Effects of furazlocillin, a beta-lactam antibiotic which binds selectively to penicillin-binding protein 3, on Escherichia coli mutants deficient in other penicillin-binding proteins

170

363

SUMMARY Escherichia coli FtsN is a bitopic membrane protein that is essential for triggering active cell constriction. A small periplasmic subdomain (EFtsN) is required and sufficient for function, but its mechanism of action is unclear. We isolated extragenic EFtsN*-suppressing mutations that restore division in cells producing otherwise non-functional variants of FtsN. These mapped to the IC domain of FtsA in the cytoplasm and to small subdomains of the FtsB and FtsL proteins in the periplasm. All FtsB and FtsL variants allowed survival without EFtsN, but many then imposed a new requirement for interaction between the cytoplasmic domain of FtsN (NFtsN) with FtsA. Alternatively, variants of FtsA, FtsB or FtsL acted synergistically to allow cell division in the complete absence of FtsN. Strikingly, moreover, substitution of a single residue in FtsB (E56) proved sufficient to rescue ΔftsN cells as well. In FtsN+ cells, EFtsN*-suppressing mutations promoted cell fission at an abnormally small cell size, and caused cell shape and integrity defects under certain conditions. This and additional evidence support a model in which FtsN acts on either side of the membrane to induce a conformational switch in both FtsA and the FtsBLQ subcomplex to derepress septal peptidoglycan synthesis and membrane invagination.

Section: Resultsmentioning

confidence: 99%

Roles for both FtsA and the FtsBLQ subcomplex in FtsN‐stimulated cell constriction in Escherichia coli

Liu

Persons

Lee

et al. 2015

170

363

“…Although mutants lacking the cell wall synthase PBP1b are viable, they lyse at an elevated frequency (Paradis-Bleau et al, 2014) and are hypersensitive to beta-lactam antibiotics (Schmidt et al, 1981), indicating they have an attenuated capacity for PG synthesis. In such mutants, PBP1a and its activator, LpoA, become essential (Paradis-Bleau et al, 2010;Typas et al, 2010).…”

Section: A Multicopy Lethal Screen For New Cell Wall Biogenesis Factorsmentioning

confidence: 99%

Identification of MltG as a potential terminase for peptidoglycan polymerization in bacteria

Yunck

Cho

Bernhardt

2015

103

171

SUMMARY Bacterial cells are fortified against osmotic lysis by a cell wall made of peptidoglycan (PG). Synthases called penicillin-binding proteins (PBPs), the targets of penicillin and related antibiotics, polymerize the glycan strands of PG and crosslink them into the cell wall meshwork via attached peptides. The average length of glycan chains inserted into the matrix by the PBPs is thought to play an important role in bacterial morphogenesis, but polymerization termination factors controlling this process have yet to be discovered. Here, we report the identification of Escherichia coli MltG (YceG) as a potential terminase for glycan polymerization that is broadly conserved in bacteria. A clone containing mltG was initially isolated in a screen for multicopy plasmids generating a lethal phenotype in cells defective for the PG synthase PBP1b. Biochemical studies revealed that MltG is an inner membrane enzyme with endolytic transglycosylase activity capable of cleaving at internal positions within a glycan polymer. Radiolabeling experiments further demonstrated MltG-dependent nascent PG processing in vivo, and bacterial two-hybrid analysis identified an MltG-PBP1b interaction. Mutants lacking MltG were also shown to have longer glycans in their PG relative to wild-type cells. Our combined results are thus consistent with a model in which MltG associates with PG synthetic complexes to cleave nascent polymers and terminate their elongation.

“…The same lytic response can be triggered when wild-type E. coli is treated together with aztreonam and the specific Slt70 inhibitor, bulgecin (Templin et al, 1992). As aztreonam, by specifically blocking septum formation, normally causes E. coli to form stable filaments (Schmidt et al, 1981), autolysis of cells that are deficient in a specific murein hydrolases (Slt70) was a puzzling result. To our excitement, it turned out that lysis does not take place when the amidase triple mutant MHD 52 is treated with a combination of aztreonam and bulgecin (Fig.…”

Section: Growth Rate and Antibiotic Resistance Of The Amidase Deletiomentioning

confidence: 99%

Involvement of N‐acetylmuramyl‐l‐alanine amidases in cell separation and antibiotic‐induced autolysis of Escherichia coli

Heidrich¹,

Templin²,

Ursinus³

et al. 2001

314

433

SummaryN-acetylmuramyl-L-alanine amidases are widely distributed among bacteria. However, in Escherichia coli, only one periplasmic amidase has been described until now, which is suggested to play a role in murein recycling. Here, we report that three amidases, named AmiA, B and C, exist in E. coli and that they are involved in splitting of the murein septum during cell division. Moreover, the amidases were shown to act as powerful autolytic enzymes in the presence of antibiotics. Deletion mutants in amiA, B and C were growing in long chains of unseparated cells and displayed a tolerant response to the normally lytic combination of aztreonam and bulgecin. Isolated murein sacculi of these chain-forming mutants showed rings of thickened murein at the site of blocked septation. In vitro, these murein ring structures were digested more slowly by muramidases than the surrounding murein. In contrast, when treated with the amidase AmiC or the endopeptidase MepA, the rings disappeared, and gaps developed at these sites in the murein sacculi. These results are taken as evidence that highly stressed murein crossbridges are concentrated at the site of blocked cell division, which, when cleaved, result in cracking of the sacculus at this site. As amidase deletion mutants accumulate trimeric and tetrameric cross-links in their murein, it is suggested that these structures mark the division site before cleavage of the septum.