Dataset Information

MltG activity antagonizes cell wall synthesis by both types of peptidoglycan polymerases in Escherichia coli.

ABSTRACT: Bacterial cells are surrounded by a peptidoglycan (PG) cell wall. This structure is essential for cell integrity and its biogenesis pathway is a key antibiotic target. Most bacteria utilize two types of synthases that polymerize glycan strands and crosslink them: class A penicillin-binding proteins (aPBPs) and complexes of SEDS proteins and class B PBPs (bPBPs). Although the enzymatic steps of PG synthesis are well characterized, the steps involved in terminating PG glycan polymerization remain poorly understood. A few years ago, the conserved lytic transglycosylase MltG was identified as a potential terminase for PG synthesis in Escherichia coli. However, characterization of the in vivo function of MltG was hampered by the lack of a growth or morphological phenotype in ΔmltG cells. Here, we report the isolation of MltG-defective mutants as suppressors of lethal deficits in either aPBP or SEDS/bPBP PG synthase activity. We used this phenotype to perform a domain-function analysis for MltG, which revealed that access to the inner membrane is important for its in vivo activity. Overall, our results support a model in which MltG functions as a terminase for both classes of PG synthases by cleaving PG glycans as they are being actively synthesized.

SUBMITTER: Bohrhunter JL

PROVIDER: S-EPMC9020800 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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MltG activity antagonizes cell wall synthesis by both types of peptidoglycan polymerases in Escherichia coli.

Bohrhunter Jessica L JL Rohs Patricia D A PDA Torres Grasiela G Yunck Rachel R Bernhardt Thomas G TG

Molecular microbiology 20201219 6

Bacterial cells are surrounded by a peptidoglycan (PG) cell wall. This structure is essential for cell integrity and its biogenesis pathway is a key antibiotic target. Most bacteria utilize two types of synthases that polymerize glycan strands and crosslink them: class A penicillin-binding proteins (aPBPs) and complexes of SEDS proteins and class B PBPs (bPBPs). Although the enzymatic steps of PG synthesis are well characterized, the steps involved in terminating PG glycan polymerization remain ...[more]

PMID: 33278861

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Project description:Bacterial morphology is largely determined by the spatial and temporal regulation of peptidoglycan (PG) biosynthesis. Ovococci possess a unique pattern of PG synthesis different from the well studied Bacillus, and the mechanism of the coordination of PG synthesis remains poorly understood. Several regulatory proteins have been identified to be involved in the regulation of ovococcal morphogenesis, among which DivIVA is an important one to regulate PG synthesis in streptococci, while its mechanism is largely unknown. Here, the zoonotic pathogen Streptococcus suis was used to investigate the regulation of DivIVA on PG synthesis. Fluorescent d-amino acid probing and 3D-structured illumination microscopy found that DivIVA deletion caused abortive peripheral PG synthesis, resulting in a decreased aspect ratio. The phosphorylation-depleted mutant (DivIVA3A) cells displayed a longer nascent PG and became longer, whereas the phosphorylation-mimicking mutant (DivIVA3E) cells showed a shorter nascent PG and became shorter, suggesting that DivIVA phosphorylation is involved in regulating peripheral PG synthesis. Several DivIVA-interacting proteins were identified, and the interaction was confirmed between DivIVA and MltG, a cell wall hydrolase essential for cell elongation. DivIVA did not affect the PG hydrolysis activity of MltG, while the phosphorylation state of DivIVA affected its interaction with MltG. MltG was mislocalized in the ΔdivIVA and DivIVA3E cells, and both ΔmltG and DivIVA3E cells formed significantly rounder cells, indicating an important role of DivIVA phosphorylation in regulating PG synthesis through MltG. These findings highlight the regulatory mechanism of PG synthesis and morphogenesis of ovococci. IMPORTANCE The peptidoglycan (PG) biosynthesis pathway provides a rich source of novel antimicrobial drug targets. However, bacterial PG synthesis and its regulation is a very complex process involving dozens of proteins. Moreover, unlike the well studied Bacillus, ovococci undergo unusual PG synthesis with unique mechanisms of coordination. DivIVA is an important regulator of PG synthesis in ovococci, while its exact role in regulating PG synthesis remains poorly understood. In this study, we determined the role of DivIVA in regulating lateral PG synthesis of Streptococcus suis and identified a critical interacting partner, MltG, in which DivIVA influenced the subcellular localizations of MltG through its phosphorylation. Our study characterizes the detailed role of DivIVA in regulating bacterial PG synthesis, which is very helpful for understanding the process of PG synthesis in streptococci.

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.

Dataset Information

MltG activity antagonizes cell wall synthesis by both types of peptidoglycan polymerases in Escherichia coli.

Publications

MltG activity antagonizes cell wall synthesis by both types of peptidoglycan polymerases in Escherichia coli.

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