Two small c-type cytochromes affect virulence gene expression in Bacillus anthracis

Regulated expression of the genes for anthrax toxin proteins is essential for the virulence of the pathogenic bacterium Bacillus anthracis . Induction of toxin gene expression depends on several factors, including temperature, bicarbonate levels, and metabolic state of the cell. To identify factors that regulate toxin expression, transposon mutagenesis was performed under non-inducing conditions and mutants were isolated that untimely expressed high levels of toxin. A number of these mutations clustered in the haem biosynthetic and cytochrome c maturation pathways. Genetic analysis revealed that two haem-dependent, small c -type cytochromes, CccA and CccB, located on the extracellular surface of the cytoplasmic membrane, regulate toxin gene expression by affecting the expression of the master virulence regulator AtxA. Deregulated AtxA expression in early exponential phase resulted in increased expression of toxin genes in response to loss of the CccA-CccB signalling pathway. This is the first function identified for these two small c -type cytochromes of Bacillus species. Extension of the transposon screen identified a previously uncharacterized protein, BAS3568, highly conserved across many bacterial and archeal species, as involved in cytochrome c activity and virulence regulation. These findings are significant not only to virulence regulation in B. anthracis , but also to analysis of virulence regulation in many pathogenic bacteria and to the study of cytochrome c activity in Gram-positive bacteria.     

Regulation of glycerol kinase by enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system.

Wild-type glycerol kinase of Escherichia coli is inhibited by both nonphosphorylated enzyme IIIGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system and fructose 1,6-diphosphate. Mutant glycerol kinase, resistant to inhibition by fructose 1,6-diphosphate, was much less sensitive to inhibition by enzyme IIIGlc. The difference between the wild-type and mutant enzymes was even greater when inhibition was measured in the presence of both enzyme IIIGlc and fructose 1,6-diphosphate. The binding of enzyme IIIGlc to glycerol kinase required the presence of the substrate glycerol.     

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.     

Molecular cloning and expression of the Bacillus anthracis edema factor toxin gene: a calmodulin-dependent adenylate cyclase.

The Bacillus anthracis exotoxin is composed of a lethal factor, a protective antigen, and an edema factor (EF). EF is a calmodulin-dependent adenylate cyclase which elevates cyclic AMP levels within cells. The entire EF gene (cya) has been cloned in Escherichia coli, but EF gene expression by its own B. anthracis promoter could not be detected in E. coli. However, when the EF gene was placed downstream from the lac or the T7 promoter, enzymatically active EF was produced. The EF gene, like the protective antigen (pag) and lethal factor (lef) genes, was present on the large B. anthracis toxin plasmid pXO1.     

Cereulide synthetase gene cluster from emetic Bacillus cereus: Structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1

Background  

Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Recently, it has been shown that this toxin is produced by a nonribosomal peptide synthetase (NRPS), but its exact genetic organization and biochemical synthesis is unknown.     

Coordinate regulation of adenylate cyclase and carbohydrate permeases by the phosphoenolpyruvate:sugar phosphotransferase system in Salmonella typhimurium.

Adenylate cyclase (EC 4.6.1.1) and several carbohydrate permeases are inhibited by D-glucose and other substrates of the phosphoenolpyruvate:sugar phosphotransferase system. These activities are coordinately altered by sugar substrates of the phosphotransferase system in a variety of bacterial strains which contain differing cellular levels of the protein components of the phosphotransferase system: Enzyme I, a small heat-stable protein, and Enzyme II. It is suggested that the activities of adenylate cyclase and the permease proteins are subject to allosteric regulation and that the allosteric effector is a regulatory protein which can be phosphorylated by the phosphotransferase system.