The two-component system ChvGI maintains cell envelope homeostasis in Caulobacter crescentus

Two-component systems (TCS) are often used by bacteria to rapidly assess and respond to environmental changes. The ChvG/ChvI (ChvGI) TCS conserved in α-proteobacteria is known for regulating expression of genes related to exopolysaccharide production, virulence and growth. The sensor kinase ChvG autophosphorylates upon yet unknown signals and phosphorylates the response regulator ChvI to regulate transcription. Recent studies in Caulobacter crescentus showed that chv mutants are sensitive to vancomycin treatment and fail to grow in synthetic minimal media. In this work, we identified the osmotic imbalance as the main cause of growth impairment in synthetic minimal media. We also determined the ChvI regulon and found that ChvI regulates cell envelope architecture by controlling outer membrane, peptidoglycan assembly/recycling and inner membrane proteins. In addition, we found that ChvI phosphorylation is also activated upon antibiotic treatment with vancomycin. We also challenged chv mutants with other cell envelope related stress and found that treatment with antibiotics targeting transpeptidation of peptidoglycan during cell elongation impairs growth of the mutant. Finally, we observed that the sensor kinase ChvG relocates from a patchy-spotty distribution to distinctive foci after transition from complex to synthetic minimal media. Interestingly, this pattern of (re)location has been described for proteins involved in cell growth control and peptidoglycan synthesis upon osmotic shock. Overall, our data support that the ChvGI TCS is mainly used to monitor and respond to osmotic imbalances and damages in the peptidoglycan layer to maintain cell envelope homeostasis. Author summary The cell envelope is the first barrier protecting cells from harsh environmental conditions, such as temperature, pH, oxidative and osmotic imbalances. It is also an obstacle for the intake of antibiotics targeting essential cellular processes. Therefore, molecular components and systems responding to cell envelope stress and maintaining cell envelope homeostasis are important targets for drug therapy. Here we show that the two-component system ChvGI, highly conserved in free-living and pathogenic α-proteobacteria, is activated upon osmotic upshift and treatment with antibiotics targeting peptidoglycan synthesis to enhance the transcription of multiple genes involved in cell envelope homeostasis in Caulobacter crescentus. We also show that the kinase sensor ChvG displays a dynamic localisation pattern that changes depending on osmotic imbalance. To our knowledge this is the first two-component system reported to change its cellular localisation upon environmental stress

The two-component system ChvGI maintains cell envelope homeostasis in Caulobacter crescentus.

Two-component systems (TCS) are often used by bacteria to rapidly assess and respond to environmental changes. The ChvG/ChvI (ChvGI) TCS conserved in α-proteobacteria is known for regulating expression of genes related to exopolysaccharide production, virulence and growth. The sensor kinase ChvG autophosphorylates upon yet unknown signals and phosphorylates the response regulator ChvI to regulate transcription. Recent studies in Caulobacter crescentus showed that chv mutants are sensitive to vancomycin treatment and fail to grow in synthetic minimal media. In this work, we identified the osmotic imbalance as the main cause of growth impairment in synthetic minimal media. We also determined the ChvI regulon and found that ChvI regulates cell envelope architecture by controlling outer membrane, peptidoglycan assembly/recycling and inner membrane proteins. In addition, we found that ChvI phosphorylation is also activated upon antibiotic treatment with vancomycin. We also challenged chv mutants with other cell envelope related stress and found that treatment with antibiotics targeting transpeptidation of peptidoglycan during cell elongation impairs growth of the mutant. Finally, we observed that the sensor kinase ChvG relocates from a patchy-spotty distribution to distinctive foci after transition from complex to synthetic minimal media. Interestingly, this pattern of (re)location has been described for proteins involved in cell growth control and peptidoglycan synthesis upon osmotic shock. Overall, our data support that the ChvGI TCS is mainly used to monitor and respond to osmotic imbalances and damages in the peptidoglycan layer to maintain cell envelope homeostasis

ChvG-dependent phosphorylation of ChvI is stimulated upon osmotic shock.

(A) Growth of WT (black) and ΔchvG (yellow) in M5GG, WT and ΔchvI (orange) mutants in M5GG with (dashed lines) or without (solid lines) 6% sucrose (Suc). The data represent the average value of biological replicates (n = 3, error bars show standard deviation). (B) In vivo phosphorylation levels of ChvI in WT and ΔchvG mutant grown in M5GG. (C) in vivo phosphorylation levels of ChvI in WT grown in M5GG exposed (+) or not (-) to sucrose (Suc) for 7 min. (D) Quantified in vivo phosphorylation levels of ChvI in the same growth conditions than in (C). The data represent the average values of biological replicates (n = 3, error bars show standard deviation). * = p < 0.05, single factor ANOVA analysis of ChvI phosphorylation.</p

Δ<i>chvI</i> is not sensitive to every cell envelope stress.

(A) Growth of WT (black) and ΔchvI (orange) cells in PYE with (dashed lines) or without (solid lines) acidic stress pH 5.5, polymixin B (Pol B) and cephalexin (Ceph). The data represent the average values of biological replicates (n = 3, error bars show standard deviation). (B) Viability of ΔchvI cells on plates supplemented with 0.001% or 0.0025% sodium dodecyl sulfate (SDS), Pol B, Ceph or A22. Images are representative of three biological replicates. (C) Viability of single (ΔchvI and ΔchvT) and double (ΔchvI ΔchvT) mutants on PYE agar with vancomycin (Van), mecillinam (Mec), cefsulodin (Cef) and moenomycin (Moe). Images represent three biological replicates. (EPS)</p

ChvI target genes determined by ChIP-seq and validated by β-galactosidase assays.

(A) Genome-wide occupancy of ChvI on the chromosome of C. crescentus determined by ChIP-seq on WT strain grown in M2G. The x-axis represents the coordinates on the genome (Mb), the y-axis shows the normalized ChIP-Seq read abundance in reads. Some top hits, corresponding to the promoter regions of chvT, CCNA_R0088, ftsN, CCNA_02715, chvR, dipM and chvI operon, are highlighted. (B) Volcano plot representing the relation between the fold change and P values on gene expression between ΔchvI and WT strains exposed to osmotic upshift in PYE 6% sucrose by RNA-seq. Genes identified are presented as dots. Significant down- and up-regulated genes are presented as green and red dots, respectively, while genes with no significant alterations are presented as black dots. chvI, chvR and chvT genes as well as genes encoding Tol (blue) and Bam (orange) complexes or TonB-dependent receptors (purple) are highlighted. (C) Percentage (%) of genes from the RNA-seq data in the following COG categories: B, Chromatin structure and dynamics; C, Energy production and conversion; D, Cell cycle control, cell division, chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; G, Carbohydrate transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism; J, Translation, ribosomal structure and biogenesis; K, Transcription; L, Replication, recombination and repair; M, Cell wall/membrane/envelope biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; T, Signal transduction mechanisms; U, Intracellular trafficking, secretion and vesicular transport; and V, Defense mechanisms. Genes classified as R, General function prediction only (of them 14% downregulated and 9.5% upregulated); and S, Unknown function (of them 15.8% downregulated and 13.0% upregulated) were not included in the data representation. (D) Activity of the dipM, ftsN, phyR and chvI promoters (Miller Units) in WT (black bars), ΔchvI (white bars) and ΔchvG (gray bars) cells grown overnight in PYE, then washed and exposed for 4h in PYE 6% sucrose. (E) WebLogo of predicted ChvI consensus sequence obtained with MEME. The data in (B) and (C) represent the average values of biological replicates (n = 3, error bars show standard deviation). * = p p < 0.01, single factor ANOVA analysis of β-galactosidase activity.</p

ChvG relocates from a patchy-spotty pattern to mid-cell upon osmotic upshift.

(A) Localisation of ChvG-eGFP in cells grown in either M2G or PYE and imaged on M2G or PYE agar pads, respectively; or cells grown in PYE, washed in M2G and imaged on M2G agarose pads (PYE ➔ M2G); or cells grown in M2G, washed in PYE and imaged on PYE agarose pads (M2G ➔ PYE). (B) Localisation of ChvI-eGFP in a ΔchvI background and (C) ChvGH309N-eGFP in a ΔchvG background grown overnight in PYE and imaged on PYE agarose pads or grown in PYE, washed in M2G and imaged on M2G agarose pads (PYE ➔ M2G). Demograph data represent cells sorted from short to long length with no less than 200 cells per sample. Liquid cultures and pads were supplemented with 0.1% xylose to allow expression of ChvG, ChvI and ChvGH309N fused to eGFP fusions. Scale bar = 1 μm.</p

<i>chvI</i> and <i>sigT</i> are interconnected.

(A) Activity of the chvI promoter PchvI (in Miller Units) in WT (black bars), ΔchvI (light grey bars) ΔsigT (dark grey bars) cells grown in PYE and exposed to different osmotic conditions. Data in M2G was obtained from cells pelleted from PYE cultures, washed twice and exposed to M2G. The data represent the average values of biological replicates (n = 3, error bars show standard deviation). ** = p 0.05, Single factor ANOVA analysis of β-galactosidase activity. (B) Viability of single ΔchvI and ΔsigT mutants in PYE supplemented with different osmolytes at different concentrations. Images are representative of three biological replicates. (EPS)</p

Plasmids used in this study.

(PDF)</p

Supplementary methods.

Construction of plasmids. (PDF)</p

ChvI regulon determined by RNA-seq.

(A) genes with expression down-regulated or (B) up-regulated in the ΔchvI mutant upon osmotic stress with 6% sucrose. (PDF)</p