<i>In vitro</i> binding of <i>met</i> leader RNA to tRNAs.

<p>(A) Schematic of the binding interaction between tRNA and T-box leader RNA according to <i>Bacillus</i> T-box systems <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003606#ppat.1003606-Green1" target="_blank">[9]</a>. On the top (‘+ Met’), system under high intracellular levels of methionine, on the bottom (‘− Met’) under methionine starvation. Indicated are tRNAs with an amino acid (‘aa’, top) or a free 3′-CCA end (bottom) and the respective pairing sequences within the T-box leader RNA. (B–D) The <i>met</i> leader RNA was transcribed <i>in vitro</i> by T7 RNA polymerase from a defined DNA template. Where indicated, preformed and radiolabeled tRNA was present during the <i>in vitro</i> transcription (IVT) reaction. Samples were analyzed by a non-denaturing PAGE. Asterisks mark IVT <i>met</i> leader RNA reactions without tRNA, but with [α³²P]-CTP present as an IVT efficiency control. The arrow indicates tRNAs bound to the <i>met</i> leader RNA. All experiments were performed at least twice. (B) Methionine-specific tRNA from the different genomic loci (tRNA_i^fMet, tRNA^Met3 and tRNA^Met4) or tRNA^Cys were present in the IVT. The leader RNA was transcribed from either the <i>S. aureus</i> COL or <i>S. epidermidis</i> RP62A sequence. (C) Increasing concentrations of each tRNA with 3′-CCA end (10 and 50 nM for tRNA^Cys and 10, 25 and 50 nM for tRNA_i^fMet, respectively) were present during IVT. Bound tRNA_i^fMet was quantified by measuring the Photo-Stimulated Luminescence (PSL), which is proportional to the amount of radiation exposed to the IP plate. The PSL values are expressed per mm² (y-axis) against the tRNA molarity (x-axis). (D) Either formylmethionine- (tRNA_i^fMet) or cysteine- (tRNA^Cys) specific tRNA was present during IVT. Two different tRNA species were used: with a free 3′-CCA end (‘cca’) or with an additional cytosine (‘AdC’) at the 3′-CCA end to mimic amino acid charging <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003606#ppat.1003606-Yousef1" target="_blank">[31]</a>.</p

Model of a regulatory cascade for methionine biosynthesis operon control.

<p>(i) With high amino acid levels, branched-chain amino acids (BCAA) and GTP are bound to the CodY repressor, increasing its affinity for target DNA binding; downstream genes are repressed (small picture, bottom left). (ii) Low amino acid levels will trigger the stringent response due to stalled ribosomes, which leads to an increase in RelA-mediated ppGpp alarmone synthesis resulting in less GTP. Subsequently, CodY dissociates from the DNA activating downstream transcription of the T-box leader RNA. The T-box acts as the crucial check-point sensing uncharged tRNA_i^fMet levels and determines transcription of the <i>met</i> biosynthesis genes in a highly methionine-dependent manner. Rapid degradation of the <i>met</i> mRNA by the RNA degradosome is an additional mechanism to limit unnecessary translation of methionine biosynthesis mRNA.</p

Effect of single nucleotide exchanges in the T-box on tRNA binding.

<p>(A) Overview on mutations introduced into the <i>met</i> leader RNA. SI Table S2 lists all mutated constructs. The model of the antiterminator structure with the conserved T-box (in grey) demonstrates deletion of the side bulge in SC2, nucleotide substitutions of constructs SC3 to SC8 and the possible loss of the side bulge in SC5 through alternative base pairing. Nucleotide positions refer to the complete length of the <i>met</i> leader RNA of <i>S. aureus</i> COL. (B) IVT with WT or mutated constructs SC1 to SC8 (lanes 1–8) of the <i>met</i> leader RNA template with radiolabeled tRNA_i^fMet present. The arrow indicates the bound tRNA_i^fMet. The asterisk marks the control IVT reaction of the WT <i>met</i> leader RNA transcribed without tRNA, but in the presence of [α³²P]-CTP. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003606#s2" target="_blank">Results</a> are representative of two independent experiments.</p

Determination of a leader RNA transcript upstream of methionine biosynthesis genes.

<p>(A) Overview of the genomic organization of the methionine biosynthesis operon. A transcript (black arrow) was detected upstream of <i>metI</i>. (B) Northern blot analysis of total RNA from three <i>S. aureus</i> strains (Newmann, COL and N315) for the intergenic region (IGR) between <i>spo0J</i> and <i>metI</i>. Radiolabeled probes were either the PCR product (DNA) or <i>in vitro</i> transcribed RNA of each strand (sense and antisense) of the IGR. The experiment was done in duplicate. Fragment sizes correspond to a high-range RNA ladder (Fermentas). The 16S rRNA is shown as a loading control in the corresponding agarose gel. (C) Sequence of the IGR upstream of <i>metI</i> in <i>S. aureus</i> COL. Transcription start of the <i>met</i> leader RNA, as experimentally determined by 5′ RACE, is indicated by a bold ‘T’; putative -35 and -10 promoter sites are boxed; the CodY binding motif is boxed in grey. The potential specifier box and the highly conserved T-box motif are shown in black; overlapping with the T-box is a predicted Rho-independent transcription terminator (in italics and dark grey). The Shine-Dalgarno sequence (SD) and the start codon of the <i>metI</i> gene (light grey) are underlined.</p

Methionine metabolism and biosynthesis control among <i>Bacillales</i> and <i>Lactobacillales</i>.

<p>Presence of genes of the methionine salvage pathway, polyamine synthesis, SAM recycling and control mechanisms of methionine biosynthesis in genomes of bacteria of the <i>Bacillales</i> and <i>Lactobacillales</i> orders as well as number of methionine-specific T-box riboswitches in these genomes. Data were obtained by querying the KEGG <a href="http://www.genome.jp/kegg/" target="_blank">http://www.genome.jp/kegg/</a> and RegPrecise <a href="http://regprecise.lbl.gov/RegPrecise/index.jsp" target="_blank">http://regprecise.lbl.gov/RegPrecise/index.jsp</a> databases <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003606#ppat.1003606-Novichkov1" target="_blank">[15]</a> and from references <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003606#ppat.1003606-GutierrezPreciado1" target="_blank">[6]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003606#ppat.1003606-Joshi1" target="_blank">[20]</a>.</p

Quantitative RT-PCRs of phagocytosed bacteria.

<p>Transcript analyses of <i>rpsB</i>, <i>infB</i> (category I), <i>ilvC</i> (category II), <i>psmα1-4</i>, <i>psmβ1,2</i> (category III) and <i>RNAIII</i> genes after phagocytosis of the <i>S. aureus</i> strain HG001 and the corresponding <i>rsh_Syn</i> mutant, <i>rsh_Syn</i>, <i>codY</i> double mutant and the complemented <i>rsh_Syn</i> mutant (compl). The transcripts were relatively quantified in reference of <i>gyrB</i> and are shown in a log₁₀ scale relative to the wild-type strain (HG001). RNA was isolated after a 60 and 90-minute interaction of <i>S. aureus</i> with PMNs. Values from four separate experiments were used to calculate the mean expression. The levels of significance compared to the wild-type strain were determined by the two-tailed Student t test (p<0.05).</p

Quantitative analysis of the intracellular ATP concentrations.

<p>The intracellular ATP pools of strain HG001, the corresponding <i>rsh_Syn</i> mutant, the <i>rsh_Syn</i>, <i>codY</i> double mutant and the complemented <i>rsh_Syn</i> mutant (compl) were determined by luciferase activity. The bacteria were grown in CDM to exponential growth phase (OD₆₀₀ = 0.5) followed by further incubation in medium with (control) or without (−) leu/val for 30 minutes. The levels of significance to the corresponding control (with leu/val) were determined by the two-tailed Student t test (p<0.05).</p

Genes positively regulated under stringent response independent of CodY.

1<p>: Fold changes are indicated for each comparison and displayed for genes showing statistically significant differential expression. Values corresponds to expression ratios, i.e. averaged expression levels from three independent replicate experiments (p<0.05).</p

Overview of the presumed impact of the stringent response on survival after phagocytosis.

<p>The induction of the stringent response (pppGpp) in phagocytosed bacteria leads to increased <i>psms</i> expression. The PSMs in turn mediate a pronounced survival after phagocytosis, most likely through phagosomal escape or intracellular lysis of the phagocytes.</p

Transcriptome analysis of (p)ppGpp dependent genes during stringent response.

<p>Transcriptional changes of <i>S. aureus</i> in response to leucine/valine starvation (−leu/val) and the overlap of the two regulons CodY and stringent response. (A) Venn diagrams showing genes which are down- (blue) and up-regulated (red) during RSH mediated stringent response. A contribution of CodY to the regulation was determined by analyzing the transcriptional changes in a <i>codY</i>-positive background (WT strains vs. <i>rsh_Syn</i> mutant) indicated as codY+ in the figure and in a <i>codY</i>-negative background (<i>codY</i> mutant vs. <i>rsh_Syn</i>, <i>codY</i> double mutant) indicated as codY−. (B) Functional classes of genes which are down- (blue) and up-regulated (red) during RSH mediated stringent response. (C) Heat maps of gene expression ratios for indicated functional classes prominently altered in their expression. The ratios were analyzed in a <i>codY-</i>positive (codY+) and a <i>codY-</i> negative (codY−) background as described for the Venn diagrams. ** saturated signal *** genes with annotated CodY binding motifs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003016#ppat.1003016-Majerczyk1" target="_blank">[16]</a>.</p