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A mutant Escherichia coli that attaches peptidoglycan to lipopolysaccharide and displays cell wall on its surface.

ABSTRACT: The lipopolysaccharide (LPS) forms the surface-exposed leaflet of the outer membrane (OM) of Gram-negative bacteria, an organelle that shields the underlying peptidoglycan (PG) cell wall. Both LPS and PG are essential cell envelope components that are synthesized independently and assembled by dedicated transenvelope multiprotein complexes. We have identified a point-mutation in the gene for O-antigen ligase (WaaL) in Escherichia coli that causes LPS to be modified with PG subunits, intersecting these two pathways. Synthesis of the PG-modified LPS (LPS*) requires ready access to the small PG precursor pool but does not weaken cell wall integrity, challenging models of precursor sequestration at PG assembly machinery. LPS* is efficiently transported to the cell surface without impairing OM function. Because LPS* contains the canonical vancomycin binding site, these surface-exposed molecules confer increased vancomycin-resistance by functioning as molecular decoys that titrate the antibiotic away from its intracellular target. This unexpected LPS glycosylation fuses two potent pathogen-associated molecular patterns (PAMPs).

SUBMITTER: Grabowicz M

PROVIDER: S-EPMC4296511 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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A mutant Escherichia coli that attaches peptidoglycan to lipopolysaccharide and displays cell wall on its surface.

Grabowicz Marcin M Andres Dorothee D Lebar Matthew D MD Malojčić Goran G Kahne Daniel D Silhavy Thomas J TJ

eLife 20141231

The lipopolysaccharide (LPS) forms the surface-exposed leaflet of the outer membrane (OM) of Gram-negative bacteria, an organelle that shields the underlying peptidoglycan (PG) cell wall. Both LPS and PG are essential cell envelope components that are synthesized independently and assembled by dedicated transenvelope multiprotein complexes. We have identified a point-mutation in the gene for O-antigen ligase (WaaL) in Escherichia coli that causes LPS to be modified with PG subunits, intersecting ...[more]

PMID: 25551294

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Project description:Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes to expand the cell wall matrix.

Project description:UNLABELLED:Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The thin PG layer of Escherichia coli resides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins [PBPs]) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles identified dd-carboxypeptidases (DD-CPases) that were more active in cells grown at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were grown at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that the enzyme is more stable and has a lower Km at acidic pH, explaining why PBP6b is more active at low pH. Hence, PBP6b is a specialized DD-CPase that contributes to cell shape maintenance at low pH, and E. coli appears to utilize redundant DD-CPases for normal growth under different conditions. IMPORTANCE:Escherichia coli requires peptidoglycan dd-carboxypeptidases to maintain cell shape by controlling the amount of pentapeptide substrates available to the peptidoglycan synthetic transpeptidases. Why E. coli has eight, seemingly redundant dd-carboxypeptidases has remained unknown. We now show that one of these dd-carboxypeptidases, PBP6b, is important for cell shape maintenance in acidic growth medium, consistent with the higher activity and stability of the enzyme at low pH. Hence, the presence of multiple dd-carboxypeptidases with different enzymatic properties may allow E. coli to maintain a normal cell shape under various growth conditions.

Dataset Information

A mutant Escherichia coli that attaches peptidoglycan to lipopolysaccharide and displays cell wall on its surface.

Publications

A mutant Escherichia coli that attaches peptidoglycan to lipopolysaccharide and displays cell wall on its surface.

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