Biology subjects

Breukink, E.

Publications and source records attributed to Breukink, E..

5 recordsLinked to original sources

bioRxiv2025

A novel peptidoglycan deacetylase modulates daughter cell separation in E. coli

Peptidoglycan hydrolases facilitate bacterial cell wall growth by creating space for insertion of new material and allowing physical separation of daughter cells. In Escherichia coli, three peptidoglycan amidases, AmiA, AmiB and AmiC, cleave septal peptidoglycan during cell division. The LytM-domain proteins EnvC and NlpD activate these amidases either from inside the cell or the outer membrane: EnvC binds to the cytoplasmic membrane-anchored divisome components FtsEX, and NlpD and ActS are outer membrane lipoproteins. Here we report the identification of a novel periplasmic deacetylase called SddA that removes acetyl groups from denuded peptidoglycan glycan strands, the products of amidases. SddA is a substrate for the periplasmic protease Prc, suggesting regulation via protein degradation. The sddA gene is co-expressed with the gene encoding EnvC, linking SddA function to amidase activation. Consistent with this link, the deletion of sddA alleviates phenotypes associated with lack of amidase activation, while overexpression of sddA alleviates phenotypes related to a defective Tol-Pal system and causes cell chaining due to reduced septum peptidoglycan cleavage unless envC is co-expressed. We present a model according to which SddA modulates the activation of the septum-splitting amidases during cell division. AUTHOR SUMMARYBacteria surround their cell membrane by the essential peptidoglycan (cell wall) layer to prevent bursting open due to their turgor. During cell division, bacteria produce a septum at midcell, which must be cleaved for daughter cells to separate. Here, we report the identification of a new enzyme, SddA, that modifies a particular type of peptidoglycan material, denuded glycan chains released during the splitting of septal peptidoglycan for daughter cell separation, in the Gram-negative Escherichia coli. We propose a model in which SddA modulates a switch in the septal peptidoglycan splitting, ensuring splitting is activated from the cell membrane in the early stages of cell division and from the outer membrane in the late stages.

microbiology↗

bioRxiv2023

A new antibiotic from an uncultured bacterium binds to an immutable target

Antimicrobial resistance is a leading mortality factor worldwide. Here we report the discovery of clovibactin, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant bacterial pathogens without detectable resistance. Using biochemical assays, solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, Lipid II, LipidWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate, but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the irreversible sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.

microbiology↗

bioRxiv2021

Brevibacillin 2V, a novel antimicrobial lipopeptide with an exceptional low hemolytic activity

Bacterial non-ribosomally produced peptides (NRPs) form a rich source of antibiotics, including more than twenty of these antibiotics that are used in the clinic, such as penicillin G, colistin, vancomycin, and chloramphenicol. Here we report the identification, purification, and characterization of a novel NRP, i.e., brevibacillin 2V, from Brevibacillus laterosporus DSM 25. Brevibacillin 2V has strong antimicrobial activity against Gram-positive bacterial pathogens, including difficult-to-treat antibiotic-resistant Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus. Notably, brevibacillin 2V has a much lower hemolytic activity to eukaryotic cells than previously reported NRPs of the lipo-tridecapeptide family, including other brevibacillins, which makes it a promising candidate for antibiotic development. In addition, our results demonstrate that brevibacillins display synergistic action with established antibiotics against Gram-negative bacterial pathogens. Thus, we identified and characterized a novel and promising antibiotic candidate (brevibacillin 2V) with a low hemolytic activity, which can be used either on its own or as a template for further total synthesis and modification.

microbiology↗

bioRxiv2020

Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as {beta}-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here we developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we demonstrate this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay allows unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high throughput screening for new antimicrobials.

biochemistry↗

bioRxiv2020

Biochemical and structural insights into the activation of PBP1b by the essential domain of FtsN

Peptidoglycan (PG) is an essential constituent of the bacterial cell wall. During cell division PG synthesis localizes at mid-cell under the control of a multiprotein complex, the divisome. In Escherichia coli, septal PG synthesis and cell constriction rely on the accumulation of FtsN at the division site. The region L75 to Q93 of FtsN (EFtsN) was shown to be essential and sufficient for its functioning in vivo but the specific target and the molecular mechanism remained unknown. Here, we show that EFtsN binds specifically to the major PG synthase PBP1b and is sufficient to stimulate its GTase activity. We also report the crystal structure of PBP1b in complex with EFtsN which provides structural insights into the mode of binding of EFtsN at the junction between the GTase and UB2H domains of PBP1b. Interestingly, the mutations R141A/R397A of PBP1b, within the EFtsN binding pocket, reduce the activation of PBP1b by FtsN. This mutant was unable to rescue {Delta}ponB-ponAts strain at nonpermissive temperature and induced a mild cell chaining phenotype and cell lysis. Altogether, the results show that PBP1b is a target of EFtsN and suggest that binding of FtsN to PBP1b contributes to trigger septal PG synthesis and cell constriction.

microbiology↗