Dual opposing regulatory axes of EnvZ/OmpR control biofilm-mediated tolerance and OmpF-dependent resistance to β-lactams in Phytobacter diazotrophicus

Background Phytobacter diazotrophicus ( P. diazotrophicus ) is an infrequent opportunistic pathogen. Its biofilm-forming potential and the regulatory function of the EnvZ/OmpR two-component system linking biofilm formation and β-lactam resistance have yet to be elucidated. Herein, we confirmed biofilm production in P. diazotrophicus and generated envZ / ompR knockout mutants to dissect the dual regulatory roles of this two-component system. Methods In this study, biofilm formation was qualitatively and quantitatively evaluated through direct visual observation, Gram staining, scanning electron microscopy (SEM), and turbidity measurement. The minimum inhibitory concentrations (MICs) of 12 kinds of β-lactam antibiotics were determined using the MH agar plate dilution method. Gene deletion strains were constructed by homologous recombination. Quantitative reverse transcription PCR (qRT-PCR) and electrophoretic mobility shift assays (EMSA) were performed to explore the regulatory mechanism of the EnvZ/OmpR system. In addition, swimming motility assays were used to characterize biofilm-associated motility phenotypes, and ethidium bromide (EB) accumulation assays were applied to assess bacterial outer membrane permeability. Results P. diazotrophicus formed robust biofilms on coverslips at the air-liquid–solid interface when cultured in BHI medium, whereas biofilm formation was absent in LB medium. Relative to planktonic counterparts, biofilm-embedded cells displayed significantly elevated MICs to piperacillin, carbenicillin, and meropenem, demonstrating that biofilm matrices confer specific tolerance to these three β-lactams. Deletion of either envZ or ompR completely abolished biofilm formation compared with the wild type. qPCR assays confirmed marked transcriptional repression of csgD and fliC in the Δ ompR mutant. EMSA assays further proved that OmpR directly interacts with the promoters of fliC and csgD . Consistent with this regulatory pattern, loss of fliC drastically compromised bacterial swimming motility, which is critical for biofilm assembly. In contrast to the biofilm-associated tolerance phenotype, the Δ envZ and Δ ompR mutants displayed increased resistance to eight other β-lactam agents. The MICs of the Δ envZ and Δ ompR mutants increased by 2-fold and 4-fold, respectively, for cefotaxime. Their MICs increased by 4-, 4-, 2-, 2-, 4-, 4-, and 2-fold against cefalotin, cefoxitin, cefuroxime, ceftazidime, cefepime, aztreonam, and ertapenem, respectively. Transcriptomic profiling revealed that four outer membrane porin genes ( ompF , ompC , ompF2 , phoE ) were significantly downregulated upon ompR deletion. Single gene knockout validation showed that only the Δ ompF mutant presented elevated MICs (4-, 8-, 4-, 16-, 4-, 2-, 8-, and 8-fold) to the above eight antibiotics; mutants lacking ompC , ompF2 , or phoE exhibited no change in antimicrobial resistance. Additional EMSA results verified direct binding of OmpR to the promoter regions of ompF . EB accumulation assays revealed drastically reduced fluorescence intensity in the Δ ompF mutant versus the wild type, confirming that OmpF governs outer membrane permeability. Conclusion This study reveals a dual antagonistic regulatory mechanism of the EnvZ/OmpR system in modulating β-lactam resistance in clinical P. diazotrophicus . EnvZ/OmpR promotes biofilm formation via the csgD - fliC axis to enhance tolerance to three specific β-lactams, while it upregulates ompF to maintain outer membrane permeability and sensitize bacteria to other eight antibiotics. This study first revealed that OmpR directly regulated fliC expression. These findings clarify the molecular basis of heterogeneous β-lactam susceptibility and provide novel insights into clinical infection control of this rare pathogen.