Dual-Specificity Anti-sigma Factor Reinforces Control of Cell-Type Specific Gene Expression in Bacillus subtilis

Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σ^Fand σ^E control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σ^F is replaced by σ^G and σ^E is replaced by σ^K. The anti-sigma factor CsfB is produced under the control of σ^F and binds to and inhibits the auto-regulatory σ^G, but not σ^F. A position in region 2.1, occupied by an asparagine in σ^G and by a glutamate in ο^F, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σ^K and that CsfB binds to and inhibits σ^E but not σ^K, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σ^E and by a conserved glutamate in σ^K suffices for discrimination by CsfB. We also show that CsfB prevents activation of σ^G in the mother cell and the premature σ^G-dependent activation of σ^K. Thus, CsfB establishes negative feedback loops that curtail the activity of σ^E and prevent the ectopic activation of σ^G in the mother cell. The capacity of CsfB to directly block σ^E activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σ^E activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σ^F/σ^G and σ^E/σ^K pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.     

High-throughput sequencing reveals suppressors of Vibrio cholerae rpoE mutations: one fewer porin is enough

Analyses of suppressor mutations have been extremely valuable in understanding gene function. However, techniques for mapping suppressor mutations are not available for most bacterial species. Here, we used high-throughput sequencing technology to identify spontaneously arising suppressor mutations that enabled disruption of rpoE (which encodes σ^E) in Vibrio cholerae, the agent of cholera. The alternative sigma factor σ^E, which is activated by envelope stress, promotes expression of factors that help preserve and/or restore cell envelope integrity. In Escherichia coli, rpoE is an essential gene that can only be disrupted in the presence of additional suppressor mutations. Among a panel of independent V. cholerae rpoE mutants, more than 75% contain suppressor mutations that reduce production of OmpU, V. cholerae’s principal outer membrane porin. OmpU appears to be a key determinant of V. cholerae’s requirement for and production of σ^E. Such dependence upon a single factor contrasts markedly with regulation of σ^E in E. coli, in which numerous factors contribute to its activation and none is dominant. We also identified a suppressor mutation that differs from all previously described suppressors in that it elevates, rather than reduces, σ^E’s activity. Finally, analyses of a panel of rpoE mutants shed light on the mechanisms by which suppressor mutations may arise in V. cholerae.     

Expression of the sigmaF-directed csfB locus prevents premature appearance of sigmaG activity during sporulation of Bacillus subtilis

During sporulation, σ^G becomes active in the prespore upon the completion of engulfment. We show that the inactivation of the σ^F-directed csfB locus resulted in premature activation of σ^G. CsfB exerted control distinct from but overlapping with that exerted by LonA to prevent inappropriate σ^G activation. The artificial induction of csfB severely compromised spore formation.     

A Mycobacterium tuberculosis Sigma Factor Network Responds to Cell-Envelope Damage by the Promising Anti-Mycobacterial Thioridazine

Background

Novel therapeutics are urgently needed to control tuberculosis (TB). Thioridazine (THZ) is a candidate for the therapy of multidrug and extensively drug-resistant TB.

Methodology/Principal Findings

We studied the impact of THZ on Mycobacterium tuberculosis (Mtb) by analyzing gene expression profiles after treatment at the minimal inhibitory (1x MIC) or highly inhibitory (4x MIC) concentrations between 1–6 hours. THZ modulated the expression of genes encoding membrane proteins, efflux pumps, oxido-reductases and enzymes involved in fatty acid metabolism and aerobic respiration. The Rv3160c-Rv3161c operon, a multi-drug transporter and the Rv3614c/3615c/3616c regulon, were highly induced in response to THZ. A significantly high number of Mtb genes co-expressed with σ^B (the σ^B regulon) was turned on by THZ treatment. σ^B has recently been shown to protect Mtb from envelope-damage. We hypothesized that THZ damages the Mtb cell-envelope, turning on the expression of the σ^B regulon. Consistent with this hypothesis, we present electron-microscopy data which shows that THZ modulates cell-envelope integrity. Moreover, the Mtb mutants in σ^H and σ^E, two alternate stress response sigma factors that induce the expression of σ^B, exhibited higher sensitivity to THZ, indicating that the presence and expression of σ^B allows Mtb to resist the impact of THZ. Conditional induction of σ^B levels increased the survival of Mtb in the presence of THZ.

Conclusions/Significance

THZ targets different pathways and can thus be used as a multi-target inhibitor itself as well as provide strategies for multi-target drug development for combination chemotherapy. Our results show that the Mtb sigma factor network comprising of σ^H, σ^E and σ^B plays a crucial role in protecting the pathogen against cell-envelope damage.