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

Oligonucleotides used in this study.

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

The <i>S</i>. <i>aureus wal</i> locus consists of two separate transcription units.

<p>(A) <i>S</i>. <i>aureus</i> total RNA was used to synthesize cDNAs and amplify intergenic regions with oligonucleotides hybridizing specifically within the upstream and downstream genes (indicated by arrows, not to scale). DNA fragments were separated by electrophoresis on ethidium bromide stained 1.5% agarose gels and their size is indicated. Lanes 1: positive PCR control using HG001 genomic DNA as the template; lanes 2: PCR using cDNA as the template; lanes 3: control PCR reaction using total RNA as the template (without reverse transcriptase treatment). M: 100 bp molecular mass ladder (Eurogentec, Angers, France). (B) Representation of the <i>wal</i> locus and the corresponding transcripts (not to scale).</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

Phylogenetic relationships within the Bacilli class and inferred losses of <i>wal</i> genes.

<p>(A) Maximum likelihood phylogeny of Bacilli based on a concatenation of 47 ribosomal proteins comprising 5,945 amino acid positions. The conservation of <i>wal</i> genes in each genome is indicated by circles: <i>walR</i> (red), <i>walK</i> (green), <i>walH</i> (blue), <i>walI</i> (yellow), <i>walJ</i> (purple). Red crosses indicate the loss of <i>walH</i> and <i>walI</i> in Streptococcaceae and of <i>walJ</i> in <i>Leuconostoc</i>. (B) Maximum likelihood phylogeny based on a concatenation of WalR, WalK, WalH, WalI and WalJ protein sequences comprising 1,102 amino acid positions. For both analyses, values at nodes represent bootstrap proportions calculated on 1,000 resamplings of the original data set. For clarity, only the values corresponding to monophyly of families and their evolutionary relationships are shown. The scale bar represents the average number of substitutions per site. For details on analyses, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151449#sec002" target="_blank">Materials and Methods</a>.</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

The <i>walRKHI</i> operon and the <i>walJ</i> gene are transcribed from σ^A promoters.

<p>(A) Primer extension analysis of the <i>walR</i> (left panel) and <i>walJ</i> (right panel) mRNA. Sanger dideoxy chain termination sequencing reactions (GATC) were carried out on PCR-generated DNA fragments corresponding to the respective upstream regions. (B) DNA sequence of the <i>walR</i> and <i>walJ</i> upstream regions with the identified transcription start sites indicated by arrows and consensus -10 and -35 sequences and extended -10 promoter TG dinucleotides shown by grey boxes. Translation and transcription initiation sites are indicated in bold letters. (C) Expression of the <i>walRKHI</i> operon and the <i>walJ</i> gene was followed using <i>lacZ</i> transcriptional fusions in <i>S</i>. <i>aureus</i> strains ST1365 (P<i>walR</i> RI + RII), ST1366 (P<i>walR</i> RII) and ST1367 (P<i>walJ</i>) and control strain ST1398 carrying plasmid pSA14 alone. β-Galactosidase assays were performed as described in Materials and Methods and measured during late exponential growth at 37°C in TSB. Cultures were spotted on TSB plates containing X-Gal as shown above the histogram bars. (D) Schematic representation of the <i>wal</i> locus genetic structure. Transcription start sites, nucleotide distances or overlap between coding sequences and transcription terminators downstream from <i>walI</i> and <i>walJ</i> are shown.</p

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