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A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination.

ABSTRACT: The Bacillus subtilis sleB gene, which codes for the enzyme homologous to the germination-specific amidase from Bacillus cereus, was cloned and its nucleotide sequence was determined. Sequence analysis showed that it had an open reading frame of 918 bp, coding for a polypeptide of 305 amino acids with a putative signal sequence of 29 residues. Enzyme activity was not found in germination exudate of B. subtilis spores, which differs from the case of B. cereus enzyme. A B. subtilis mutant with an insertionally inactivated sleB gene revealed normal behavior in growth and sporulation. However, the sleB mutant was unable to complete germination mediated by L-alanine.

SUBMITTER: Moriyama R

PROVIDER: S-EPMC178467 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

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A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination.

Moriyama R R Hattori A A Miyata S S Kudoh S S Makino S S

Journal of bacteriology 19961001 20

The Bacillus subtilis sleB gene, which codes for the enzyme homologous to the germination-specific amidase from Bacillus cereus, was cloned and its nucleotide sequence was determined. Sequence analysis showed that it had an open reading frame of 918 bp, coding for a polypeptide of 305 amino acids with a putative signal sequence of 29 residues. Enzyme activity was not found in germination exudate of B. subtilis spores, which differs from the case of B. cereus enzyme. A B. subtilis mutant with an ...[more]

PMID: 8830707

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Project description:Spore-forming bacteria of the orders Bacillales and Clostridiales play a major role in food spoilage and foodborne diseases. When environmental conditions become favorable, these spores can germinate as the germinant receptors located on the spore's inner membrane are activated via germinant binding. This leads to the formation of vegetative cells via germination and subsequent outgrowth and potential deleterious effects on foods. The present report focuses on analysis of the synthesis of the MalS (malic enzyme) protein during Bacillus subtilis spore germination by investigating the dynamics of the presence and fluorescence level of a MalS-GFP (MalS-green fluorescent protein) fusion protein using time-lapse fluorescence microscopy. Our results show an initial increase in MalS-GFP fluorescence intensity within the first 15 min of germination, followed by a discernible drop and stabilization of the fluorescence throughout spore outgrowth as reported previously (L. Sinai, A. Rosenberg, Y. Smith, E. Segev, and S. Ben-Yehuda, Mol Cell 57:695-707, 2015, https://doi.org/10.1016/j.molcel.2014.12.019). However, in contrast to the earlier report, both Western blotting and SILAC (stable isotopic labeling of amino acids in cell culture) analysis showed there was no increase in MalS-GFP levels during the 15 min after the addition of germinants and that MalS synthesis did not begin until more than 90 min after germinant addition. Thus, the increase in MalS-GFP fluorescence early in germination is not due to new protein synthesis but is perhaps due to a change in the physical environment of the spore cores. Our findings also show that different sporulation conditions and spore maturation times affect expression of MalS-GFP and the germination behavior of the spores, albeit to a minor extent, but still result in no changes in MalS-GFP levels early in spore germination.IMPORTANCE The spores formed by Bacillus subtilis remain in a quiescent state for extended periods due to their dormancy and resistance features. Dormancy is linked to a very low level of core water content and a phase-bright state of spores. The present report, focusing on proteins MalS and PdhD (pyruvate dehydrogenase subunit D) and complementary to our companion report published in this issue, aims to shed light on a major dilemma in the field, i.e., whether protein synthesis, in particular that of MalS, takes place in phase-bright spores. Clustered MalS-GFP in dormant spores diffuses throughout the spore as germination proceeds. However, fluorescence intensity measurements, supported by Western blot analysis and SILAC proteomics, confirm that there is no new MalS protein synthesis in bright-phase dormant spores.

Project description:When Bacillus subtilis spores detect nutrients, they exit dormancy through the processes of germination and outgrowth. A key step in germination is the activation of two functionally redundant cell wall hydrolases (SleB and CwlJ) that degrade the specialized cortex peptidoglycan that surrounds the spore. How these enzymes are regulated remains poorly understood. To identify additional factors that affect their activity, we used transposon sequencing to screen for synthetic germination defects in spores lacking SleB or CwlJ. Other than the previously characterized protein YpeB, no additional factors were found to be specifically required for SleB activity. In contrast, our screen identified SafA and YlxY (renamed SwsB) in addition to the known factors GerQ and CotE as proteins required for CwlJ function. SafA is a member of the spore's proteinaceous coat and we show that, like GerQ and CotE, it is required for accumulation and retention of CwlJ in the dormant spore. SwsB is broadly conserved among spore formers, and we show that it is required for CwlJ to efficiently degrade the cortex during germination. Intriguingly, SwsB resembles polysaccharide deacetylases, and its putative catalytic residues are required for its role in germination. However, we find no chemical signature of its activity on the spore cortex or in vitro While the precise, mechanistic role of SwsB remains unknown, we explore and discuss potential activities.IMPORTANCE Spore formation in Bacillus subtilis has been studied for over half a century, and virtually every step in this developmental process has been characterized in molecular detail. In contrast, how spores exit dormancy remains less well understood. A key step in germination is the degradation of the specialized cell wall surrounding the spore called the cortex. Two enzymes (SleB and CwlJ) specifically target this protective layer, but how they are regulated and whether additional factors promote their activity are unknown. Here, we identified the coat protein SafA and a conserved but uncharacterized protein YlxY as additional factors required for CwlJ-dependent degradation of the cortex. Our analysis provides a more complete picture of this essential step in the exit from dormancy.

Dataset Information

A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination.

Publications

A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination.

Similar Datasets

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