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Roles of germination-specific lytic enzymes CwlJ and SleB in Bacillus anthracis.

ABSTRACT: 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.

SUBMITTER: Heffron JD

PROVIDER: S-EPMC2655533 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Roles of germination-specific lytic enzymes CwlJ and SleB in Bacillus anthracis.

Heffron Jared D JD Orsburn Benjamin B Popham David L DL

Journal of bacteriology 20090130 7

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 ...[more]

PMID: 19181808

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Project description:BackgroundBacillus cells faced with unfavorable environmental conditions undergo an asymmetric division process ultimately leading to the formation of the bacterial spore. In some instances the spore serves as an infectious agent; such is the case with the spore of Bacillus anthracis and the disease anthrax. Spores are resistant to a variety of environment conditions including traditional decontamination techniques due to the formation of specialized cellular structures. One such structure, the spore cortex, is a thick layer of modified peptidoglycan that contributes to spore dormancy through maintenance of the dehydrated state of the spore core. During spore germination, degradation of the cortex is required to facilitate complete hydration of the core and a return to vegetative growth. Degradation of the cortex is accomplished through the action of germination-specific lytic enzymes. One of these enzymes, SleB, has been previously shown to require the presence of the YpeB protein for its stable incorporation and subsequent function in spores of B. anthracis. The focus of the present study is to identify protein interactions of YpeB through in vivo chemical cross-linking and two-hybrid analysis.ResultsConserved residues within YpeB PepSY domains were altered to facilitate implementation of a site-specific chemical cross-linker, 4-Azidophenacyl bromide. Analyses of crosslinked-spore extracts suggests that YpeB exists as a dimer or larger multimer within the spore, potentially mediated through interactions of the C-terminal domains. Spores expressing stable truncated forms of YpeB were crosslinked and corresponding truncated dimers were detected. Further characterization of individual YpeB domains using bacterial two-hybrid analysis indicated a possible role for both N-and C-terminal domains in YpeB oligomerization.ConclusionsThe YpeB protein likely exists as dimer or higher-order multimer in the dormant spore. Both the N- and C-terminal YpeB domains contribute to multimerization. SleB likely also exists as an oligomer, and SleB and YpeB may be found together within a protein complex. Disassembly of this complex during spore germination likely allows SleB to become active in spore cortex degradation. Further study of this protein complex may contribute to the development of methods to inhibit or stimulate germination, allowing more effective spore decontamination.

Dataset Information

Roles of germination-specific lytic enzymes CwlJ and SleB in Bacillus anthracis.

Publications

Roles of germination-specific lytic enzymes CwlJ and SleB in Bacillus anthracis.

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