Initiation of Endospore Formation in Clostridia

Endospore formation in the model organism, Bacillus subtilis, is now rather well understood. The decision to initiate spore formation is not taken lightly. It is controlled by the phosphorylation status of the Spo0A master regulator and information about the internal and external environment is conveyed to this transcription factor by a number of proteins comprising a phosphorelay. Several autophosphorylating kinases, whose phosphorylation status is controlled by unknown ligands, transfer their phosphate to the Spo0F response regulator. This may either be dephosphorylated, or it may transfer its phosphate onwards to the Spo0B response regulator phosphotransferase, which then, in turn, passes it on to Spo0A. Phosphate transfer through the phosphorelay is under complex regulation at several points, which makes the decision to sporulate sensitive to multiple inputs indicative of nutrient starvation. Using an elegant combination of site-directed mutagenesis, X-ray crystallography, and biochemical analyses, the residues forming the interaction surfaces between the various phosphorelay components have now been identified. Bacterial genome sequencing projects have shown that the clostridia (and related anaerobic spore-forming organisms) do not contain a recognisable phosphorelay. Some organisms contain a protein similar to B. subtilis Spo0B, but none of them appear to contain Spo0F. <P>This raises an intriguing question: how is Spo0A phosphorylated in these organisms? A potential answer to this question is provided by recently acquired knowledge of protein-protein interactions in the phosphorelay. In B. subtilis, all the sensor kinases that feed into the phosphorelay show substantial sequence conservation in an alpha-helix that makes contact with Spo0F. The corresponding region of B. subtilis Spo0B (which contacts Spo0A) lacks sequence conservation in this helix, but shares conservation, instead, with the B. subtilis YufL (malate) and CitS (citrate) sensors and also, significantly, with the corresponding region of Spo0B found in some clostridia (and relatives). This suggests that the phosphorelay may have arisen during evolution (as oxygen became more abundant in the Earths atmosphere) from genes resembling the YufLM CitSB sensor-regulator couples in the ancestral organism. This moreover suggests the testable hypothesis that kinase resembling B. subtilis YufL and CitS may phosphorylate Spo0A in clostridia.<P> To test this, we will inactivate the Clostridium acetobutylicum and Clostridium botulinum kinases that resemble B. subtilis YufL CitS and determine whether either singly, or in combination, these genetic defects reduce the phosphorylation status of Spo0A, engendering phenotypes such as the loss of the ability to form spores and solvents or toxins and the loss of motility, characteristic of the inactivation of spo0A itself. There is also direct experimental evidence that sporulation of some clostridia can be stimulated by exposure to (traces of) oxygen, suggesting, perhaps, that one or more of their redox-sensitive PAS domain-containing kinases phosphorylates Spo0A. This will also be explored by systematic inactivation of the encoding genes. In the case of C. botulinum, the global effects of kinase gene inactivation will also be explored using microarray analysis. Inactivation will be achieved by gene replacement and by using antisense technology. <P>Methods for undertaking gene replacement in clostridia have been developed in the authors laboratories and also in the laboratory of Philippe Soucaille, INSA, Toulouse, France. Gene replacement will be the method of choice in C. acetobutylicum, while in C. botulinum, the antisense approach will be favoured. To confirm these in vivo experiments, recombinant forms of the YufL CitS-like and PAS domain-containing kinases will be produced and their ability to transfer phosphate to clostridial Spo0A in vitro will be explored. Joint with BB/D001498/1.