Figure 1.
Determination of a leader RNA transcript upstream of methionine biosynthesis genes.
(A) Overview of the genomic organization of the methionine biosynthesis operon. A transcript (black arrow) was detected upstream of metI. (B) Northern blot analysis of total RNA from three S. aureus strains (Newmann, COL and N315) for the intergenic region (IGR) between spo0J and metI. Radiolabeled probes were either the PCR product (DNA) or in vitro 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 metI in S. aureus COL. Transcription start of the met 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 metI gene (light grey) are underlined.
Figure 2.
In vitro binding of met leader RNA to tRNAs.
(A) Schematic of the binding interaction between tRNA and T-box leader RNA according to Bacillus T-box systems [9]. 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 met leader RNA was transcribed in vitro by T7 RNA polymerase from a defined DNA template. Where indicated, preformed and radiolabeled tRNA was present during the in vitro transcription (IVT) reaction. Samples were analyzed by a non-denaturing PAGE. Asterisks mark IVT met leader RNA reactions without tRNA, but with [α32P]-CTP present as an IVT efficiency control. The arrow indicates tRNAs bound to the met leader RNA. All experiments were performed at least twice. (B) Methionine-specific tRNA from the different genomic loci (tRNAifMet, tRNAMet3 and tRNAMet4) or tRNACys were present in the IVT. The leader RNA was transcribed from either the S. aureus COL or S. epidermidis RP62A sequence. (C) Increasing concentrations of each tRNA with 3′-CCA end (10 and 50 nM for tRNACys and 10, 25 and 50 nM for tRNAifMet, respectively) were present during IVT. Bound tRNAifMet 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- (tRNAifMet) or cysteine- (tRNACys) 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 [31].
Figure 3.
Effect of single nucleotide exchanges in the T-box on tRNA binding.
(A) Overview on mutations introduced into the met 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 met leader RNA of S. aureus COL. (B) IVT with WT or mutated constructs SC1 to SC8 (lanes 1–8) of the met leader RNA template with radiolabeled tRNAifMet present. The arrow indicates the bound tRNAifMet. The asterisk marks the control IVT reaction of the WT met leader RNA transcribed without tRNA, but in the presence of [α32P]-CTP. Results are representative of two independent experiments.
Figure 4.
Role of CodY in met leader RNA/metICFE-mdh transcription control.
Northern blot analysis with radiolabeled probes. Total RNA from S. aureus strain Newman and its isogenic codY mutant was sampled at three different points of growth: early exponential (E1), exponential (E2) and early stationary phase (S). Cultures were grown in CDM without (‘−’) or supplemented with 1 mM L-methionine (‘+’). (A) Hybridization with the met leader RNA-specific probe (low range RNA ladder (Fermentas) indicated on the left). The blot was re-hybridized with a 16S rDNA-specific probe, for loading control. (B) Hybridization with metI-specific probe (high range RNA ladder (Fermentas) indicated on the left). The 16S rRNA signal on the gel is shown below. Results are representative of two independent experiments.
Figure 5.
Stringent response relay and involvement of RNases.
(A) Northern blot analysis of total RNA from S. aureus strain Newman (‘Wt’) and different isogenic mutants (‘Δ’) sampled at exponential growth phase. The cultures were grown in CDM without (‘−’) or supplemented with 1 mM L-methionine (‘+’). DIG-labeled DNA probes for brnQ-1 (branched-chain amino acid transport), the met leader RNA or metI were used to hybridize RNA from WT, the rsh (ppGpp synthetase) and a codY mutant. The data are representative of two independent experiments. (B) RNA stability assay. Northern blot analyses of met leader RNA (upper panel) and metICFE-mdh mRNA (lower panel) upon rifampicin exposure of S. aureus strain Newman (‘Wt’) and isogenic RNase J2 and RNase III deletion mutants (‘ΔRnJ2’, ‘ΔRnIII’), respectively. Bacteria were grown in CDM without methionine and total RNA was prepared from cells before (0 min) and after the addition of 500 µg ml−1 rifampicin at the time points indicated in the figure. Blots were hybridized with DIG-labeled met leader- and metI-specific DNA probes, respectively. Bottom panels in (A) and (B) show the 16S rRNA as loading controls in the corresponding agarose gels.
Table 1.
Methionine metabolism and biosynthesis control among Bacillales and Lactobacillales.
Figure 6.
Model of a regulatory cascade for methionine biosynthesis operon control.
(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 tRNAifMet levels and determines transcription of the met biosynthesis genes in a highly methionine-dependent manner.
Rapid degradation of the met mRNA by the RNA degradosome is an additional mechanism to limit unnecessary translation of methionine biosynthesis mRNA.