Cytoplasmic pentapeptide repeat proteins (PRPs) protect bacterial DNA gyrase from quinolone antibiotics. While some secreted PRPs are essential upon quinolone exposure, their role in the regulation of antibiotic resistance remains to be fully characterized. We show that a YjbI-type secreted PRP regulates antibiotic sensitivity, bimodally for small or large molecules, via modulation of the Caulobacter crescentus outer membrane (OM). YjbI silences two converging envelope-stress pathways that globally reprogram the OM proteome via TonB-dependent receptors (TBDRs), periplasmic proteases, and AcrAB-NodT, a multidrug efflux pump whose induction by small molecules and antibiotics is lethal to yjbI mutant cells. Loss of YjbI also confers sensitivity to vancomycin and bacitracin, two large peptidoglycan-targeting and zinc-binding antibiotics that permeate the outer membrane via the previously uncharacterized TBDR BugA and its orthologs. Zinc stress triggers rapid proteolytic removal of Yjbl, activates expression of TBDRs, including BugA, and ultimately leads to replenishment of YjbI. Molecular dynamics simulations and reactive thiol probing imply an asymmetric surface disposition of YjbI, explaining the differential accessibility of its conserved cysteine pairs that flank the quadrilateral β-helix. Taken together, our findings identify a role of YjbI as a cell surface-regulator of outer membrane composition and antibiotic sensitivity in a Gram-negative bacterium.
© 2025. The Author(s).

Cell wall peptidoglycan is a heteropolymeric mesh that protects the bacterium from internal turgor and external insults. In many rod-shaped bacteria, peptidoglycan synthesis for normal growth is achieved by two distinct pathways: the Rod complex, comprised of MreB, RodA, and a cognate class B penicillin-binding protein (PBP), and the class A PBPs (aPBPs). In contrast to laterally growing bacteria, pole-growing mycobacteria do not encode an MreB homolog and do not require SEDS protein RodA for in vitro growth. However, RodA contributes to the survival of Mycobacterium tuberculosis in some infection models, suggesting that the protein could have a stress-dependent role in maintaining cell wall integrity. Under basal conditions, we find here that the subcellular distribution of RodA largely overlaps that of the aPBP PonA1 and that both RodA and the aPBPs promote polar peptidoglycan assembly. Upon cell wall damage, RodA fortifies Mycobacterium smegmatis against lysis and, unlike aPBPs, contributes to a shift in peptidoglycan assembly from the poles to the sidewall. Neither RodA nor PonA1 relocalize; instead, the redistribution of nascent cell wall parallels that of peptidoglycan precursor synthase MurG. Our results support a model in which mycobacteria balance polar growth and cell-wide repair via spatial flexibility in precursor synthesis and extracellular insertion. IMPORTANCE Peptidoglycan synthesis is a highly successful target for antibiotics. The pathway has been extensively studied in model organisms under laboratory-optimized conditions. In natural environments, bacteria are frequently under attack. Moreover, the vast majority of bacterial species are unlikely to fit a single paradigm of cell wall assembly because of differences in growth mode and/or envelope structure. Studying cell wall synthesis under nonoptimal conditions and in nonstandard species may improve our understanding of pathway function and suggest new inhibition strategies. Mycobacterium smegmatis, a relative of several notorious human and animal pathogens, has an unusual polar growth mode and multilayered envelope. In this work, we challenged M. smegmatis with cell wall-damaging enzymes to characterize the roles of cell wall-building enzymes when the bacterium is under attack.

Bacterial species have diverse cell shapes that enable motility, colonization and virulence. The cell wall defines bacterial shape and is primarily built by two cytoskeleton-guided synthesis machines, the elongasome and the divisome. However, the mechanisms producing complex shapes, like the curved-rod shape of Vibrio cholerae, are incompletely defined. Previous studies have reported that species-specific regulation of cytoskeleton-guided machines enables formation of complex bacterial shapes such as cell curvature and cellular appendages. In contrast, we report that CrvA and CrvB are sufficient to induce complex cell shape autonomously of the cytoskeleton in V. cholerae. The autonomy of the CrvAB module also enables it to induce curvature in the Gram-negative species Escherichia coli, Pseudomonas aeruginosa, Caulobacter crescentus and Agrobacterium tumefaciens. Using inducible gene expression, quantitative microscopy and biochemistry, we show that CrvA and CrvB circumvent the need for patterning via cytoskeletal elements by regulating each other to form an asymmetrically localized, periplasmic structure that binds directly to the cell wall. The assembly and disassembly of this periplasmic structure enables dynamic changes in cell shape. Bioinformatics indicate that CrvA and CrvB may have diverged from a single ancestral hybrid protein. Using fusion experiments in V. cholerae, we find that a synthetic CrvA/B hybrid protein is sufficient to induce curvature on its own, but that expression of two distinct proteins, CrvA and CrvB, promotes more rapid curvature induction. We conclude that morphological complexity can arise independently of cell-shape specification by the core cytoskeleton-guided synthesis machines.