Figure 1. RsaA regulates the synthesis of several metabolic enzymes.

(A) 2-D fluorescence difference gel electrophoresis (DiGE) performed on HG001 and ΔrsaA-HG001 strains reveals proteins whose synthesis is affected by the deletion of RsaA. The proteins were identified by mass spectroscopy analysis. Ratios correspond to the quantification obtained for HG001 versus ΔrsaA strain (data from four independent experiments). In the last column are given the proteins, which belong to the MgrA regulon as shown by Luong et al. [26]. Statistical differences (p<0.05, except for the bifunctional GMP synthase : p<0.01) between the two strains were obtained for each protein. (B) Potential base pairings between RsaA and potential mRNA targets are shown. The Shine and Dalgarno sequence (SD) of target mRNAs are shown in green while the conserved C rich motif in RsaA is in red. Other predicted RsaA-mRNA complexes are shown in Table S1. (C) Relative MgrA protein expression level was defined by LC-MS-MS followed by a spectral counting strategy. Protein extracts were prepared from HG001 (WT), ΔrsaA mutant strain, and the same strain complemented with a plasmid expressing RsaA. In parallel to MgrA, we analyzed ribosomal protein L21 as internal control of constitutively expressed protein. The spectral count was normalized to the value determined for the WT strain. (D) Western blot performed with monoclonal antibodies against MgrA performed on protein extracts prepared from HG001, ΔrsaA mutant strain and the same strain complemented with a plasmid expressing RsaA. As a control, a gel run in parallel with the same samples was stained with Coomassie blue to verify that each lane contained comparable amounts of protein. Protein size markers were run in parallel. (E) The expression of RsaA and of mgrA mRNA was quantified and normalized to the level of gyrB mRNA expression from total RNA extracts prepared from in vitro culture to the mid- or late-exponential phase of HG001 (WT), the ΔrsaA mutant strain, and the same strain complemented with a plasmid expressing RsaA. (F) Northern experiments showing the steady state levels of mgrA mRNA in HG001 and in the ΔrsaA mutant strains. Total RNAs were prepared after 2, 4 and 6 h of culture in BHI at 37°C. We used two different RNA probes to visualize the expression of RsaA and of mgrA mRNA. We have previously shown that a longer transcript including RsaA was also expressed [20]. This longer form of RsaA is known to be processed by RNase Y [37].