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Warzecha, D. J.

Publications and source records attributed to Warzecha, D. J..

2 recordsLinked to original sources
bioRxiv2025

Chain-length regulation by WzzE is necessary for, but genetically separable from, cyclic enterobacterial common antigen synthesis

Enterobacterial common antigen (ECA) is a conserved glycan that supports outer membrane impermeability and intrinsic antibiotic resistance in Enterobacterales. ECA exists in outer membrane diacylglycerol-phosphate- and lipopolysaccharide-linked forms, and a cyclic periplasmic form (ECACYC). Intriguingly, ECACYC both affects the outer-membrane permeability barrier and functions in regulation of diacylglycerol-phosphate-linked ECA levels. While the length of linear ECA polymers generated by WzyE is regulated by the co-polymerase WzzE, WzzE is also required for ECACYC biogenesis and no ECACYC is synthesized in its absence. To uncover WzzE functions necessary for ECACYC biosynthesis, we generated plasmid-borne and chromosomal wzzE mutants in Escherichia coli K-12 and quantified their effects on linear chain-length regulation and on ECACYC synthesis. Mutations affecting linear ECA chain-length regulation in either transmembrane helix 2 or the periplasmic domain abolished ECACYC synthesis. Notably, two variants at residue F104 exhibited identical linear ECA chain-length regulation which was similar to wild type, but resulted in sharply different ECACYC production: chromosomal WzzEF104Y produced near wild-type levels of ECACYC, whereas WzzEF104H produced approximately two-fold less ECACYC. This difference in ECACYC production vs. linear chain-length regulation demonstrates ECACYC synthesis is genetically separable from this regulation. Thus, WzzE-mediated chain-length regulation is necessary for ECACYC biogenesis, but not sufficient.

microbiology↗
bioRxiv2025

Impaired envelope integrity in the absence of SanA is linked to increased Lipid II availability and an imbalance of FtsI and FtsW activities

In Gram-negative bacteria, the outer membrane (OM) acts in conjunction with the peptidoglycan (PG) cell wall as a barrier against physical, osmotic, and chemical environmental stressors including antibiotics. SanA, an inner membrane protein in Escherichia coli K-12, is required for vancomycin resistance at high temperatures (>42 {degrees}C) and impacts sodium dodecyl sulfate (SDS) resistance during stationary phase reached from carbon limitation. However, its function remains unknown. Here, we show that {Delta}sanA has a synthetic genetic interaction with {Delta}wecA, a mutation that increases the availability of the isoprenoid carrier for PG synthesis. Specifically, the {Delta}sanA {Delta}wecA strain demonstrated heightened SDS-EDTA sensitivity, activation of the Rcs stress response, and increased cell length. Further investigation tied the SDS-EDTA sensitivity to increased lipid II available for PG synthesis. Spontaneous suppressor mutants of this phenotype harbored point mutations in prc, which encodes tail specific protease, or ftsI, which encodes the cell division DD-transpeptidase, a target of Prc. We focused on the ftsI mutations and demonstrated that the ftsI mutations had increased cell length but nevertheless enhanced PG incorporation at the septum compared to the {Delta}sanA mutant, returning PG incorporation to wild-type levels. Moreover, other mutations affecting septal PG synthesis, but not divisome assembly, also suppressed the SDS-EDTA sensitivity. These findings suggest that, in the absence of SanA, increased lipid II availability perturbs the balance between septal PG synthesis, lateral PG elongation, and other envelope biogenesis pathways, which leads to increased OM permeability. IMPORTANCEThe Gram-negative cell envelope is a barrier that protects the cell from environmental stress. Therefore, the synthesis of each layer of this envelope needs to be closely coordinated throughout growth and division. Here, we investigated SanA, a protein in Escherichia coli K-12 that affects envelope permeability under cellular stress, including nutrient limitation and high temperature. We found that SanA plays a key role in maintaining the permeability barrier when precursor levels for peptidoglycan (PG) synthesis are elevated, linking envelope integrity to balanced septal PG production during cell division. Our results suggest that SanA modulates substrate availability to preserve envelope function, and that in its absence, imbalanced substrate flux to septal PG synthesis disrupts septum formation and compromises barrier integrity.

microbiology↗