Initiation of endospore formation in clostridia

Some bacteria are capable of producing profoundly dormant and highly resistant spores in nature, making them difficult to eradicate from hospital environments, foodstuffs and pharmaceutical preparations. The ability to form spores is shared by members of the bacterial families Bacillus and Clostridium. The former require air for growth, whereas the latter can only grow in the absence of oxygen and are known as anaerobes. A number of Clostridium species have achieved notoriety because they cause human disease, largely as a consequence of spore production. For example, Clostridium difficile is highly resistant to many antibiotics and causes serious problems in our hospitals, sometimes leading to mortality. Once an infection breaks out in a hospital ward the presence of spores make it very difficult to control. Wards frequently have to be closed for rigorous disinfection. Similarly, the spores of Clostridium botulinum remain a potentially serious problem in the food preservation industry because they are able to withstand high temperatures and pressures. Moreover, public concern about spore-forming organisms have been heightened post 9/11, as they pose a potential bioterrorism threat. Paradoxically, it is the ability to form spores that presents Clostridium with, perhaps, its greatest potential benefit to mankind. Spores can be exploited as a drug delivery system for treating cancer. When injected into the bloodstream, Clostridium spores are unable to germinate and grow because normal healthy tissues contain oxygen. However, the central mass of solid tumors is devoid of oxygen and those spores that enter a tumour are able to germinate there. They then proliferate and establish an actively growing bacterial population, specifically restricted to the tumour mass. This unique feature provides the opportunity to deliver therapeutic agents selectively to solid tumours, simply by endowing the organism with genes able to direct the production of anticancer drugs. The well-studied model organism, Bacillus subtilis, contains a group of interacting protein molecules (called a phosphorelay) responsible for sensing environmental changes as a prelude to launching the process of spore development. It was assumed that all spore-formers would possess a similar phosphorelay. Recently, the entire DNA sequence of the chromosomes of a number of Clostridium family members have been determined. To our surprise, they do not make phosphorelay proteins. The clostridia are believed to resemble the more primitive life forms that were present on Earth before our atmosphere contained oxygen. Little is known about the environmental changes that might signal the onset of spore formation in this important group of organisms and the objective of this research project, is to discover what triggers sporulation in the absence of a phosphorelay. Should we solve this riddle, then scientists may be able to devise strategies that interfere with spore formation by clostridia. This will open up new ways of preventing spore formation and reducing the incidence of C. botulinum spores in foods and the spore load of C. difficile in hospital wards. It should also result in the more effective production of spore preparations for use in anticancer therapies. Joint with BB/D522797/1.

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. 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. subtilils 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 Earth's atmosphere) from genes resembling the YufLM / CitSB sensor-regulator couples in the ancestral organism. This moreover suggests the testable hypothesis that kinases resembling B. subtilis YufL and CitS may phosphorylate Spo0A in clostridia. To test this, we will inactivate the Clostridium acetobutylicum and Clostridium botulinum kinases that resemble B. subtlis 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 & 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. 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 replacment 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/D522797/1.