While large membrane-impermeable antibiotics cannot traverse a lipid barrier, spacious importers such as TonB-dependent receptors (TBDRs) can mistakenly ferry antibiotics across the bacterial outer membrane (OM). We discovered that loss of PerA, an enigmatic β-helix protein in the OM of the oligotrophic α-proteobacterium Caulobacter crescentus, reprograms the OM TBDR proteome from ChvT that imports the glycopeptide antibiotic vancomycin to an uncharacterized TBDR (BugA) that confers sensitivity to the polypeptide antibiotic bacitracin. Both antibiotics are large zinc-binding molecules that target the peptidoglycan, echoing the zinc stress response induces destabilization of PerA. Inactivation of PerA launches two conserved and interwoven envelope stress programs that remodel the OM with TBDRs, a tripartite multidrug efflux pump and periplasmic proteases. Thus, unanticipated entry routes for antibiotics emerge in stressed diderm bacteria that may be treatable with membrane impermeable antimicrobials owing to an underlying transcriptional stress response pathways coordinated by a novel type of OM regulator.

The acquisition of multi-drug resistance (MDR) determinants jeopardizes treatment of bacterial infections with antibiotics. The tripartite efflux pump AcrAB-NodT confers adaptive MDR in the non-pathogenic α-proteobacterium Caulobacter crescentus via transcriptional induction by first-generation quinolone antibiotics. We discovered that overexpression of AcrAB-NodT by mutation or exogenous inducers confers resistance to cephalosporin and penicillin (β-lactam) antibiotics. Combining two-step mutagenesis-sequencing (Mut-Seq) and cephalosporin-resistant point mutants, we dissected how TipR uses a common operator of the divergent tipR and acrAB-nodT promoter for adaptive and/or potentiated AcrAB-NodT-directed efflux. Chemical screening identified compounds that interfere with DNA-binding by TipR or induce its dependent proteolytic turnover. We found that long-term induction of AcrAB-NodT disfigures the envelope and that homeostatic control by TipR includes co-induction of the DnaJ-like co-chaperone DjlA, to boost pump assembly and/or capacity in anticipation of envelope stress. Thus, the adaptive MDR regulatory circuitry reconciles drug efflux with co-chaperone function for trans-envelope assemblies and maintenance.

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