Biology subjects

Weaver, A. I.

Publications and source records attributed to Weaver, A. I..

5 recordsLinked to original sources

bioRxiv2026

Staphylococcus aureus uses a eukaryotic-like uridyltransferase to make UDP-GlcNAc for cell wall synthesis

UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite used to build the peptidoglycan cell wall that protects bacteria against lysis caused by high turgor pressure. More than thirty years ago, GlmU, which contains both acetyltransferase and uridyltransferase activities, was discovered to make UDP-GlcNAc in E. coli, and it is now widely considered essential for UDP-GlcNAc synthesis across bacteria. Here we report that the clinically important pathogen Staphylococcus aureus relies on a previously uncharacterized uridyltransferase called NagU to synthesize most of its UDP-GlcNAc for cell wall synthesis. We also report that NagU binds to and negatively regulates DacA, the diadenylate cyclase that makes the essential second messenger cyclic di-AMP, which regulates turgor pressure. We propose a model in which increasing NagU expression simultaneously increases flux into peptidoglycan and increases turgor pressure to enable cell wall expansion. Finally, an evolutionary analysis shows that many bacteria beyond Staphylococcaceae encode NagU homologues but lack GlmU either entirely or in part, suggesting that alternative UDP-GlcNAc biosynthesis pathways are not rare.

microbiology↗

bioRxiv2026

The transcription factor Vca0578 (DsvR) mediated expression of ZapC is required to promote cell division during lytic transglycosylase insufficiency in Vibrio cholerae

The bacterial peptidoglycan (PG) cell wall, a polymer made of amino-acid-bearing glycan strands, maintains cell shape, provides structural integrity, and protects against osmotic lysis. PG maintenance is an active process that requires regulated PG breakdown to make space for insertion of new PG strands. PG breakdown is accomplished by autolysins, i.e. endogenous enzymes with cell wall cleavage activity. The lytic transglycosylases (LTGs), a class of autolysins, for example, cleave glycan strands during PG remodelling. LTGs are broadly conserved and are highly redundant in bacteria, but their physiological role is poorly-defined. In this study, we interrogated physiological consequences of LTG insufficiency in Vibrio cholerae using TnSeq to gain insights about roles of these enzymes. We identify an uncharacterized transcription factor, Vca0578, which alleviates defects associated with the {Delta}6LTG mutant. We demonstrate that Vca0578 positively regulates the expression of zapC, a typically non-essential Z-ring associated protein. In the absence of zapC, cell division was impaired during perturbations of cell envelope homeostasis caused by absence of LTGs, or by exposure to antibiotics inhibiting cell elongation; either condition rendered zapC conditionally essential. This essentiality could be overcome by increasing FtsZ levels. Lastly, we found that ZapC also contributes to both width and length homeostasis during normal growth. This work thus uncovers a novel transcriptional circuit that contributes to effective cell division in{Delta} 6LTG cells, and suggests an essential role for ZapC in cell division under stress conditions that cause perturbation of cell width homeostasis. AUTHOR SUMMARYBacteria must maintain their outer shell (the cell envelope) in the face of changes in the environment. For this, they use elaborate systems that remodel the cell envelope. How some of these systems work is not well understood. In this study, we describe a new gene circuit that is required to keep cells dividing when the cell envelope is compromised. We found that Vca0578, a putative transcription factor, controls expression of the zapC gene. The protein ZapC then helps bacteria grow and divide when the cell envelope is under stress, for example, in the presence of certain antibiotics. Thus, we have discovered a regulatory circuit that promotes bacterial growth and antibiotic resistance under stress.

microbiology↗

bioRxiv2021

Lytic transglycosylases mitigate periplasmic crowding by degrading soluble cell wall turnover products

The peptidoglycan cell wall is a predominant structure of bacteria, determining cell shape and supporting survival in diverse conditions. Peptidoglycan is dynamic and requires regulated synthesis of new material, remodeling, and turnover - or autolysis - of old material. Despite exploitation of peptidoglycan synthesis as an antibiotic target, we lack a fundamental understanding of how peptidoglycan synthesis and autolysis intersect to maintain the cell wall. Here, we uncover a critical physiological role for a widely misunderstood class of autolytic enzymes, lytic transglycosylases (LTGs). We demonstrate that LTG activity is essential to survival by contributing to periplasmic processes upstream and independent of peptidoglycan recycling. Defects accumulate in Vibrio cholerae LTG mutants due to generally inadequate LTG activity, rather than absence of specific enzymes, and essential LTG activities are likely independent of protein-protein interactions, as heterologous expression of a non-native LTG rescues growth of a conditionally LTG-null mutant. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains, also detected in the wild-type, are enriched in LTG mutants, and that LTG mutants are hypersusceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that LTGs prevent toxic crowding of the periplasm with synthesis-derived peptidoglycan polymers and contrary to prevailing models, that this autolytic function can be temporally separate from peptidoglycan synthesis.

microbiology↗

bioRxiv2019

Lytic transglycosylases RlpA and MltC assist in Vibrio cholerae daughter cell separation

The cell wall is a crucial structural feature in the vast majority of bacteria and comprises a rigid, covalently closed, mesh-like network of peptidoglycan (PG) strands. While PG synthesis is important for bacterial survival under many conditions, the cell wall is also a dynamic structure, undergoing degradation and remodeling by so-called \"autolysins\", enzymes that break bonds in the PG network. Cell division, for example, requires extensive PG remodeling and separation of daughter cells, which depends primarily upon the activity of amidases. However, in V. cholerae, we have found that amidase activity alone is insufficient for daughter cell separation and that the lytic transglycosylases RlpA and MltC both contribute to this process. MltC and RlpA both localize to the septum and are functionally redundant under normal laboratory conditions; however, only RlpA can support normal cell separation in low salt media. The division-specific activity of lytic transglycosylases has implications for the local structure of septal PG, suggesting that there may be glycan bridges between daughter cells that cannot be resolved by amidases. We propose that lytic transglycosylases at the septum serve as a back-up mechanism to cleave rare, stochastically produced PG strands that are crosslinked beyond the reach of the highly spatio-temporally limited activity of the amidase and to clear PG debris that may block the completion of outer-membrane invagination.

microbiology↗

bioRxiv2018

Genetic determinants of penicillin tolerance in Vibrio cholerae

Many bacteria are resistant to killing (\"tolerant\") by typically bactericidal antibiotics due to their ability to counteract drug-induced cell damage. Vibrio cholerae, the cholera agent, displays an unusually high tolerance to diverse inhibitors of cell wall synthesis. Exposure to these agents, which in other bacteria leads to lysis and death, results in a breakdown of the cell wall and subsequent sphere formation in V. cholerae. Spheres readily recover to rod-shaped cells upon antibiotic removal, but the mechanisms mediating the recovery process are not well-characterized. Here, we found that the mechanisms of recovery are dependent on environmental conditions. Interestingly, on agarose pads, spheres undergo characteristic stages during the restoration of rod shape. Drug inhibition and microscopy experiments suggest that class A Penicillin Binding Proteins (aPBPs) play a more active role than the Rod system, especially early in sphere recovery. TnSeq analyses revealed that LPS and cell wall biogenesis genes as well as the sigma E cell envelope stress response were particularly critical for recovery. LPS core and O-antigen appear to be more critical for sphere formation/integrity and viability than Lipid A modifications. Overall, our findings demonstrate that the outer membrane is a key contributor to beta lactam tolerance and suggest a role for aPBPs in cell wall biogenesis in the absence of rod-shape cues. Factors required for post-antibiotic recovery could serve as targets for antibiotic adjuvants that enhance the efficacy of antibiotics that inhibit cell wall biogenesis.

microbiology↗