Figure 3. Cell growth effects of RraA-mediated inhibition of SOS.

Physiological production of RraA at the level required for down-regulation of SOS. In stationary phase, RraA is produced at levels that can interfere with SOS response induction, demonstrated by western blot (A) and β-galactosidase activity (B). (A) A small amount of RraA protein expressed from the lacZ promoter of the pTrc-rraA plasmid was sufficient to suppress the SOS response; the level mediating such suppression was compared to the level of RraA protein expressed in stationary phase (after 24 h growth) by untreated SY2 cells harboring pTrc-99A (empty plasmid). Aliquots were assayed by Western blot at various times with and without MTC addition (samples were separated by Criterion 12% Bis-Tris gel electrophoresis using XT-MOPS buffer and were immunoblotted using anti-RraA and anti-S1 antibodies. Specific protein bands were imaged by VersaDoc 1000 (Bio-Rad) and quantified by Quantity One (Bio-Rad). (B) Repression of SOS response by leaky expression of RraA from pTrc-rraA plasmid in SY2 cells cultured in the absence of IPTG. SOS response was induced at 0 time in SY2 cells harboring pTrc99A (○, empty plasmid) or pTrc-rraA (•) by addition of MTC (0.1 µg/ml).