Initiation of endospore formation in clostridia
Lead Research Organisation:
University of Nottingham
Department Name: Inst of Infections and Immunity
Abstract
Some bacteria are capable of producing profoundly dormant and highly resistant spores in nature, making them difficult to eradicate form hospital environments, foodstuffs and pharmaceutical preparations. The ability to form spores is shared by members of the bacterial families Bacillus and Clostridium. Whereas the former require atmospheric air for growth (aerobes), oxygen is poisonous to the latter. Thus, clostridia can only grow in the absence of oxygen and are therefore classified 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 and frequently, wards 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, through the exploitation of their spores as a delivery system for treating cancer. When spores are injected into the bloodstream, the bacteria are unable to grow because normal healthy tissues contain oxygen. However, the central mass of solid tumors are devoid of oxygen. Those spores that enter a tumour are therefore able to germinate, and bring about the establishment of an actively growing population, specifically restricted to the tumour mass. This unique feature provides the opportunity to deliver therapeutic agents selectively to solid tumours, by endowing the organism used with genes able to direct the production of the desired 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 have 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, in the absence of a phosphorelay, triggers sporulation. Should we solve this riddle, then scientists may be able to devise strategies that interfere with spore formation by clostridia. This will reveal 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/D001498/1.
Technical Summary
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. 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 Earth¿s 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. 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. 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.
People |
ORCID iD |
Nigel Minton (Principal Investigator) |
Publications
Cartman ST
(2010)
The emergence of 'hypervirulence' in Clostridium difficile.
in International journal of medical microbiology : IJMM
Cooksley CM
(2010)
Regulation of neurotoxin production and sporulation by a Putative agrBD signaling system in proteolytic Clostridium botulinum.
in Applied and environmental microbiology
Heap JT
(2010)
ClosTron-targeted mutagenesis.
in Methods in molecular biology (Clifton, N.J.)
Heap JT
(2009)
A modular system for Clostridium shuttle plasmids.
in Journal of microbiological methods
Heap JT
(2007)
The ClosTron: a universal gene knock-out system for the genus Clostridium.
in Journal of microbiological methods
Heap JT
(2010)
The ClosTron: Mutagenesis in Clostridium refined and streamlined.
in Journal of microbiological methods
Sebaihia M
(2007)
Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes.
in Genome research
Steiner E
(2011)
Multiple orphan histidine kinases interact directly with Spo0A to control the initiation of endospore formation in Clostridium acetobutylicum.
in Molecular microbiology
Description | 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. Whereas the former require atmospheric air for growth ('aerobes'), oxygen is poisonous to the latter. Thus, clostridia can only grow in the absence of oxygen and are therefore classified 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 and frequently, wards 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. The well-studied model organism, Bacillus 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. However, when the genetic blueprint of a number of Clostridia was determined, the genes responsible for this phosphorelay were missing. How then does the bacterium sense unfavourable external conditions and then transmit that information to the genetic machinery inside the cell and start producing spores? The traditional approach to solving such conundrums in biology is to systematically inactivate selected genes, thereby preventing production of the protein they encode, and then testing to see what effect the absence of that protein/gene has on the process under investigation -in this instance ability to form spores. Prior to this project, methods of inactivating genes in Clostridia were unavailable. During the course of this work, we have developed a revolutionary tool (the ClosTron) that may be used to directly target specific genes and bring about their deactivation. We have use this tool to identify those proteins that are located on the cells surface that most likely sense the external changes and then directly pass this information on to the cells genetic machinery. |
Exploitation Route | On a general level, our work has resulted in the development of a technology (the ClosTron) that has a myriad of applications to bring about a better understanding of how Clostridia function through targeted gene inactivation. This will lead to better ways of treating the diseases they cause and/or exploiting their useful properties for the benefit of humankind. On a specific level, the identification of those proteins pivotal to spore formation could lead to 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. |
Sectors | Aerospace, Defence and Marine,Energy,Healthcare |
Description | Historically, the bacterial genus Clostridium is most often associated with debilitating and life-threatening diseases such as that studied here, botulism, but equally infamous are tetanus, gangrene, antibiotic associated diarrhoea and food poisoning. However, the vast majority of clostridia are entirely benign, and far from being the scourge of humankind, may well be its saviour. Clostridia occupy many specialised biological niches and have evolved a plethora of bio-catalytic abilities that can be exploited for humankind's benefit. Many species are being pursued as cell factories for production of biofuels and chemicals through alternative processes to traditional petro-chemical routes. Yet others are being explored as tumour delivery vehicles for anti-cancer drugs. Tools for manipulating clostridial genomes (for gene knock-out and knock-in) are essential for generating the functional knowledge needed to combat clostridial pathogens, and for genetic enhancement of strains used in chemical production and cancer therapy. Without those tools, in the 20 years prior to breakthrough research at Nottingham, just five mutants had ever been made in the biobutanol organism C.acetobutylicum, only one in the pathogen C.difficile and none in the pathogens C.botulinum or C.sporogenes. The genetic method developed here, ClosTron technology, provides a simple and rapid method for generating mutants, a prerequisite for ascribing function to the organism's genes/ gene products, itself a requirement for the rational development of effective countermeasures against clostridial pathogens. These include the formulation of more effective means for preventing food spoilage and poisoning by Clostridium botulinum. The paper describing the ClosTron is the most cited paper in the Journal of Microbiology Methods since its publication in 2007. ClosTron has revolutionised clostridial molecular biology and it is now the most widely used gene tool within the clostridial community. A patent protecting the system was filed in 2006. |
First Year Of Impact | 2007 |
Sector | Agriculture, Food and Drink,Chemicals,Energy,Healthcare,Pharmaceuticals and Medical Biotechnology,Other |
Description | BBSRC Responsive Mode |
Amount | £643,519 (GBP) |
Funding ID | BB/E021271/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2007 |
End | 03/2011 |
Description | ERANET SysMO1 |
Amount | £364,436 (GBP) |
Funding ID | BB/F003390/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 04/2007 |
End | 06/2010 |
Description | HORIZON2020 Marie Curie ETN, CLOSPORE |
Amount | € 3,946,605 (EUR) |
Funding ID | 642068 |
Organisation | European Commission |
Department | Horizon 2020 |
Sector | Public |
Country | European Union (EU) |
Start | 03/2015 |
End | 02/2018 |
Description | Ipsen Contract Sequencing of Industrial Master Seed Banks |
Amount | £35,000 (GBP) |
Organisation | Ipsen |
Sector | Private |
Country | Global |
Start | 11/2013 |
End | 09/2014 |
Description | Marie Curie Initial Training Network (ITN) |
Amount | £4,111,621 (GBP) |
Funding ID | 215697-2 |
Organisation | Marie Sklodowska-Curie Actions |
Sector | Charity/Non Profit |
Country | Global |
Start | 08/2009 |
End | 08/2013 |
Description | PhD Studentship in Synthetic Biology (Patrick Budd) |
Amount | £58,000 (GBP) |
Organisation | Scott and White Healthcare |
Sector | Charity/Non Profit |
Country | United States |
Start | 10/2011 |
End | 09/2015 |
Description | ZonMw Studentship |
Amount | £54,000 (GBP) |
Funding ID | P. Lambin order 30943121N |
Organisation | Maastricht University (UM) |
Sector | Academic/University |
Country | Netherlands |
Start | 02/2009 |
End | 07/2012 |