New insights into the WalK/WalR (YycG/YycF) essential signal transduction pathway reveal a major role in controlling cell wall metabolism and biofilm formation in Staphylococcus aureus

Abstract

The highly conserved WalK/WalR (also known as YycG/YycF) two-component system is specific to low-G+C gram-positive bacteria. While this system is essential for cell viability, both the nature of its regulon and its physiological role have remained mostly uncharacterized. We observed that, unexpectedly, Staphylococcus aureus cell death induced by WalKR depletion was not followed by lysis. We show that WalKR positively controls autolytic activity, in particular that of the two major S. aureus autolysins, AtlA and LytM. By using our previously characterized consensus WalR binding site and carefully reexamining the genome annotations, we identified nine genes potentially belonging to the WalKR regulon that appeared to be involved in S. aureus cell wall degradation. Expression of all of these genes was positively controlled by WalKR levels in the cell, leading to high resistance to Triton X-100-induced lysis when the cells were starved for WalKR. Cells lacking WalKR were also more resistant to lysostaphin-induced lysis, suggesting modifications in cell wall structure. Indeed, lowered levels of WalKR led to a significant decrease in peptidoglycan biosynthesis and turnover and to cell wall modifications, which included increased peptidoglycan cross-linking and glycan chain length. We also demonstrated a direct relationship between WalKR levels and the ability to form biofilms. This is the first example in S. aureus of a regulatory system positively controlling autolysin synthesis and biofilm formation. Taken together, our results now define this signal transduction pathway as a master regulatory system for cell wall metabolism, which we have accordingly renamed WalK/WalR to reflect its true function.

FIG. 1.

Viability assay of WalKR-depleted cells. (A) IPTG-dependent growth of the conditional ST1000 mutant strain. An overnight culture in TSB plus 1 mM IPTG was diluted to an OD₆₀₀ of 0.005 in TSB medium with (squares) or without (circles) 1 mM IPTG. At times T₁, T₂, and T₃, aliquots of the culture in TSB without IPTG were sampled for fluorescent staining. (B) Fluorescent staining of cells with the LIVE/DEAD BacLight viability kit, followed by fluorescence microscopy. SYTO 9-stained bacteria were alive and appear green, while propidium iodide-stained bacteria were dead and appear red.

FIG. 2.

Zymographic analyses of SDS-extracted proteins from strain ST1000 grown without (lane1) or with (lane 2) 1 mM IPTG. Zymographic analysis was performed following SDS-PAGE on a 10% acrylamide gel containing heat-inactivated S. aureus cells. The positions of molecular mass standards are indicated on the left.

FIG. 3.

Sequence logo from the putative WalR binding sites, generated using WebLogo (http://weblogo.berkeley.edu/) (8, 47). (B) Domain architecture of cell wall hydrolases encoded by putative WalKR-regulated genes based on the graphical output of the SMART web interface (http://smart.embl-heidelberg.de) (48). The red fragments represent signal peptides, and the pink sections correspond to low-complexity regions. The definitions of the boxed domains are as follows: Ami-2, N-acetylmuramoyl-l-alanine amidase; LYZ2, mannosyl-glycoprotein endo-β-N-acetylglucosamidase; LysM, peptidoglycan binding function; CHAP, cysteine, histidine-dependent aminohydrolase/peptidase; peptidase-M23, glycyl-glycine endopeptidase; transglycosylas, transglycosylase. (C) Schematic representation of S. aureus peptidoglycan and the known hydrolytic activities by which it is cleaved. Peptidoglycan hydrolase activities encoded by potential members of the WalKR regulon are listed.

FIG. 4.

Effect of walKR induction on transcription of genes involved in cell wall degradation. Total RNA was extracted from ST1000 cells grown in TSB supplemented with 0.05 mM or 1 mM IPTG. After reverse transcription, specific cDNAs were quantified by qRT-PCR. The results are expressed as the means and standard deviations of quadruplicate experiments using primers specific for target genes and 16S rRNA (normalizing gene).

FIG. 5.

Primer extension analysis of the lytM transcript. (A) Total RNA was extracted from cultures of strain ST1000 grown in TSB supplemented with 0.05 mM IPTG (lane 1) or 1 mM IPTG (lane 2) to an OD₆₀₀ of 1.2. Primer extensions were performed using a lytM-specific primer (OSA91). Sanger sequencing reactions (GATC) were carried out on a PCR fragment (OSA90-OSA91) corresponding to the lytM upstream region. (B) Nucleotide sequence of the lytM upstream region. The ATG codon is indicated. The transcription start site is labeled +1, and the consensus −10 and −35 sequences are boxed. The extent of the previously characterized WalR DNase I footprint is shaded, and the WalR binding site is indicated by arrows.

FIG. 6.

Phase-contrast micrographs (magnification, ×1,000) of strain ST1000 cultivated in TSB medium without (A) or with (B) 1 mM IPTG. Fields for the cultures grown without inducer showed large numbers of clusters; a representative field is shown.

FIG. 7.

Autolysis profiles upon walKR induction. Strain ST1000 was grown in TSB without IPTG (⧫) or with 0.05 mM IPTG (▪), or 1 mM IPTG (•) to an OD₆₀₀ of 1. The cells were then pelleted and resuspended in PBS to test the effects of autolysis-inducing agents. Triton- and lysostaphin-mediated lysis of staphylococci was measured as the decline of OD₆₀₀ over time. (A) Autolysis determined at 30°C in the presence of 0.1% Triton X-100. (B) Autolysis determined at 37°C in the presence of 200 ng/ml lysostaphin.

FIG. 8.

Effect of WalKR depletion on S. aureus peptidoglycan structure. Structural analysis was performed by determining the muropeptide composition (A) and glycan strand length distribution (B). ST1000 cells were grown with (white bars) or without (black bars) 1 mM IPTG and harvested immediately after the cessation of growth in the absence of inducer. (A) Muropeptide nomenclature was as previously described. The upper graph represents the HPLC muropeptide elution profile of the ST1000 strain grown with 1 mM IPTG and the identification of the muropeptide species (10). Briefly, muropeptides 1 to 7 correspond to monomeric muropeptides, 11 to 14 to dimeric muropeptides, 15 to trimers, 16 to tetramers, and 17 to pentamers. Highly oligomeric muropeptides elute as an unresolved “hump.” (B) Glycan strand species nomenclature corresponds to the number of disaccharide N-acetylglucosamine-β(1, 4)-N-acetylmuramic acid repeating units (5). The HPLC elution profile and identification of glycan strand species are shown in the upper graph. Satellite peaks were observed in low abundance and were excluded to simplify the analysis, since they did not change under the conditions tested (data not shown).

FIG. 9.

Effect of WalKR depletion on cell wall biosynthesis and turnover. (A) Cell wall biosynthesis of strain ST1000 grown with (black bars) and without (gray bars) 1 mM IPTG. Cell wall biosynthesis was measured as the incorporation of [³H]GlcNAc over time. (B) Cell wall turnover of strain ST1000 grown with (black bars) or without (gray bars) IPTG. Turnover was measured following [³H]GlcNAc labeling as the decrease of incorporated radioactivity over time. The results are expressed as the percentage of the initial quantity of cell-associated radioactivity.

FIG. 10.

Biofilm formation of strain ST1000 in the presence of increasing concentrations of IPTG. Biofilm assays were performed on microtiter plates in TSB plus 0.25% glucose with increasing concentrations of IPTG: 0.05 mM (A), 0.5 mM (B), and 1 mM (C). Quantifications were performed by measuring the OD₅₉₅ following crystal violet staining and resuspension in ethanol-acetone (80:20). All cultures had the same growth rate, and the data were normalized to the OD₆₀₀ of each cell culture and presented as variation (n-fold) compared to condition A. The results represent the mean values ± standard deviations of duplicate quantifications. Similar results were obtained in three independent experiments.