Biology subjects

Arthur, M.

Publications and source records attributed to Arthur, M..

8 recordsLinked to original sources

bioRxiv2026

Polysaccharide release factors drive lipid-carrier recycling in bacteria

Gram-negative bacteria build their cell envelope by transporting various sugar-based precursors across the inner membrane on the shared lipid carrier undecaprenyl (also known as C55). Because the cellular pool of undecaprenyl is finite and used by multiple competing pathways, it must be efficiently regenerated to sustain envelope assembly. Yet the enzymes that release polysaccharide cargo from the lipid carrier have remained elusive, leaving a major gap in our understanding of bacterial envelope biogenesis. Here we identify three periplasmic enzymes in Escherichia coli - UprA, UprB, and UprC - that hydrolyze the bond linking polysaccharide precursors to undecaprenyl. UprA releases elongating peptidoglycan glycan strands from the inner membrane, catalyzing the long-sought terminal step of peptidoglycan synthesis. UprB cleaves the undecaprenyl-linked initiating unit common to the enterobacterial common antigen and O-antigen pathways, while UprC is the likely release factor for the colanic acid pathway. These enzymes define a widespread Upr-like protein family (formerly DUF218) as polysaccharide release factors, revealing a unifying mechanism by which bacteria coordinate envelope assembly. Because all three Upr-like proteins share a conserved catalytic core, this family represents a potential target for antibiotics that simultaneously disrupt multiple envelope pathways.

microbiology↗

bioRxiv2024

Inner membrane protein OutB is covalently attached to peptidoglycan in the γ-proteobacterium Dickeya dadantii

Gram-negative bacteria possess a multilayered envelope comprising an inner membrane (IM), a thin peptidoglycan (PG) layer, and an outer membrane (OM). In Escherichia coli and certain other {gamma}-proteobacteria, Braun lipoprotein (Lpp) covalently tethers the OM to PG. Only a few other OM proteins have been found to be covalently linked to PG in Gram-negative bacteria. Here, we showed that in the phytopathogenic {gamma}-proteobacterium Dickeya dadantii, an IM protein, OutB, is covalently attached to PG, thereby tethering itself and the associated type 2 secretion system to the cell wall. In contrast to Lpp, OutB reaches the PG layer from the IM side. By modifying the length of Lpp, which would displace the PG layer in the periplasm, we found that the elongated Lpp+21 improved OutB attachment to PG, whereas the shortened Lpp{Delta}21 reduced it. We showed that two L,D-transpeptidases, Ldt03 and Ldt84, tether Lpp and OutB to PG by the same catalytic mechanism involving the formation of an amide bond between their C-terminal lysine and the stem peptide. Ldt03 and Ldt84, each display substrate specificity for the type of peptide stem and preferentially cross-link Lpp to monomeric and dimeric muropeptide, respectively. The C-terminal Lpp-like box of OutB is almost identical to that of Lpp; it tolerates substantial amino acid substitutions and allows PG-tethering of a bona fide periplasmic protein. Thus, it seems possible that the repertoire of periplasmic and membrane proteins tethered to PG may be more extensive than currently assumed.

microbiology↗

bioRxiv2023

Peptidoglycan-tethered and free forms of the Braun lipoprotein are in dynamic equilibrium in Escherichia coli

Peptidoglycan (PG) is a giant macromolecule that completely surrounds bacterial cells and prevents lysis in hypo-osmotic environments. This net-like macromolecule is made of glycan strands linked to each other by two types of transpeptidases that form either 4[->]3 (PBPs) or 3[->]3 (LDTs) cross-links. Previously, we devised a heavy isotope-based PG full labeling method coupled to mass spectrometry to determine the mode of insertion of new-subunits into the expanding PG network (Atze et al., 2022). We showed that PG polymerization operates according to different modes for the formation of the septum and of the lateral cell walls, as well as for bacterial growth in the presence or absence of {beta}-lactams in engineered strains that can exclusively rely on LDTs for PG cross-linking when drugs are present. Here, we apply our method to the resolution of the kinetics of the reactions leading to the covalent tethering of the Braun lipoprotein (Lpp) to PG and the subsequent hydrolysis of that same covalent link. We find that Lpp and disaccharide-peptide subunits are independently incorporated into the expanding lateral cell walls. Newly synthesized septum PG appears to contain small amounts of tethered Lpp. LDTs did mediate intense shuffling of Lpp between PG stems leading to a dynamic equilibrium between the PG-tethered and free forms of Lpp.

microbiology↗

bioRxiv2022

Spatial and social structure of rewilded laboratory mice

Social behavior and social organization have major influences on individual health and fitness. Yet, biomedical research focuses on studying a few genotypes under impoverished social conditions. Understanding how lab conditions have modified social organizations of model organisms, such as lab mice, relative to natural populations is a missing link between socioecology and biomedical science. Using a common garden design, we describe the formation of social structure in the well-studied laboratory mouse strain, C57BL/6J, in replicated mixed-sex populations over 10-day trials compared to control trials with wild-derived outbred house mice in outdoor field enclosures. We focus on three key features of mouse social systems: (i) territory establishment in males, (ii) female social relationships, and (iii) the social networks formed by the populations. Male territorial behaviors were similar but muted in C57 compared to wild-derived mice. Female C57 sharply differed from wild-derived females, showing little social bias toward cage mates and exploring substantially more of the enclosures compared to all other groups. Female behavior consistently generated denser social networks in C57 than in wild-derived mice. The repeatable societies formed under field conditions highlights opportunities to experimentally study the interplay between society and individual biology using model organisms.

animal behavior and cognition↗

bioRxiv2021

Heavy isotope labeling and mass spectrometry reveal unexpected remodeling of bacterial cell wall expansion in response to drugs

Antibiotics of the {beta}-lactam (penicillin) family inactivate target enzymes called D,D-transpeptidases or penicillin-binding proteins (PBPs) that catalyze the last cross-linking step of peptidoglycan synthesis. The resulting net-like macromolecule is the essential component of bacterial cell walls that sustains the osmotic pressure of the cytoplasm. In Escherichia coli, bypass of PBPs by the YcbB L,D-transpeptidase leads to resistance to these drugs. We developed a new method based on heavy isotope labeling and mass spectrometry to elucidate PBP- and YcbB-mediated peptidoglycan polymerization. PBPs and YcbB similarly participated in single-strand insertion of glycan chains into the expanding bacterial side wall. This absence of any transpeptidase-specific signature suggests that the peptidoglycan expansion mode is determined by other components of polymerization complexes. YcbB did mediate {beta}-lactam resistance by insertion of multiple strands that were exclusively cross-linked to existing tripeptide-containing acceptors. We propose that this unprecedented mode of polymerization depends upon accumulation of linear glycan chains due to PBP inactivation, formation of tripeptides due to cleavage of existing cross-links by a {beta}-lactam-insensitive endopeptidase, and concerted cross-linking by YcbB.

biochemistry↗

bioRxiv2021

Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli

Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of {beta}-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3[->]3 cross-links compared to 4[->]3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify amongst eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4[->]3 (MepM) and 3[->]3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4[->]3 and 3[->]3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4[->]3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of {beta}-lactams.

microbiology↗

bioRxiv2018

Tryptophan fluorescence quenching in β-lactam-interacting proteins is modulated by the structure of intermediates and final products of the acylation reaction

In most bacteria, {beta}-lactam antibiotics inhibit the last cross-linking step of peptidoglycan synthesis by acylation of the active-site Ser of D,D-transpeptidases belonging to the penicillin-binding protein (PBP) family. In mycobacteria, cross-linking is mainly ensured by L,D-transpeptidases (LDTs), which are promising targets for the development of {beta}-lactam-based therapies for multidrug-resistant tuberculosis. For this purpose, fluorescence spectroscopy is used to investigate the efficacy of LDT inactivation by {beta}-lactams but the basis for fluorescence quenching during enzyme acylation remains unknown. In contrast to what has been reported for PBPs, we show here using a model L,D-transpeptidase (Ldtfm) that fluorescence quenching of Trp residues does not depend upon direct hydrophobic interaction between Trp residues and {beta}-lactams. Rather, Trp fluorescence was quenched by the drug covalently bound to the active-site Cys residue of Ldtfm. Fluorescence quenching was not quantitatively determined by the size of the drug and was not specific of the thioester link connecting the {beta}-lactam carbonyl to the catalytic Cys as quenching was also observed for acylation of the active-site Ser of {beta}-lactamase BlaC from M. tuberculosis. Fluorescence quenching was extensive for reaction intermediates containing an amine anion and for acylenzymes containing an imine stabilized by mesomeric effect, but not for acylenzymes containing a protonated {beta}-lactam nitrogen. Together, these results indicate that the extent of fluorescence quenching is determined by the status of the {beta}-lactam nitrogen. Thus, fluorescence kinetics can provide information not only on the efficacy of enzyme inactivation but also on the structure of the covalent adducts responsible for enzyme inactivation.

biochemistry↗

bioRxiv2018

In vitro and intracellular activity of imipenem combined with tedizolid, rifabutin, and avibactam against Mycobacterium abscessus

Mycobacterium abscessus has emerged as a significant pathogen responsible for chronic pulmonary infections in cystic fibrosis (CF) patients, which are difficult to treat due to resistance to a broad range of antibiotics. The initial phase of the recommended treatment in CF patients includes imipenem used without any {beta}-lactamase inhibitor in spite of the production of the {beta}-lactamase BlaMab. Here, we determine whether the addition of tedizolid, a once-daily oxazolidinone, improves the activity of imipenem alone or in combination with a {beta}-lactamase inhibitor, avibactam, and rifabutin.\n\nThe activity of the drugs was evaluated against M. abscessus CIP104536 by determining in vitro and intracellular antibacterial activities. The impact of BlaMab inhibition by avibactam on antibiotic activity was assessed by comparing CIP104536 and its {beta}-lactamase-deficient derivative ({Delta}blaMab).\n\nThe minimal inhibitory concentrations (MICs) of tedizolid against M. abscessus CIP104536 and {Delta}blaMab were 4 g/mL. Tedizolid combined with imipenem showed a moderate synergistic effect with fractional inhibitory concentration (FIC) indexes of 0.41 and 0.38 for CIP104536 and {Delta}blaMab, respectively. For both strains, the addition of tedizolid at 2 g/mL, corresponding to the peak serum concentration, increased the intracellular efficacy of imipenem at 8 and 32 g/mL. Addition of avibactam and rifabutin improved the activity of the imipenem-tedizolid combination against CIP104536S.\n\nThe imipenem-tedizolid combination should be further considered for the treatment of M. abscessus pulmonary infections in CF patients. The efficacy of the treatment might benefit from the use of a {beta}-lactamase inhibitor, such as avibactam, and the addition of rifabutin.

microbiology↗