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      Functional Characterisation of Germinant Receptors in Clostridium botulinum and Clostridium sporogenes Presents Novel Insights into Spore Germination Systems

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          Abstract

          Clostridium botulinum is a dangerous pathogen that forms the highly potent botulinum toxin, which when ingested causes a deadly neuroparalytic disease. The closely related Clostridium sporogenes is occasionally pathogenic, frequently associated with food spoilage and regarded as the non-toxigenic equivalent of Group I C. botulinum. Both species form highly resistant spores that are ubiquitous in the environment and which, under favourable growth conditions germinate to produce vegetative cells. To improve the control of botulinum neurotoxin-forming clostridia, it is imperative to comprehend the mechanisms by which spores germinate. Germination is initiated following the recognition of small molecules (germinants) by a specific germinant receptor (GR) located in the spore inner membrane. The present study precisely defines clostridial GRs, germinants and co-germinants. Group I C. botulinum ATCC3502 contains two tricistronic and one pentacistronic GR operons, while C. sporogenes ATCC15579 has three tricistronic and one tetracistronic GR operons. Insertional knockout mutants, allied with characterisation of recombinant GRs shows for the first time that amino acid stimulated germination in C. botulinum requires two tri-cistronic encoded GRs which act in synergy and cannot function individually. Spore germination in C. sporogenes requires one tri-cistronic GR. Two other GRs form part of a complex involved in controlling the rate of amino-acid stimulated germination. The suitability of using C. sporogenes as a substitute for C. botulinum in germination studies and food challenge tests is discussed.

          Author Summary

          Clostridium botulinum is a dangerous pathogen that forms the deadly botulinum neurotoxin. Strains of C. botulinum are present in the environment as spores. Under suitable conditions, the dormancy of the bacterial spore is broken, and germination occurs. Germination is initiated following the recognition of small molecules by a specific germinant receptor (GR) located within spores. Currently, the identification and characterisation of these GRs remains unknown, but is critical if strategies are to be developed to either prevent spore germination altogether, or to germinate all the spores and then inactivate the emergent sensitive vegetative cells. The present study has characterised two functionally active GRs in C. botulinum which act in synergy and cannot function individually, and a related functionally active GR in C. sporogenes. These GRs respond to amino acids. Other GRs appear to form part of a complex involved in controlling the speed of germination, or are not functionally active. This study provides new insights into the mechanisms involved in germination and will allow us to develop new strategies to control this deadly pathogen.

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          Most cited references55

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          A modular system for Clostridium shuttle plasmids.

          Despite their medical and industrial importance, our basic understanding of the biology of the genus Clostridium is rudimentary in comparison to their aerobic counterparts in the genus Bacillus. A major contributing factor has been the comparative lack of sophistication in the gene tools available to the clostridial molecular biologist, which are immature, and in clear need of development. The transfer and maintenance of recombinant, replicative plasmids into various species of Clostridium has been reported, and several elements suitable as shuttle plasmid components are known. However, these components have to-date only been available in disparate plasmid contexts, and their use has not been broadly explored. Here we describe the specification, design and construction of a standardized modular system for Clostridium-Escherichia coli shuttle plasmids. Existing replicons and selectable markers were incorporated, along with a novel clostridial replicon. The properties of these components were compared, and the data allow researchers to identify combinations of components potentially suitable for particular hosts and applications. The system has been extensively tested in our laboratory, where it is utilized in all ongoing recombinant work. We propose that adoption of this modular system as a standard would be of substantial benefit to the Clostridium research community, whom we invite to use and contribute to the system.
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            Clostridium difficile spore biology: sporulation, germination, and spore structural proteins.

            Clostridium difficile is a Gram-positive, spore-forming obligate anaerobe and a major nosocomial pathogen of worldwide concern. Owing to its strict anaerobic requirements, the infectious and transmissible morphotype is the dormant spore. In susceptible patients, C. difficile spores germinate in the colon to form the vegetative cells that initiate Clostridium difficile infections (CDI). During CDI, C. difficile induces a sporulation pathway that produces more spores; these spores are responsible for the persistence of C. difficile in patients and horizontal transmission between hospitalized patients. Although important to the C. difficile lifecycle, the C. difficile spore proteome is poorly conserved when compared to members of the Bacillus genus. Further, recent studies have revealed significant differences between C. difficile and Bacillus subtilis at the level of sporulation, germination, and spore coat and exosporium morphogenesis. In this review, the regulation of the sporulation and germination pathways and the morphogenesis of the spore coat and exosporium will be discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.
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              The ClosTron: Mutagenesis in Clostridium refined and streamlined.

              The recent development of the ClosTron Group II intron directed mutagenesis tool for Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using FLP/FRT in Clostridium acetobutylicum; tested the capacity of the system for the delivery of transgenes to the chromosome of Clostridium sporogenes, which proved feasible for 1.0kbp transgenes in addition to a marker; and extended the host range of the system, constructing mutants in Clostridium beijerinckii and, for the first time, in a B1/NAP1/027 'epidemic' strain of Clostridium difficile. Automated intron design bioinformatics are now available free-of-charge at our website http://clostron.com; the out-sourced construction of re-targeted intron plasmids has become cost-effective as well as rapid; and the combination of constitutive intron expression with direct selection for intron insertions has made mutant isolation trivial. These developments mean mutants can now be constructed with very little time and effort for the researcher. Those who prefer to construct plasmids in-house are no longer reliant on a commercial kit, as a mixture of two new plasmids provides unlimited template for intron re-targeting by Splicing by Overlap Extension (SOE) PCR. The new ClosTron plasmids also offer blue-white screening and other options for identification of recombinant plasmids. The improved ClosTron system supersedes the prototype plasmid pMTL007 and the original method, and exploits the potential of Group II introns more fully. Copyright 2009 Elsevier B.V. All rights reserved.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                September 2014
                11 September 2014
                : 10
                : 9
                : e1004382
                Affiliations
                [1]Gut Health and Food Safety, Institute of Food Research (IFR), Norwich Research Park, Colney, Norwich, Norfolk, United Kingdom
                Tufts University, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JB MWP ATC. Performed the experiments: JB JP MI ATC. Analyzed the data: JB MWP. Contributed reagents/materials/analysis tools: DJHG. Contributed to the writing of the manuscript: JB MWP ATC.

                Article
                PPATHOGENS-D-14-01032
                10.1371/journal.ppat.1004382
                4161481
                25210747
                849e74e9-86be-4484-81ad-f3ae19dd0349
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 2 May 2014
                : 4 August 2014
                Page count
                Pages: 14
                Funding
                This research was funded by the BBSRC Institute Strategic Programme on Gut Health and Food Safety (BB/J004529/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Biotechnology
                Applied Microbiology
                Genetics
                Genomics
                Microbial Genomics
                Bacterial Genomics
                Bacterial Genomes
                Microbiology
                Bacteriology
                Bacterial Physiology
                Bacterial Spores
                Bacterial Genes
                Microbial Mutation
                Microbial Physiology
                Molecular biology
                Molecular biology techniques
                Cloning
                DNA cloning
                Complementary DNA cloning
                Gene Cloning
                Molecular Cloning
                Mutagenesis and Gene Deletion Techniques
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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