Temporal regulation of σB by partner-switching mechanism at a distinct growth stage in Bacillus cereus.

The alternative transcription factor σB in Bacillus cereus governs the transcription of a number of genes that confer protection against general stress. This transcription factor is regulated by protein-protein interactions among RsbV, RsbW, σB, RsbY, RsbM and RsbK, all encoded in the sigB cluster. Among these regulatory proteins, RsbV, RsbW and σB comprise a partner-switching mechanism. Under normal conditions, σB remains inactive by associating with anti-sigma factor RsbW, which prevents σB from binding to the core RNA polymerase. During environmental stress, RsbK activates RsbY to hydrolyze phosphorylated RsbV, and the dephosphorylated RsbV then sequesters RsbW to liberate σB from RsbW. Although the σB partner-switching module is thought to be the core mechanism for σB regulation, the actual protein-protein interactions among these three proteins in the cell remain to be investigated. In the current study, we show that RsbW and RsbV form a long-lived complex under transient stress treatment, resulting in high persistent expression of RsbV, RsbW and σB from mid-log phase to stationary phase. Full sequestration of RsbW by excess RsbV and increased RsbW:RsbV complex stability afforded by cellular ADP contribute to the prolonged activation of σB. Interestingly, the high expression levels of RsbV, RsbW and σB were dramatically decreased beginning from the transition stage to the stationary phase. Thus, protein interactions among σB partner-switching components are required for the continued induction of σB during environmental stress in the log phase and significant down-regulation of σB is observed in the stationary phase. Our data show that σB is temporally regulated in B. cereus.

Methylatable Signaling Helix Coordinated Inhibitory Receiver Domain in Sensor Kinase Modulates Environmental Stress Response in Bacillus Cereus.

σB, an alternative transcription factor, controls the response of the cell to a variety of environmental stresses in Bacillus cereus. Previously, we reported that RsbM negatively regulates σB through the methylation of RsbK, a hybrid sensor kinase, on a signaling helix (S-helix). However, RsbK comprises a C-terminal receiver (REC) domain whose function remains unclear. In this study, deletion of the C-terminal REC domain of RsbK resulted in high constitutive σB expression independent of environmental stimuli. Thus, the REC domain may serve as an inhibitory element. Mutagenic substitution was employed to modify the putative phospho-acceptor residue D827 in the REC domain of RsbK. The expression of RsbKD827N and RsbKD827E exhibited high constitutive σB, indicating that D827, if phosphorylatable, possibly participates in σB regulation. Bacterial two-hybrid analyses demonstrated that RsbK forms a homodimer and the REC domain interacts mainly with the histidine kinase (HK) domain and partly with the S-helix. In particular, co-expression of RsbM strengthens the interaction between the REC domain and the S-helix. Consistently, our structural model predicts a significant interaction between the HK and REC domains of the RsbK intradimer. Here, we demonstrated that coordinated the methylatable S-helix and the REC domain of RsbK is functionally required to modulate σB-mediated stress response in B. cereus and maybe ubiquitous in microorganisms encoded RsbK-type sensor kinases.

Differential regulation and activity against oxidative stress of Dps proteins in Bacillus cereus.

In this study, the sequence similarity, structure, ferroxidase activity and efficacy in antagonizing oxidative stress of three Dps-like proteins, Dps1, Dps2 and Dps3, encoded by Bacillus cereus were comparatively analyzed. The three Dps-like proteins are homologous to other bacterial Dps proteins that exhibit ferroxidase activity. Both Dps1 and Dps2 have a typical Dps spherical structure, but Dps3 has a unique filamentous structure. Several dps mutant strains were generated to investigate the functional role of dps genes in cell protection. The dps1 null strain was the most labile to oxidative stress in the stationary phase, and the loss of dps2 resulted in greater sensitivity to peroxide exposure compared with the other mutant strains in the log phase. Interestingly, after simultaneous deletion of dps1 and dps2, the survival rate was dramatically reduced by approximately 5 log in the stationary phase. Immunoblotting analysis demonstrated that Dps1 and Dps2 in the wild-type strain were induced by oxidative stress, and Dps3 responded to general stress in the log phase. Constitutively high expression of Dps2 in a perR null mutant and PerR-specific binding of the promoter region of dps2 confirmed Dps2 as a member of the PerR regulon. In addition, the expression of Dps1 and Dps2, absent any stress, was initiated in the log phase and was abundant in the stationary phase, suggesting that the expression of Dps1 and Dps2 was dependent on the bacterial growth stage. In summary, the three Dps proteins conferred cellular protection, particularly from oxidative stress, and were differentially regulated in response to varied stress conditions.

Interplay of RsbM and RsbK controls the σ(B) activity of Bacillus cereus.

The alternative transcription factor σ(B) of Bacillus cereus controls the expression of a number of genes that respond to environmental stress. Four proteins encoded in the sigB gene cluster, including RsbV, RsbW, RsbY (RsbU) and RsbK, are known to be essential in the σ(B)-mediated stress response. In the context of stress, the hybrid sensor kinase RsbK is thought to phosphorylate the response regulator RsbY, a PP2C serine phosphatase, leading to the dephosphorylation of the phosphorylated RsbV. The unphosphorylated RsbV then sequesters the σ(B) antagonist, RsbW, ultimately liberating σ(B). The gene arrangement reveals an open reading frame, bc1007, flanked immediately downstream by rsbK within the sigB gene cluster. However, little is known about the function of bc1007. In this study, the deletion of bc1007 resulted in high constitutive σ(B) expression independent of environmental stimuli, indicating that bc1007 plays a role in σ(B) regulation. A bacterial two-hybrid analysis demonstrated that BC1007 interacts directly with RsbK, and autoradiographic studies revealed a specific C(14)-methyl transfer from the radiolabelled S-adenosylmethionine to RsbK when RsbK was incubated with purified BC1007. Our data suggest that BC1007 (RsbM) negatively regulates σ(B) activity by methylating RsbK. Additionally, mutagenic substitution was employed to modify 12 predicted methylation residues in RsbK. Certain RsbK mutants were able to rescue σ(B) activation in a rsbK-deleted bacterial strain, but RsbK(E439A) failed to activate σ(B), and RsbK(E446A) only moderately induced σ(B). These results suggest that Glu439 is the preferred methylation site and that Glu446 is potentially a minor methylation site. Gene arrays of the rsbK orthologues and the neighbouring rsbM orthologues are found in a wide range of bacteria. The regulation of sigma factors through metylation of RsbK-like sensor kinases appears to be widespread in the microbial world.