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