bDuring the early stages of sporulation, a subpopulation of Bacillus subtilis cells secrete toxins that kill their genetically identical siblings in a process termed cannibalism. One of these toxins is encoded by the sdpC gene of the sdpABC operon. The active form of the SDP toxin is a 42-amino-acid peptide with a disulfide bond which is processed from an internal fragment of pro-SdpC. The factors required for the processing of pro-SdpC into mature SDP are not known. We provide evidence that pro-SdpC is secreted via the general secretory pathway and that signal peptide cleavage is a required step in the production of SDP. We also demonstrate that SdpAB are essential to produce mature SDP, which has toxin activity. Our data indicate that SdpAB are not required for secretion, translation, or stability of SdpC. Thus, SdpAB may participate in a posttranslation step in the production of SDP. The mature form of the SDP toxin contains a disulfide bond. Our data indicate that while the disulfide bond does increase activity of SDP, it is not essential for SDP activity. We demonstrate that the disulfide bond is formed independently of SdpAB. Taken together, our data suggest that SDP production is a multistep process and that SdpAB are required for SDP production likely by controlling, directly or indirectly, cleavage of SDP from the pro-SdpC precursor.

Bacteria produce chemical signals (pheromones) to coordinate behaviors across a population in a process termed quorum sensing (QS). QS systems comprising peptide pheromones and their corresponding Rgg receptors are widespread among Firmicutes and may be useful targets for manipulating microbial behaviors, like suppressing virulence. The Rgg2/3 QS circuit of the human pathogen Streptococcus pyogenes controls genes affecting resistance to host lysozyme in response to short hydrophobic pheromones (SHPs). Considering that artificial activation of a QS pathway may be as useful in the objective of manipulating bacteria as inhibiting it, we sought to identify small-molecule inducers of the Rgg2/3 QS system. We report the identification of a small molecule, P516-0475, that specifically induced expression of Rgg2/3-regulated genes in the presence of SHP pheromones at concentrations lower than typically required for QS induction. In searching for the mode of action of P516-0475, we discovered that an S. pyogenes mutant deficient in pepO, a neprilysin-like metalloendopeptidase that degrades SHP pheromones, was unresponsive to the compound. P516-0475 directly inhibited recombinant PepO in vitro as an uncompetitive inhibitor. We conclude that this compound induces QS by stabilizing SHP pheromones in culture. Our study indicates the usefulness of cell-based screens that modulate pathway activities to identify unanticipated therapeutic targets contributing to QS signaling. This work was supported by National Institutes of Health Grants AI091779 and AI125452, the Burroughs Wellcome Fund PATH award, and the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Table S1 and Figs. S1 and S2.

No abstract

Bacillus subtilis is a Gram-positive spore-forming soil bacterium. Under environmental stress conditions such as starvation, B. subtilis enters the pathway of sporulation. Earlier work demonstrated that B. subtilis can delay sporulation by undergoing cannibalism. Sporulating cells secrete toxins that kill nearby siblings, thus allowing cells to feed on the released contents. One of these toxins, SdpC, is encoded by the sdpABC operon. To uncover the requirements for SdpC toxic activity during cannibalism, all proteins in the sdpABC operon were analyzed. We report that mutations of SdpC which block signal peptidase cleavage also block toxin production. In addition, production and secretion of SdpC do not require SdpA and SdpB. Our results indicate that SdpC secretion is indispensable for induction of the immunity operon sdpRI. Furthermore, SdpC secreted from an sdpAB strain does not fully induce sdpRI expression and has decreased toxicity to cells that are sensitive to wild type SdpC. Lastly, differences in SdpC mobility are observed in the presence of SdpA and SdpB. Thus, we propose that SdpA and SdpB may function by post-translationally modifying SdpC into the active form of the toxin.

No abstract