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Journal of Bacteriology, September 2009, p. 5569-5576, Vol. 191, No. 18
0021-9193/09/$08.00+0     doi:10.1128/JB.00408-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The Germination-Specific Lytic Enzymes SleB, CwlJ1, and CwlJ2 Each Contribute to Bacillus anthracis Spore Germination and Virulence{triangledown}

Jonathan D. Giebel, Katherine A. Carr, Erica C. Anderson, and Philip C. Hanna*

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109

Received 26 March 2009/ Accepted 22 June 2009

The bacterial spore cortex is critical for spore stability and dormancy and must be hydrolyzed by germination-specific lytic enzymes (GSLEs), which allows complete germination and vegetative cell outgrowth. We created in-frame deletions of three genes that encode GSLEs that have been shown to be active in Bacillus anthracis germination: sleB, cwlJ1, and cwlJ2. Phenotypic analysis of individual null mutations showed that the removal of any one of these genes was not sufficient to disrupt spore germination in nutrient-rich media. This finding indicates that these genes have partially redundant functions. Double and triple deletions of these genes resulted in more significant defects. Although a small subset of {Delta}sleB {Delta}cwlJ1 spores germinate with wild-type kinetics, for the overall population there is a 3-order-of-magnitude decrease in the colony-forming efficiency compared with wild-type spores. {Delta}sleB {Delta}cwlJ1 {Delta}cwlJ2 spores are unable to complete germination in nutrient-rich conditions in vitro. Both {Delta}sleB {Delta}cwlJ1 and {Delta}sleB {Delta}cwlJ1 {Delta}cwlJ2 spores are significantly attenuated, but are not completely devoid of virulence, in a mouse model of inhalation anthrax. Although unable to germinate in standard nutrient-rich media, spores lacking SleB, CwlJ1, and CwlJ2 are able to germinate in whole blood and serum in vitro, which may explain the persistent low levels of virulence observed in mouse infections. This work contributes to our understanding of GSLE activation and function during germination. This information may result in identification of useful therapeutic targets for the disease anthrax, as well as provide insights into ways to induce the breakdown of the protective cortex layer, facilitating easier decontamination of resistant spores.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Michigan Medical School, 5641 Medical Science Building II, Box 0620, Ann Arbor, MI 48109-062. Phone: (734) 615-3706. Fax: (734) 764-3562. E-mail: pchanna{at}umich.edu

{triangledown} Published ahead of print on 6 July 2009.

Journal of Bacteriology, September 2009, p. 5569-5576, Vol. 191, No. 18
0021-9193/09/$08.00+0     doi:10.1128/JB.00408-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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