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Journal of Bacteriology, December 2009, p. 7296-7305, Vol. 191, No. 23
0021-9193/09/$08.00+0     doi:10.1128/JB.00882-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of the Sporulation Initiation Pathway of Clostridium difficile and Its Role in Toxin Production {triangledown} ,{dagger}

Sarah Underwood,1 Shuang Guan,1 Vinod Vijayasubhash,1 Simon D. Baines,1,2 Luke Graham,3 Richard J. Lewis,3 Mark H. Wilcox,1,2 and Keith Stephenson1*

Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, Leeds University, Leeds LS2 9JT, United Kingdom,1 Department of Microbiology, Leeds General Infirmary, Leeds LS1 3EX, United Kingdom,2 Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom3

Received 7 July 2009/ Accepted 17 September 2009

Clostridium difficile is responsible for significant mortality and morbidity in the hospitalized elderly. C. difficile spores are infectious and are a major factor contributing to nosocomial transmission. The Spo0A response regulator is the master regulator for sporulation initiation and can influence many other cellular processes. Using the ClosTron gene knockout system, we inactivated genes encoding Spo0A and a putative sporulation-associated sensor histidine kinase in C. difficile. Inactivation of spo0A resulted in an asporogeneous phenotype, whereas inactivation of the kinase reduced C. difficle sporulation capacity by 3.5-fold, suggesting that this kinase also has a role in sporulation initiation. Furthermore, inactivation of either spo0A or the kinase resulted in a marked defect in C. difficile toxin production. Therefore, Spo0A and the signaling pathway that modulates its activity appear to be involved in regulation of toxin synthesis in C. difficile. In addition, Spo0A was directly phosphorylated by a putative sporulation-associated kinase, supporting the hypothesis that sporulation initiation in C. difficile is controlled by a two-component signal transduction system rather than a multicomponent phosphorelay. The implications of these findings for C. difficile sporulation, virulence, and transmission are discussed.

* Corresponding author. Mailing address: Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, Leeds University, Leeds LS2 9JT, United Kingdom. Phone: 44(0)113 3435581. Fax: 44(0)113 3435638. E-mail: k.stephenson{at}leeds.ac.uk

{triangledown} Published ahead of print on 25 September 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, December 2009, p. 7296-7305, Vol. 191, No. 23
0021-9193/09/$08.00+0     doi:10.1128/JB.00882-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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