrane or peptidoglycan layer). The functions of these proteins were related to food supply (phospholipases, proteases, toxins), cell protection (bacteriocins, toxins, transporters, cell wall biogenesis) and environment-sensing (two-component sensors, chemotaxis proteins, GGDEF family regulators). Four genes coded for cytoplasmic regulators. The PlcR regulon appears to integrate a large range of environmental signals, including food deprivation and self cell-density, and regulate the transcription of genes designed to overcome obstacles that hinder B. cereus growth within the host: food supply, host barriers, host immune defenses, and competition with other bacterial species. PlcR appears to be a key component in the efficient adaptation of B. cereus to its host environment.