FIG. 5.

Analysis of csb7 (SACOL2484) transcription in the absence of RsbU, RsbV, and RsbW. S. aureus GP269 expresses chromosomally encoded SigB and the whole set of Rsb proteins (RsbU, RsbV, and RsbW). SG003 is an RsbU-, RsbV-, RsbW-, and SigB-deficient strain in the 8325-4 genetic background transformed with a plasmid expressing sigB under the control of a tetracycline-inducible promoter. The different S. aureus strains were grown in LB medium. At an OD₅₄₀ of 0.7, the culture was split into three parts. The first part served as an unstressed control and was sampled at the beginning of the experiment (co1) and 20 min later (co2). The second part of the culture was stressed with potassium hydroxide (30 mM final concentration), and samples were taken 10 and 20 min after stress exposure. The third part was supplemented with tetracycline (50 ng ml⁻¹) to induce expression of the plasmid-encoded SigB. Samples from this culture were harvested at 10 and 20 min. In addition, at 10 min after tetracycline exposure one part of the tetracycline-treated culture was transferred to a new Erlenmeyer flask and stressed with potassium hydroxide (30 mM final concentration) for another 10 min. For Northern blot analyses of csb7 (SACOL2484) transcription, 10 μg of total RNA was blotted per lane and the membrane was probed with a digoxigenin-labeled csb7 (SACOL2484)-specific RNA probe. All experiments were performed in triplicate.