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Activity and regulation of various forms of CwlJ, SleB, and YpeB proteins in degrading cortex peptidoglycan of spores of Bacillus species in vitro and during spore germination.

ABSTRACT: Germination of Bacillus spores requires degradation of a modified layer of peptidoglycan (PG) termed the spore cortex by two redundant cortex-lytic enzymes (CLEs), CwlJ and SleB, plus SleB's partner protein, YpeB. In this study, in vitro and in vivo analyses have been used to clarify the roles of individual SleB and YpeB domains in PG degradation. Purified mature Bacillus cereus SleB without its signal sequence (SleB(M)) and the SleB C-terminal catalytic domain (SleB(C)) efficiently triggered germination of decoated Bacillus megaterium and Bacillus subtilis spores lacking endogenous CLEs; previously, SleB's N-terminal domain (SleB(N)) was shown to bind PG but have no enzymatic activity. YpeB lacking its putative membrane anchoring sequence (YpeB(M)) or its N- and C-terminal domains (YpeB(N) and YpeB(C)) alone did not exhibit degradative activity, but YpeB(N) inhibited SleB(M) and SleB(C) activity in vitro. The severe germination defect of B. subtilis cwlJ sleB or cwlJ sleB ypeB spores was complemented by ectopic expression of full-length sleB [sleB(FL)] and ypeB [ypeB(FL)], but normal levels of SleB(FL) in spores required normal spore levels of YpeB(FL) and vice versa. sleB(FL) or ypeB(FL) alone, sleB(FL) plus ypeB(C) or ypeB(N), and sleB(C) or sleB(N) plus ypeB(FL) did not complement the cortex degradation defect in cwlJ sleB ypeB spores. In addition, ectopic expression of sleB(FL) or cwlJ(FL) with a Glu-to-Gln mutation in a predicted active-site residue failed to restore the germination of cwlJ sleB spores, supporting the role of this invariant glutamate as the key catalytic residue in SleB and CwlJ.

SUBMITTER: Li Y

PROVIDER: S-EPMC3676066 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Activity and regulation of various forms of CwlJ, SleB, and YpeB proteins in degrading cortex peptidoglycan of spores of Bacillus species in vitro and during spore germination.

Li Yunfeng Y Butzin Xuan Y XY Davis Andrew A Setlow Barbara B Korza George G Üstok Fatma Işik FI Christie Graham G Setlow Peter P Hao Bing B

Journal of bacteriology 20130329 11

Germination of Bacillus spores requires degradation of a modified layer of peptidoglycan (PG) termed the spore cortex by two redundant cortex-lytic enzymes (CLEs), CwlJ and SleB, plus SleB's partner protein, YpeB. In this study, in vitro and in vivo analyses have been used to clarify the roles of individual SleB and YpeB domains in PG degradation. Purified mature Bacillus cereus SleB without its signal sequence (SleB(M)) and the SleB C-terminal catalytic domain (SleB(C)) efficiently triggered ge ...[more]

PMID: 23543708

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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

Activity and regulation of various forms of CwlJ, SleB, and YpeB proteins in degrading cortex peptidoglycan of spores of Bacillus species in vitro and during spore germination.

Publications

Activity and regulation of various forms of CwlJ, SleB, and YpeB proteins in degrading cortex peptidoglycan of spores of Bacillus species in vitro and during spore germination.

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