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Journal of Bacteriology, November 2006, p. 7905-7913, Vol. 188, No. 22
0021-9193/06/$08.00+0     doi:10.1128/JB.00997-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

ExsY and CotY Are Required for the Correct Assembly of the Exosporium and Spore Coat of Bacillus cereus{triangledown}

Matt J. Johnson,1,{dagger} Sarah J. Todd,1 David A. Ball,1 Andrew M. Shepherd,1 Patricia Sylvestre,2 and Anne Moir1*

Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom,1 Unité Toxine et Pathogénie Bactériennes, Institut Pasteur, Paris, France2

Received 7 July 2006/ Accepted 4 September 2006

The exosporium-defective phenotype of a transposon insertion mutant of Bacillus cereus implicated ExsY, a homologue of B. subtilis cysteine-rich spore coat proteins CotY and CotZ, in assembly of an intact exosporium. Single and double mutants of B. cereus lacking ExsY and its paralogue, CotY, were constructed. The exsY mutant spores are not surrounded by an intact exosporium, though they often carry attached exosporium fragments. In contrast, the cotY mutant spores have an intact exosporium, although its overall shape is altered. The single mutants show altered, but different, spore coat properties. The exsY mutant spore coat is permeable to lysozyme, whereas the cotY mutant spores are less resistant to several organic solvents than is the case for the wild type. The exsY cotY double-mutant spores lack exosporium and have very thin coats that are permeable to lysozyme and are sensitive to chloroform, toluene, and phenol. These spore coat as well as exosporium defects suggest that ExsY and CotY are important to correct formation of both the exosporium and the spore coat in B. cereus. Both ExsY and CotY proteins were detected in Western blots of purified wild-type exosporium, in complexes of high molecular weight, and as monomers. Both exsY and cotY genes are expressed at late stages of sporulation.

* Corresponding author. Mailing address: Department of Molecular Biology & Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom. Phone: 44 114 2224418. Fax: 44 1142222800. E-mail: a.moir{at}sheffield.ac.uk.

{triangledown} Published ahead of print on 15 September 2006.

{dagger} Present address: Abcam, 332 Cambridge Science Park, Milton Rd., Cambridge CB4 0FW, United Kingdom.

Journal of Bacteriology, November 2006, p. 7905-7913, Vol. 188, No. 22
0021-9193/06/$08.00+0     doi:10.1128/JB.00997-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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