The Rcs phosphorelay is a cell envelope stress response activated by peptidoglycan stress and contributes to intrinsic antibiotic resistance

Abstract

Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the beta-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to beta-lactam antibiotics.

FIG. 1.

General overview of the roles of essential PBPs in E. coli and the effects of inhibition of specific PBPs by antibiotics used in these studies. PBPs 1a and 1b are required for insertion of new strands and cross-linking of the peptidoglycan layer during elongation and septation, while PBP 2 is required for elongation and PBP 3 for septation. Cefsulodin inhibits PBPs 1a and 1b, causing cell lysis, while amdinocillin inhibits PBP 2, causing cells to become round and eventually stop dividing.

FIG. 2.

Effect of antibiotics on growth of E. coli. Growth curves (CFU/ml as a function of time relative to antibiotic addition) are shown for Cef-Amd-treated (Cef-Amd; circles), amdinocillin-treated (Amd, squares), cefsulodin-treated (Cef, triangles), and untreated (diamonds) cultures. Overnight cultures of E. coli strain MG1655 (SEA113) were diluted to an OD₆₀₀ of 0.02 in fresh LB and grown in a gyratory water bath at 37°C with shaking. When the cultures reached an OD₆₀₀ of 0.2 (time zero), antibiotics were added to give the final concentrations indicated in the text. The numbers of CFU/ml are the averages for two replicate platings from each sample, and the error bars represent the standard deviations. Representative growth curves are shown. Samples were collected for microarray analysis immediately before growth stopped due to lysis or stasis. Cefsulodin-treated cells were collected at 10 min, Cef-Amd-treated cells were collected at 40 min, and amdinocillin-treated cells were collected at 60 min following addition of the respective antibiotics.

FIG. 3.

Venn diagram of genes with increased expression due to inhibition of PBPs 1a and 1b by cefsulodin, PBP 2 by amdinocillin, and all three PBPs by the Cef-Amd combination. The numbers in the intersecting sections refer to the number of genes induced in common, while the numbers outside of these sections refer to the number of genes induced by that treatment alone. Genes whose expression levels were increased by two or more treatments are listed under the indicated headings (all treatments, Cef-Amd versus amdinocillin, and Cef-Amd versus cefsulodin). Genes in bold are members of the Rcs regulon.

FIG. 4.

Inhibition of peptidoglycan synthesis by Cef-Amd, amdinocillin, and cefsulodin activates the Rcs pathway via RcsF. WT and ΔrcsF strains with the RcsB₂-dependent rprA-lacZ reporter were treated with antibiotics at the concentrations used for the microarray experiments. Samples were collected at 40, 60, and 20 min following Cef-Amd, amdinocillin, and cefsulodin treatment, respectively. The change in β-galactosidase activity was determined with respect to the untreated WT culture, and average values with standard deviations for a minimum of five experiments are shown. The basal level of reporter gene activity is lower in the ΔrcsF strain than in the WT strain and is no longer inducible.

FIG. 5.

The Rcs phosphorelay. Signals, indicated by lightening bolts, activate the pathway at RcsF or RcsC and initiate the phosphorelay. The direction of phosphate transfer from RcsC to RcsD to RcsB is shown. OM, outer membrane; IM inner membrane; Pgn, peptidoglycan.

FIG. 6.

The Rcs phosphorelay increases survival in the presence of antibiotics, independently of capsule synthesis and σ^S. Plating efficiencies comparing numbers of CFU/ml in the presence and absence of antibiotic are displayed. Average values and standard deviations for at least three experiments are presented. Antibiotics were used in LB agar plates at the following concentrations: 0.075 μg/ml amdinocillin and 10 μg/ml cefsulodin (top), 0.15 μg/ml amdinocillin (middle), and 45 μg/ml cefsulodin (bottom). Strains marked with an asterisk did not form colonies on plates containing antibiotics (plating efficiency = 0).