# Asymmetric envelope surface disposition of secreted protein YjbI controls bimodal antibiotic susceptibilities in C. crescentus

## TL;DR

The protein YjbI in Caulobacter crescentus controls how the bacteria respond to different antibiotics by regulating the outer membrane and stress pathways.

## Contribution

YjbI is shown to asymmetrically regulate outer membrane composition and antibiotic susceptibility in a Gram-negative bacterium.

## Key findings

- Loss of YjbI activates envelope stress pathways and increases antibiotic sensitivity.
- YjbI's asymmetric surface disposition explains its role in controlling outer membrane protein accessibility.
- BugA, a newly identified TBDR, mediates sensitivity to vancomycin and bacitracin when YjbI is absent.

## Abstract

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.

While cytoplasmic pentapeptide repeat proteins (PPRs) protect bacteria against quinolone antibiotics, the role of secreted PPRs of the YjbI family is less well investigated. This study reveals YjbI from Caulobacter crescentus as an envelope stress sensor and antibiotic susceptibility regulator that is asymmetrically embedded in the outer membrane.

Loss of YjbI activates envelope remodelling pathways and causes antibiotic sensitivities via induction of outer membrane proteins.YjbI adopts a polarized disposition in the LPS layer and is rapidly proteolyzed upon exposure to zinc stress.Expression of BugA, a newly discovered zinc-inducible TonB-dependent importer, confers sensitivity to bacitracin and vancomycin upon loss of YjbI.Induction of the AcrAB-NodT efflux pump by quinolone antibiotics is lethal to yjbI mutant cells.

Loss of YjbI activates envelope remodelling pathways and causes antibiotic sensitivities via induction of outer membrane proteins.

YjbI adopts a polarized disposition in the LPS layer and is rapidly proteolyzed upon exposure to zinc stress.

Expression of BugA, a newly discovered zinc-inducible TonB-dependent importer, confers sensitivity to bacitracin and vancomycin upon loss of YjbI.

Induction of the AcrAB-NodT efflux pump by quinolone antibiotics is lethal to yjbI mutant cells.

The pentapeptide repeat protein YjbI protects against antibiotics by suppressing envelope stress responses in a Gram-negative bacterium.