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Journal of Bacteriology, June 2005, p. 3800-3806, Vol. 187, No. 11
0021-9193/05/$08.00+0     doi:10.1128/JB.187.11.3800-3806.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The ExsA Protein of Bacillus cereus Is Required for Assembly of Coat and Exosporium onto the Spore Surface

Karen Bailey-Smith,1,{dagger} Sarah J. Todd,1 Thomas W. Southworth,1 John Proctor,2 and Anne Moir1*

Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom,1 Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom2

Received 26 October 2004/ Accepted 24 February 2005

The outermost layer of spores of the Bacillus cereus family is a loose structure known as the exosporium. Spores of a library of Tn917-LTV1 transposon insertion mutants of B. cereus ATCC 10876 were partitioned into hexadecane; a less hydrophobic mutant that was isolated contained an insertion in the exsA promoter region. ExsA is the equivalent of SafA (YrbA) of Bacillus subtilis, which is also implicated in spore coat assembly; the gene organizations around both are identical, and both proteins contain a very conserved N-terminal cortex-binding domain of ca. 50 residues, although the rest of the sequence is much less conserved. In particular, unlike SafA, the ExsA protein contains multiple tandem oligopeptide repeats and is therefore likely to have an extended structure. The exsA gene is expressed in the mother cell during sporulation. Spores of an exsA mutant are extremely permeable to lysozyme and are blocked in late stages of germination, which require coat-associated functions. Two mutants expressing differently truncated versions of ExsA were constructed, and they showed the same gross defects in the attachment of exosporium and spore coat layers. The protein profile of the residual exosporium harvested from spores of the three mutants—two expressing truncated proteins and the mutant with the original transposon insertion in the promoter region—showed some differences from the wild type and from each other, but the major exosporium glycoproteins were retained. The exsA gene is extremely important for the normal assembly and anchoring of both the spore coat and exosporium layers in spores of B. cereus.

* Corresponding author. Mailing address: Department of Molecular Biology and 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.

{dagger} Present address: School of Science and Mathematics, Sheffield Hallam University, Sheffield S1 1WB, United Kingdom.

Journal of Bacteriology, June 2005, p. 3800-3806, Vol. 187, No. 11
0021-9193/05/$08.00+0     doi:10.1128/JB.187.11.3800-3806.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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