Raphael, S. D. R. | bioRxiv Science

bioRxiv2026

Three catalase-peroxidases promote extended stationary phase survival of Vibrio natriegens during ecologically relevant exposures to exogenous hydrogen peroxide

Vibrio natriegens is an emerging model organism in laboratory and biotechnology research that is known for its fast growth rate and diverse metabolic capabilities. However, little is known about its response to oxidative stress. Reactive oxygen species (ROS) are ubiquitous stressors for most aerobic life and without mitigation can lead to cellular damage and sometimes death. V. natriegens is unusual amongst the Vibrio genus and Proteobacterial phylum in having three copies of katG encoding the bifunctional ROS defense enzyme catalase-peroxidase. The biological significance of having three copies of this protective gene was investigated with phylogenetics and gene inactivation studies. We determined that two of the katG copies arose recently via duplication and subsequent divergence. Analysis of single, double, and triple {Delta}katG constructs revealed each katG gene product contributes to survival during exposure to ecologically relevant concentrations of exogenous hydrogen peroxide (HOOH) under stationary phase conditions but found the genes were dispensable for HOOH resistance during exponential growth. We also demonstrated the involvement of the RpoS regulon in V. natriegens oxidative stress response in stationary phase. As an outcome of this investigation, we identified and repaired several spontaneous loss-of-function mutations of rpoS in laboratory cultures of the V. natriegens type strain ATCC 14048. Together, these results provide physiological and evolutionary insights into V. natriegens ROS response.

microbiology↗

bioRxiv2023

Fitness Tradeoffs of Multidrug Efflux Pumps in Escherichia coli K-12 in Acid or Base, and with Aromatic Phytochemicals

Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness tradeoffs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH is important as it contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-cultured Escherichia coli K-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with CCCP, or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemicals lipophilicity (logP). MdtEF-TolC and EmrAB-TolC each conferred a fitness cost at pH 5.5, but salicylate and benzoate conferred a net positive fitness contribution for the pump. Expression of pump genes was measured by digital PCR. Between pH 5.5 - 8.0, acrA and emrA were upregulated in log phase, whereas mdtE expression was upregulated in transition-to-stationary phase and at pH 5.5 in log phase. Methyl salicylate did not affect pump gene expression, despite selecting against AcrAB-TolC. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without positive section for others. IMPORTANCEFor drugs that are administered orally, we need to understand how ingested phytochemicals modulate intrinsic drug resistance in our gut microbiome. Intrinsic drug resistance of bacteria is mediated by PMF-driven pumps that efflux many different antibiotics and cell waste products. These pumps play a key role in bacterial defense by conferring low-level resistance to antimicrobial agents at first exposure, while providing time for a pathogen to evolve resistance to higher levels of the antibiotic exposed. Nevertheless, efflux pumps confer energetic costs due to gene expression and pump energy expense. The bacterial PMF includes the transmembrane pH difference ({Delta}pH) which may be depleted by permeant acids and membrane disruptors. Understanding the fitness costs of efflux pumps may enable us to develop resistance breakers, that is, molecules that work together with antibiotics to potentiate their effect. We show that different pumps have distinct selection criteria, and we identified non-acidic aromatic molecules as promising candidates for drug resistance breakers.

microbiology↗