Transcriptional Analysis and Subcellular Protein Localization Reveal Specific Features of the Essential WalKR System in Staphylococcus aureus

International audienceThe WalKR two-component system, controlling cell wall metabolism, is highly conserved among Bacilli and essential for cell viability. In Staphylococcus aureus, walR and walK are followed by three genes of unknown function: walH, walI and walJ. Sequence analysis and transcript mapping revealed a unique genetic structure for this locus in S. aureus: the last gene of the locus, walJ, is transcribed independently, whereas transcription of the tetra-cis-tronic walRKHI operon occurred from two independent promoters located upstream from walR. Protein topology analysis and protein-protein interactions in E. coli as well as subcel-lular localization in S. aureus allowed us to show that WalH and WalI are membrane-bound proteins, which associate with WalK to form a complex at the cell division septum. While these interactions suggest that WalH and WalI play a role in activity of the WalKR regulatory pathway, deletion of walH and/or walI did not have a major effect on genes whose expression is strongly dependent on WalKR or on associated phenotypes. No effect of WalH or WalI was seen on tightly controlled WalKR regulon genes such as sle1 or saouhsc_00773, which encodes a CHAP-domain amidase. Of the genes encoding the two major S. aureus autolysins, AtlA and Sle1, only transcription of atlA was increased in the ΔwalH or ΔwalI mutants. Likewise, bacterial autolysis was not increased in the absence of WalH and/or WalI and biofilm formation was lowered rather than increased. Our results suggest that contrary to their major role as WalK inhibitors in B. subtilis, the WalH and WalI proteins have evolved a different function in S. aureus, where they are more accessory. A phylogenomic analysis shows a striking conservation of the 5 gene wal cluster along the evolutionary history of Bacilli, supporting the key importance of this signal transduction system, and indicating that the walH and walI genes were lost in the ancestor of Streptococcaceae, leading to their atypical 3 wal gene cluster, walRKJ

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p

Expression of <i>atlA</i> is increased in the Δ<i>walH</i>, Δ<i>walI</i> and Δ<i>walHI</i> mutants.

<p>The HG001 parental strain and the Δ<i>walH</i>, Δ<i>walI</i> and Δ<i>walHI</i> mutant strains were grown in TSB rich medium until OD_{600 nm} = 1. Total RNA was extracted and quantitative real time PCR was used to compare gene expression. Expression levels were normalized using 16S rRNA as an internal standard and are indicated as the <i>n</i>-fold change with respect to the HG001 parental strain, expressed as means and standard deviations. Dark grey bars indicate expression levels in the parental and mutant strains and light grey bars correspond to values in the complemented strains carrying pMK4-Pprot<i>walHI</i>. * <i>P</i><0.05 as determined using Student’s <i>t</i>-test.</p

Biofilm formation is decreased in the Δ<i>walH</i>, Δ<i>walI</i> and Δ<i>walHI</i> mutants.

<p>Biofilm assays were performed in microtiter plates after growth at 37°C for 24 h. Adherent biomass was quantified, normalized to the OD_{600 nm} of each cell culture and represented as n-fold variation compared to the parental strain. Dark grey bars indicate biomass levels in the parental and mutant strains and light grey bars correspond to values in the complemented strains carrying pMK4-Pprot<i>walHI</i>. Experiments were carried out in quadruplicate and standard deviations are indicated. ** <i>P</i><0.01 as determined using Student’s <i>t</i>-test.</p

Triton-induced autolysis is decreased in the absence of WalH.

<p>Bacteria were grown in TSB at 37°C with shaking until OD_{600 nm} ≈ 1, pelleted (10 min; 5,400 x <i>g</i>), resuspended in phosphate buffered saline (PBS) with Triton X-100 (0.1%), and incubated at 37°C with shaking. Lysis was determined as the decrease in OD_{600 nm} over time and indicated as a percentage of the initial OD (measured OD_{600 nm} / initial OD_{600 nm}). Results are shown as the mean and standard deviation of three independent experiments. Strains: HG001 (■); ST1397 Δ<i>walH</i> (○); ST1410 Δ<i>walHI</i> (△); ST1415 Δ<i>walH</i> pMK4Pprot-<i>walHI</i> (●); ST1417 Δ<i>walHI</i> pMK4Pprot-<i>walHI</i> (▲).</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

WalK and WalH are localized at the cell division septum.

<p>(A) <i>S</i>. <i>aureus</i> HG001 strains producing fluorescent Wal protein fusions (WalK-GFP, WalH-GFP, WalI-GFP, WalJ-GFP) were grown in TSB and observed in mid-exponential phase by fluorescence microscopy. White arrows and zoomed views indicate septal enrichment. (B) Fluorescence ratios (septum/lateral membrane) were quantified for strains producing membrane protein fusions using ImageJ software and plotted using GraphPad Prism. Horizontal lines correspond to average fluorescence ratios with values greater than 2, indicating preferential septal localization (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151449#sec002" target="_blank">Materials and Methods</a>). ** <i>P</i><0.002, *** <i>P</i><0.001 as determined by the Wilcoxon signed-rank test.</p

WalH and WalI are membrane-anchored extracellular proteins.

<p>(A) <i>E</i>. <i>coli</i> DH5α cells producing ‘PhoA’-‘LacZ’ fusion proteins (WalH_1-40-PhoA_21-471-LacZ_4-60, WalI_1-40-PhoA_21-471-LacZ_4-60, or WalJ_1-40-PhoA_21-471-LacZ_4-60) were plated on indicator medium with two chromogenic substrates, Red-Gal (for β-galactosidase activity) and X-Pho (for phosphatase activity). Blue coloring of the colonies (high phosphatase activity) indicates a membrane or extracellular localization of the fusion point. Red coloring of the colonies (high β-galactosidase activity) indicates cytosolic localization of the fusion point. (B) Schematic representation of Wal protein localization and topology with respect to the cell membrane. In the case of WalK and WalR, topology and localization were deduced from primary sequence analysis using the Phobius Hidden Markov Model (indicated by stars).</p