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Journal of Bacteriology, April 2009, p. 2237-2247, Vol. 191, No. 7
0021-9193/09/$08.00+0     doi:10.1128/JB.01598-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Roles of Germination-Specific Lytic Enzymes CwlJ and SleB in Bacillus anthracis ^,

Jared D. Heffron, Benjamin Orsburn, and David L. Popham^*

Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061

Received 11 November 2008/ Accepted 16 January 2009

The structural characteristics of a spore enable it to withstand stresses that typically kill a vegetative cell. Spores remain dormant until small molecule signals induce them to germinate into vegetative bacilli. Germination requires degradation of the thick cortical peptidoglycan by germination-specific lytic enzymes (GSLEs). Bacillus anthracis has four putative GSLEs, based upon sequence similarities with enzymes in other species: SleB, CwlJ1, CwlJ2, and SleL. In this study, the roles of SleB, CwlJ1, and CwlJ2 were examined. The expression levels of all three genes peak 3.5 h into sporulation. Genetic analysis revealed that, similar to other known GSLEs, none of these gene products are individually required for growth, sporulation, or triggering of germination. However, later germination events are affected in spores lacking CwlJ1 or SleB. Compared to the wild type, germinating spores without CwlJ1 suffer a delay in optical density loss and cortex peptidoglycan release. The absence of SleB also causes a delay in cortex fragment release. A double mutant lacking both SleB and CwlJ1 is completely blocked in cortex hydrolysis and progresses through outgrowth to produce colonies at a frequency 1,000-fold lower than that of the wild-type strain. A null mutation eliminating CwlJ2 has no effect on germination. High-performance liquid chromatography and mass spectroscopy analysis revealed that SleB is required for lytic transglycosylase activity. CwlJ1 also clearly participates in cortex hydrolysis, but its specific mode of action remains unclear. Understanding the lytic germination activities that naturally diminish spore resistance can lead to methods for prematurely inducing them, thus simplifying the process of treating contaminated sites.

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* Corresponding author. Mailing address: Department of Biological Sciences, Virginia Tech, Life Sciences I, MC0910, Washington St., Blacksburg, VA 24061. Phone: (540) 231-2529. Fax: (540) 231-4043. E-mail: dpopham{at}vt.edu

Published ahead of print on 30 January 2009.

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

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Journal of Bacteriology, April 2009, p. 2237-2247, Vol. 191, No. 7
0021-9193/09/$08.00+0     doi:10.1128/JB.01598-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Giebel, J. D., Carr, K. A., Anderson, E. C., Hanna, P. C. (2009). The Germination-Specific Lytic Enzymes SleB, CwlJ1, and CwlJ2 Each Contribute to Bacillus anthracis Spore Germination and Virulence. J. Bacteriol. 191: 5569-5576 [Abstract] [Full Text]