Dataset Information

Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis. .

ABSTRACT: DNA sequencing of a region upstream of the mms223 gene of Bacillus subtilis showed the presence of two open reading frames, orf1 and orf2, which may encode 18- and 27-kDa polypeptides, respectively. The predicted amino acid sequence of the latter shows high similarity to a major autolysin of B. subtilis, CwlB, with 35% identity over 191 residues, as well as to other autolysins (CwlC, CwlM, and AmiB). The gene was tentatively named cwlD. Bright spores produced by a B. subtilis mutant with an insertionally inactivated cwlD gene were committed to germination by the addition of L-alanine, and spore darkening, a slow and partial decrease in A580, and 72% dipicolinic acid release compared with that of the wild-type strain were observed. However, degradation of the cortex was completely blocked. Spore germination of the cwlD mutant measured by colony formation after heat treatment was less than 3.7 x 10(-8). The germination deficiency of the cwlD mutant was only partially removed when the spores were treated with lysozyme. Analysis of the chromosomal transcription of cwlD demonstrated that a transcript (RNA2) appearing 3 h after initiation of sporulation may have originated from an internal sigma E-dependent promoter of the cwlD operon, and a longer transcript (RNA1) appearing 4.5 h after sporulation may have originated from a sigma G-dependent promoter upstream of the orf1 gene. The cwlD mutant harboring a B. subtilis vector plasmid containing the intact cwlD gene recovered germination at a frequency 26% of the wild-type level.

SUBMITTER: Sekiguchi J

PROVIDER: S-EPMC177368 | biostudies-other | 1995 Oct

REPOSITORIES: biostudies-other

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Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis. .

Sekiguchi J J Akeo K K Yamamoto H H Khasanov F K FK Alonso J C JC Kuroda A A

Journal of bacteriology 19951001 19

DNA sequencing of a region upstream of the mms223 gene of Bacillus subtilis showed the presence of two open reading frames, orf1 and orf2, which may encode 18- and 27-kDa polypeptides, respectively. The predicted amino acid sequence of the latter shows high similarity to a major autolysin of B. subtilis, CwlB, with 35% identity over 191 residues, as well as to other autolysins (CwlC, CwlM, and AmiB). The gene was tentatively named cwlD. Bright spores produced by a B. subtilis mutant with an inse ...[more]

PMID: 7559346

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Project description:Most bacteria are surrounded by their cell wall, containing a highly cross-linked protective envelope of peptidoglycan. To grow, bacteria must continuously remodel their wall, inserting new material and breaking old bonds. Bond cleavage is performed by cell wall hydrolases, allowing the wall to expand. Understanding the functions of individual hydrolases has been impeded by their redundancy: single knockouts usually present no phenotype. We used an exhaustive multiple-knockout approach to determine the minimal set of hydrolases required for growth in Bacillus subtilis. We identified 42 candidate hydrolases. Strikingly, we were able to remove all but two of these genes in a single strain; this "∆40" strain shows only a mild reduction in growth rate, indicating that none of the 40 hydrolases are necessary for growth. The ∆40 strain does not detectably shed old wall, suggesting that turnover is not essential for growth. The remaining hydrolases in the ∆40 strain are LytE and CwlO, previously shown to be synthetically lethal. Either can be removed in ∆40, indicating that either hydrolase alone is sufficient for cell growth. Screening of environmental conditions and biochemistry revealed that LytE activity is inhibited by Mg2+ and that RlpA-like proteins may stimulate LytE activity. Together, these results suggest that the only essential function of cell wall hydrolases in B. subtilis is to enable cell growth by expanding the wall and that LytE or CwlO alone are sufficient for this function. These experiments introduce the ∆40 strain as a tool to study hydrolase activity and regulation in B. subtilis.IMPORTANCEIn order to grow, bacterial cells must both create and break down their cell wall. The enzymes that are responsible for these processes are the target of some of our best antibiotics. Our understanding of the proteins that break down the wall- cell wall hydrolases-has been limited by redundancy among the large number of hydrolases many bacteria contain. To solve this problem, we identified 42 cell wall hydrolases in Bacillus subtilis and created a strain lacking 40 of them. We show that cells can survive using only a single cell wall hydrolase; this means that to understand the growth of B. subtilis in standard laboratory conditions, it is only necessary to study a very limited number of proteins, simplifying the problem substantially. We additionally show that the ∆40 strain is a research tool to characterize hydrolases, using it to identify three "helper" hydrolases that act in certain stress conditions.

Dataset Information

Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis. .

Publications

Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis. .

Similar Datasets

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