Stress-response regulators tune enterobacterial common antigen synthesis to support acid and reactive nitrogen species stress resistance

Enteric bacteria must survive envelope-damaging environmental conditions in both host and environmental niches (e.g., acidic environments, reactive oxygen and nitrogen species). The Gram-negative outer membrane (OM) is central to the cell’s stress resistance, acting as a permeability barrier to antimicrobials and as an adaptable structure remodeled in response to environmental changes. Enterobacterales ( e.g. , Escherichia coli , Salmonella enterica , Klebsiella pneumoniae ) produce an invariant glycan, enterobacterial common antigen (ECA), which is localized in the OM and periplasm and contributes to the OM permeability barrier. Yet, how ECA biosynthesis is controlled and the conditions under which ECA is important remain poorly defined. Therefore, we investigated transcriptional regulation of the ECA biosynthesis wec operon in E. coli K-12. We assayed wec operon promoter activity across deletions of 36 bioinformatically identified potential regulator genes and identified four for which deletion significantly altered wec promoter activity. Of these, two repressors, GadX, a regulator of the glutamate-dependent acid stress response system, and MetJ, the master regulator of methionine biosynthesis and a reactive nitrogen species (RNS) stress regulator, bound to the wec promoter in vitro , consistent with direct transcriptional regulation. In accordance with a link between ECA and these regulators, wec promoter activity increased under acidic pH and under RNS stress, and loss of ECA impaired growth under both conditions and fitness in acidic pH. Together, these findings reveal that biosynthesis of a conserved OM glycan is embedded within dedicated environmental stress regulons and identify previously unrecognized roles for ECA in bacterial stress adaptation.