Strains and plasmids.

<p>Strains and plasmids.</p

Protein interaction network between SpdC and <i>S</i>. <i>aureus</i> histidine kinases.

<p>The BACTH assay was used to test protein-protein interactions between SpdC and each of the <i>S</i>. <i>aureus</i> TCS histidine kinases. A. DHT1 <i>E</i>. <i>coli</i> strains co-transformed with each combination of plasmids (pUT18c-<i>spdC</i> and pKT25 derivatives carrying the genes for the designated kinases) were spotted on LB agar plates with X-gal as an indicator of β-galactosidase activity. Histidine kinases showing a positive result for interaction with SpdC are underlined. B. β-galactosidase activity of DHT1 strains co-transformed with the pUT18c-<i>spdC</i> and pKT25 derivatives carrying each of the histidine kinase encoding genes. β-galactosidase activity (Miller Units) of the strain carrying the empty vectors (pKT25-pUT18c) was arbitrarily fixed at 0 and that of the positive control (pKT25zip-pUT18czip) at 1000.</p

SpdC is required for biofilm formation.

<p>Biofilm assays were performed by growing cells in static cultures in PVC microtiter plates (TSB plus 0.75% glucose and 3.5% NaCl). Plates were incubated at 37°C for 24h and adherent biomass was quantified as described in Materials and Methods. Data were normalized to the OD_600nm of each culture and indicated as the <i>n-</i>fold change of the Δ<i>spdC</i> mutant strain compared to the HG001 parental strain. ***, <i>P</i><0.0005 (Student’s <i>t</i>-test).</p

Genes differentially expressed in the Δ<i>spdC</i> mutant compared to the parental HG001 strain.

<p>Genes differentially expressed in the Δ<i>spdC</i> mutant compared to the parental HG001 strain.</p

Domains involved in interaction between SpdC and the WalK and SaeS histidine kinases.

<p>A. Domains fused to either pUT18c or pKT25 for the BACTH assay. Hatched bars indicate transmembrane regions and functional domains are annotated according to the UniProt database (<a href="http://www.uniprot.org/uniprot/Q2G2U4" target="_blank">http://www.uniprot.org/uniprot/Q2G2U4</a> for WalK, <a href="http://www.uniprot.org/uniprot/Q2G2U1" target="_blank">http://www.uniprot.org/uniprot/Q2G2U1</a> for SaeS and <a href="http://www.uniprot.org/uniprot/Q2FVT1" target="_blank">http://www.uniprot.org/uniprot/Q2FVT1</a> for SpdC). B. β-galactosidase activities of DHT1 <i>E</i>. <i>coli</i> strains co-transformed with pUT18c containing either the full-length <i>spdC</i> coding sequence or a fragment encoding only the first 252 amino-acids of SpdC, and pKT25 derivatives carrying DNA fragments encoding the indicated proteins. Positive interaction results are shaded. ND: Not determined.</p

The WalKR two-component system controls expression of <i>spdC</i>, encoding a Abi-domain membrane protein.

<p>A. Predicted membrane topology of the SpdC protein generated using the Protter prediction tool (<a href="http://wlab.ethz.ch/protter/start/" target="_blank">http://wlab.ethz.ch/protter/start/</a>). The eight predicted transmembrane segments are numbered. Amino acids forming the putative Abi domain (based on Pfam database annotation) are shaded in green. B. Relative levels of <i>walR</i> and <i>spdC</i> transcripts were measured by qRT-PCR during growth of the HG001 Pspac<i>walRKHI</i> strain (ST1017). Bacteria were grown to exponential phase in TSB with or without increasing concentrations of IPTG to induce expression from the Pspac promoter. Expression levels were normalized using 16S rRNA as an internal standard and are indicated as <i>n</i>-fold change with respect to the control condition (absence of IPTG).</p

Oligonucleotides used in this study.

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

SpdC is a novel <i>S</i>. <i>aureus</i> virulence factor.

<p>Kaplan-Meier survival curves of <i>RjOrl</i>:<i>SWISS</i> mice infected with either the HG001 parental strain (circles) or the Δ<i>spdC</i> strain (triangles) by i.v. route (5.10⁷ cfu/injection). A total of 14 mice were used in each group in two independent experiments. *, <i>P</i><0.05 (Wilcoxon test).</p

SpdC impacts cell wall homeostasis.

<p>A. The Δ<i>spdC</i> mutant displays increased resistance to lysostaphin-induced lysis. Cells were grown in TSB until mid-exponential phase, harvested and incubated in PBS with lysostaphin (200 ng/ml) with aeration at 37°C. Bacterial lysis was measured by monitoring OD_600nm over time. Results are shown as the mean and standard deviation of three independent assays. HG001 parental strain (); Δ<i>spdC</i> mutant (■); Δ<i>spdC</i>/pMK4Pprot-<i>spdC</i> complemented strain (▲). B. The absence of SpdC leads to sensitivity to oxacillin and tunicamycin. Dilution series of the HG001, Δ<i>spdC</i> and Δ<i>spdC</i>/ pMK4Pprot-<i>spdC</i> strains on TSA plates with or without antibiotics. Oxacillin: 0.1 μg/ml; fosfomycin: 4 μg/ml; tunicamycin: 1 μg/ml.</p

SpdC is involved in gene regulation.

<p>A. Ontological grouping of SpdC-regulated genes according to their annotated functions. Variations in gene expression were identified by RNA-Seq analysis of a Δ<i>spdC</i> strain compared to the HG001 parental strain grown in TSB until mid-exponential phase. B. qRT-PCR comparison of gene expression in a Δ<i>spdC</i> strain and the HG001 parental strain. Strains were grown in TSB until mid-exponential phase and RNA was extracted and treated as described in Materials and Methods. Expression levels were normalized using 16S rRNA as internal standard and presented as the <i>n-</i>fold change of the Δ<i>spdC</i> mutant strain compared to the HG001 parental strain. C. Western blot analysis of LytM (upper panel) and Spa (lower panel) production. Crude extracts (LytM) and cell wall extracts (Spa) were prepared from stationary phase cultures. Lanes: 1: Purified <i>Staphylococcus aureus</i> Protein A (50 ng) 2: HG001 strain; 3: Δ<i>spdC</i> mutant strain; 4: Δ<i>spdC</i>/pMK4Pprot-<i>spdC</i> complemented strain.</p