Production of and Response to the Cannibalism Peptide SDP in Bacillus subtilis

The Gram positive soil dwelling bacteria Bacillus subtilis produces spores when encountered with a low nutrient environment. However, B. subtilis can delay spore production by a mechanism known as cannibalism. Cannibalism is a process by which B. subtilis delays commitment to sporulation by killing a subpopulation of its cells. This process involves production of two toxins, SDP and SKF. SDP is a 42 amino acid peptide with a disulfide bond derived from the internal cleavage of its precursor protein pro-SdpC. pro-SdpC is part of the sdpABC operon. Production of extracellular SDP induces expression of the sdpRI operon. Encoded in this operon is the negative regulator SdpR and SdpI. SdpI is a dual function protein which acts both as a signal transduction protein and the immunity factor against SDP. The current model states that production of SDP is sensed via SdpI. SdpI will sequester SdpR to the membrane in response and allow for sdpRI expression. The aims of this dissertation are to establish the requirements for SDP production and its response via SdpI/SdpR during cannibalism.

Studies in Chapter II were carried out to determine the factors required for production of the antimicrobial peptide SDP. Site directed mutagenesis of the leader signal peptide sequence in pro-SdpC demonstrated that proper signal peptide cleavage was required for SDP production. Additional site directed mutants of the cysteine residues in pro-SdpC revealed that these are not required for SDP toxic activity. These studies also included deletions within the sdpABC operon and revealed that the two proteins of unknown function, SdpA and SdpB are required for SDP production. Using mass spectrometry analysis, we found that SdpA and SdpB together are required to produce the active 42 amino acid peptide SDP. Taken together we concluded that SDP production was a multi step process which required proteins encoded within the operon and additional processing supplemented in the cell.

In Chapter III we investigated the role of SdpI, specifically what residues were required for the signaling and immunity functions observed. Our initial screen, included site directed mutagenesis of highly conserved residues between the 4th and 5th transmembrane domains of SdpI. These resulted in over 20 SdpI mutants generated. From these, only two SdpI mutants had defects in either signal transduction or SDP immunity. Additional localized mutagenesis was used to isolate two other mutants in SdpI which only affected signal transduction or SDP immunity. SdpI signaling-immunity+ mutants presented a defect in SdpR membrane sequestration and sdpRIinduction. Our findings suggest these types of SdpI mutants may be important for the downstream effect of SdpR membrane sequestration. SdpI signaling+ immunity- mutants revealed defects in SDP protection. Some of the residues mutated were conserved in other SdpI homologs. Site directed mutagenesis of these conserved residues in the SdpI ortholog YfhL showed these are also required for SDP resistance. For the first time, we were able to identify mutations which affected only SDP immunity and gained further insight into how SdpI signaling-immunity+ mutants play a role during signal transduction.

In Chapter IV we initiated studies to define what regions of the negative regulator SdpR are important for its function during cannibalism. We employed localized mutagenesis to identify SdpR mutants which decreased sdpRIexpression even in the presence of inducing signal. We isolated three such SdpR mutants, referred to as super repressors. We expect these SdpR super repressors are unable to be sequestered to the membrane in the presence of SDP.