SummaryExpression of genes for Bacillus anthracis toxin and capsule virulence factors are dependent upon the AtxA transcription factor. The mechanism by which AtxA regulates the transcription of its target genes is unknown. Here we report that bioinformatic analyses suggested the presence in AtxA of two PTS (phosphenolpyruvate : sugar phosphotransferase system) regulation domains (PRD) generally regulated by phosphorylation/dephosphorylation at conserved histidine residues. By means of amino acid substitutions that mimic the phosphorylated (H to D) or the unphosphorylated (H to A) state of the protein, we showed that phosphorylation of H199 of PRD1 is likely to be necessary for AtxA activation while phosphorylation of H379 in PRD2 is inhibitory to toxin gene transcription. In vivo labelling experiments with radioactive phosphate allowed us to propose that H199 and H379 are AtxA residues subject to regulated phosphorylation. In support to these notions, we also show that deletion of ptsHI, encoding the HPr intermediate and the EI enzymes of PTS, or growth in the presence of glucose affect positively and negatively, respectively, the activity of AtxA. Our results link virulence factor production in B. anthracis to carbohydrate metabolism and, for the first time, provide a mechanistic explanation for AtxA transcriptional activity.

The 11 Rap proteins of Bacillus subtilis comprise a conserved family of tetratricopeptide (TPR)-containing regulatory proteins. Their activity is inhibited by specific Phr pentapeptides produced from the product of phr genes through an export-import maturation process. We found that one of the proteins, namely RapF, is

SummaryIn bacterial differentiation, mechanisms have evolved to limit cells to a single developmental pathway. The establishment of genetic competence in Bacillus subtilis is controlled by a complex regulatory circuit that is highly interconnected with the developmental pathway for spore formation, and the two pathways appear to be mutually exclusive. Here we show by in vitro and in vivo analyses that a member of the Rap family of proteins, RapH, is activated directly by the late competence transcription factor ComK, and is capable of inhibiting both competence and sporulation. Importantly, RapH is the first member of the Rap family that demonstrates dual specificity, by dephosphorylating the Spo0F~P response regulator and inhibiting the DNA-binding activity of ComA. The protein thus acts at the stage where competence is well initiated, and prevents initiation of sporulation in competent cells as well as contributing to the escape from the competent state. A deletion of rapH induces both differentiation pathways and interferes with their temporal separation. Together, these results indicate that RapH is an integral part of a multifactorial regulatory circuit affecting the cell's decision between distinct developmental pathways.

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This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis.Bacillus anthracis, the etiological agent of anthrax, is a grampositive spore-forming organism that primarily infects ruminants but can also be highly pathogenic to other mammals, including humans. The intrinsic spore resistance to extreme stresses such as desiccation, solvents, extreme pH, temperature, UV, and ionizing radiation plays a major role in anthrax pathogenesis. Sporulation is essential for survival in the environment, and it evidently contributes to anthrax diffusion, because spores are usually present when the infection is initiated (27).The process of sporulation has been extensively studied in Bacillus subtilis and shown to be the result of a complex differentiation pathway that has its onset in a signal transduction system called phosphorelay. The phosphorelay is a more complex version of the two-component signal transduction systems, because it is composed of multiple central elements and it is modulated by a variety of ancillary factors (40,41).In B. subtilis, five histidine sensor kinases (KinA, -B, -C, -D, and -E) can respond to a multiplicity of inducing signals and activate the pathway by autophosphorylating and transferring the activating phosphoryl group to an intermediate response regulator acceptor called Spo0F. From Spo0F, the phosphoryl group is then transferred to the Spo0A response regulator through the Spo0B phosphotransferase. Spo0A is the critical transcription regulator for sporulation initiation. Accumulation of its activated form, Spo0AϳP, during growth progressively results in the repression of genes not required for sporulation and the activation of genes necessary for spore formation (7,19,28,53).Negative inputs into the phosphorelay are brought about mainly by aspartyl phosphate phosphatases that specifically dephosphorylate the response regulator components of the system. The three members of the Spo0E family of phosphatases dephosphorylate Spo0AϳP, while three members of the Rap family of phosphatases dephosphorylate the Spo0FϳP intermediate (18,36,38). Rap p...