Loss of Bacterial Cell Pole Stabilization inCaulobacter crescentusSensitizes to Outer Membrane Stress and Peptidoglycan-Directed Antibiotics

Abstract

<jats:title>Abstract</jats:title><jats:p>Rod-shaped bacteria frequently localise proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the role of such polar factors in responses to extracellular stimuli has been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from <jats:italic>Caulobacter crescentus</jats:italic>, TipN, and extracellular stress and found that TipN is required for normal tolerance of cell envelope-directed antibiotics, including vancomycin that does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin tolerance. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive Δ<jats:italic>tipN</jats:italic> strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in Δ<jats:italic>tipN</jats:italic> cells relative to wild type under some, but not all, cell wall stress conditions that this strain was sensitised to, in particular cefixime and detergent exposure. Together, these results indicate that the ChvIG two-component system has been co-opted as a sensor of cell wall stress and that TipN can influence cell envelope stability and ChvIG-mediated signaling in addition to its roles in intracellular development.</jats:p><jats:sec><jats:title>Author summary</jats:title><jats:p>Maintenance of an intact cell envelope is essential for free-living bacteria to survive harsh conditions they may encounter in their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and re-form parts of the cell envelope at the division plane in order to form new poles as the cells replicate and divide. We have found that TipN, a factor required for correct division and cell pole development in the rod-shaped bacterium, <jats:italic>Caulobacter crescentus</jats:italic>, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. We also identified an outer membrane receptor, ChvT, that was responsible for allowing vancomycin access to the cells and found that the two-component system that negatively regulates ChvT production was activated by various kinds of cell wall stress. Presence or absence of TipN influenced how active this system was in the presence of cefixime or of the membrane-disrupting detergent sodium deoxycholate. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilisation of these weak points of the cell envelope as well as its other roles inside the cell.</jats:p></jats:sec>

The ChvG–ChvI and NtrY–NtrX two-component systems coordinately regulate growth ofCaulobacter crescentus

“…Of those, 140 were upregulated and 22 were downregulated, indicating that ChvI primarily serves as a transcriptional activator. Consistent with previous work, chvR was upregulated while chvT was downregulated by overexpression of chvI(D52E) (6, 47). In addition, expression of both chvG and hprK was enhanced in cells expressing chvI(D52E) , pointing to positive autoregulation of the chvIG-hprK operon.…”

“…Consistent with previous reports, the reporter for the chvR promoter (PchvR ) was expressed in M2X in a chvI-dependent manner (Fig. 6B) (6,47). The remaining reporters, apart from PnstA, which was expressed at low levels, also exhibited clear chvI dependence, supporting our RNA-seq results (Fig.…”

“…To our knowledge, the only reported physiological phenotype for chvGI mutants in C. crescentus is sensitivity to the antibiotic vancomycin (47). However, previous work indicated that ChvGI might be important for growth in defined medium (6).…”

“…However, the molecule(s) transported by ChvT remain undefined. High ChvT levels may contribute to defects in transport in defined medium and/or alter membrane integrity, impacting the viability of cells lacking ChvGI (47). It is clear, though, that altered chvT expression cannot fully explain the growth deficiency of ∆chvI cells in M2X medium.…”

“…C. crescentus ChvGI activates transcription of the small regulatory RNA chvR, which post-transcriptionally represses the TonB-dependent receptor gene chvT (6). In addition, examination of reporters of chvR transcription indicated that ChvGI is activated by growth in defined medium, acidic pH, DNA damage, growth at stationary phase, and cell envelope stress (6,47). However, aside from chvR, genes regulated by ChvGI have not been identified.…”