Methionine Biosynthesis in Staphylococcus aureus Is Tightly Controlled by a Hierarchical Network Involving an Initiator tRNA-Specific T-box Riboswitch

In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine de novo. Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5′-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA (tRNAi fMet) while binding of elongator tRNAMet proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5′ met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence tRNAi fMet becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development.


Monitoring of cell growth and viability
Bacterial strains used are listed in Table S1. Staphylococcus strains were grown in either complex medium TSB (tryptone soya broth, Oxoid) or in a chemically defined medium (CDM). The latter consisted of a basic salt medium (12.5 mM Na 2 HPO 4 , 10 mM KH 2  For strains carrying resistance genes, antibiotics were used at the following concentrations: 100 μg ml -1 for ampicillin, 10 μg ml -1 for erythromycin and 5 μg ml -1 for tetracycline. Overnight cultures were diluted in fresh medium to an optical density at 600 nm (OD 600 ) of 0.05 and a flaskto-medium ratio of 5:1. Cultures were grown at 37°C shaking (220 rpm). Initial growth was monitored as the OD 600 of the culture over time, whereas long-term cell survival was measured by determining the CFU ml -1 at each time point indicated. For Figure 5 strains were first grown in CDM with Met. The cultures were filtered through a 0.22-µm filter with vacuum and washed twice with sterile phosphate-buffered saline (PBS). Bacteria were then resuspended in an equal volume of CDM with or without Met (with a flask-to-medium ratio of 2:1) and grown for another 60 min before sampled for RNA extraction.

Construction of strains
The conditional RNase J2 (rnjB) and RNase III (rnc) mutants were generated using the pMUTIN vector [1]. With this system, conditional mutants can be obtained by integrating the vector upstream of the target gene, which falls under the control of the isopropyl IPTG-inducible promoter P spac . For this purpose a region encompassing the first 600/900 bp of the 5´ coding region of RNase J2 and RNase III was amplified from strain RN6390 by employing the primers listed in Table   S5. The amplicons were then cloned into the EcoRI/BamHI digested pMUTIN yielding vectors pCG106 and pCG107, respectively. The vectors were transferred into the restriction-deficient strain RN4220 under IPTG induction. pMUTIN insertion into the chromosome was verified by PCR and pulsed-field gel electrophoresis (PFGE). The mutations were then transduced into S. aureus strain Newman.
Transductants were verified by PCR and PFGE.

RNA isolation and Northern blot analysis
Samples from bacterial cultures were mixed with 1X Vol. RNA protection solution (Qiagen) for immediate RNA stabilization. Cells were disrupted mechanically (Bertin technologies) and total RNA was purified using mini-scale, silica-membrane based spin-columns (Qiagen).
Northern blot analysis by agarose/ formaldehyde gels of two microgramm total RNA each sample followed standard procedures. Sequence-specific probes were generated by PCR with oligonucleotides listed in Table S3 (SI) and radioactively labeled with the DNA labeling system (Amersham, GE Healthcare) and [α 32 P]-dCTP. For Figure 1B  For Figure 5 RNA isolation and Northern blot analysis were performed as described [2]. Briefly, bacteria were lysed in Trizol reagent (Invitrogen) with zirconia-silica beads (0.1-mm diameter) in a high-speed homogenizer (Savant Instruments, Farming-dale, NY). RNA was isolated as described by the manufacturer of Trizol. Digoxigenin (DIG)-labeled probes were generated with a DIG-labeling PCR kit following the manufacturer's instructions (Roche Biochemicals) using oligonucleotides listed in Table S3. The numbers in the last column indicate the nucleotide position based on the met leader RNA sequence of S. aureus COL. The sequence of the oligonucleotides used for each construct are listed in Table S5.