Dataset Information

SweC and SweD are essential co-factors of the FtsEX-CwlO cell wall hydrolase complex in Bacillus subtilis.

ABSTRACT: The peptidoglycan (PG) sacculus is composed of long glycan strands cross-linked together by short peptides forming a covalently closed meshwork that protects the bacterial cell from osmotic lysis and specifies its shape. PG hydrolases play essential roles in remodeling this three-dimensional network during growth and division but how these autolytic enzymes are regulated remains poorly understood. The FtsEX ABC transporter-like complex has emerged as a broadly conserved regulatory module in controlling cell wall hydrolases in diverse bacterial species. In most characterized examples, this complex regulates distinct PG hydrolases involved in cell division and is intimately associated with the cytokinetic machinery called the divisome. However, in the gram-positive bacterium Bacillus subtilis the FtsEX complex is required for cell wall elongation where it regulates the PG hydrolase CwlO that acts along the lateral cell wall. To investigate whether additional factors are required for FtsEX function outside the divisome, we performed a synthetic lethal screen taking advantage of the conditional essentiality of CwlO. This screen identified two uncharacterized factors (SweD and SweC) that are required for CwlO activity. We demonstrate that these proteins reside in a membrane complex with FtsX and that amino acid substitutions in residues adjacent to the ATPase domain of FtsE partially bypass the requirement for them. Collectively our data indicate that SweD and SweC function as essential co-factors of FtsEX in controlling CwlO during cell wall elongation. We propose that factors analogous to SweDC function to support FtsEX activity outside the divisome in other bacteria.

SUBMITTER: Brunet YR

PROVIDER: S-EPMC6705773 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

SweC and SweD are essential co-factors of the FtsEX-CwlO cell wall hydrolase complex in Bacillus subtilis.

Brunet Yannick R YR Wang Xindan X Rudner David Z DZ

PLoS genetics 20190822 8

The peptidoglycan (PG) sacculus is composed of long glycan strands cross-linked together by short peptides forming a covalently closed meshwork that protects the bacterial cell from osmotic lysis and specifies its shape. PG hydrolases play essential roles in remodeling this three-dimensional network during growth and division but how these autolytic enzymes are regulated remains poorly understood. The FtsEX ABC transporter-like complex has emerged as a broadly conserved regulatory module in cont ...[more]

PMID: 31437162

Similar Datasets

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.

Project description:In this study, a sporulation-specific gene (tentatively named cwlC) involved in mother cell lysis in Bacillus thuringiensis was characterized. The encoded CwlC protein consists of an N-terminal N-acetylmuramoyl-l-alanine amidase (MurNAc-LAA) domain and a C-terminal amidase02 domain. The recombinant histidine-tagged CwlC proteins purified from Escherichia coli were able to directly bind to and digest the B. thuringiensis cell wall. The CwlC point mutations at the two conserved glutamic acid residues (Glu-24 and Glu-140) shown to be critical for the catalytic activity in homologous amidases resulted in a complete loss of cell wall lytic activity, suggesting that CwlC is an N-acetylmuramoyl-l-alanine amidase. Results of transcriptional analyses indicated that cwlC is transcribed as a monocistronic unit and that its expression is dependent on sporulation sigma factor K (σK). Deletion of cwlC completely blocked mother cell lysis during sporulation without impacting the sporulation frequency, Cry1Ac protein production, and insecticidal activity. Taken together, our data suggest that CwlC is an essential cell wall hydrolase for B. thuringiensis mother cell lysis during sporulation. Engineered B. thuringiensis strains targeting cwlC, which allows the crystal inclusion to remain encapsulated in the mother cell at the end of sporulation, may have the potential to become more effective biological control agents in agricultural applications since the crystal inclusion remains encapsulated in the mother cell at the end of sporulation.IMPORTANCE Mother cell lysis has been well studied in Bacillus subtilis, which involves three distinct yet functionally complementary cell wall hydrolases. In this study, a novel cell wall hydrolase, CwlC, was investigated and found to be essential for mother cell lysis in Bacillus thuringiensis CwlC of B. thuringiensis only shows 9 and 21% sequence identity with known B. subtilis mother cell hydrolases CwlB and CwlC, respectively, suggesting that mechanisms of mother cell lysis may differ between B. subtilis and B. thuringiensis The cwlC gene deletion completely blocked the release of spores and crystals from the mother cell without affecting insecticidal activity. This may provide a new effective strategy for crystal encapsulation against UV light inactivation.

Dataset Information

SweC and SweD are essential co-factors of the FtsEX-CwlO cell wall hydrolase complex in Bacillus subtilis.

Publications

SweC and SweD are essential co-factors of the FtsEX-CwlO cell wall hydrolase complex in Bacillus subtilis.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets