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The Escherichia coli soxRS regulon activates oxidative stress and antibiotic resistance genes in two transcriptional stages. SoxR protein becomes activated in cells exposed to excess superoxide or nitric oxide and then stimulates transcription of the soxS gene, whose product in turn activates>/=10 regulon promoters. Purified SoxR protein is a homodimer containing a pair of [2Fe-2S] centers essential for soxS transcription in vitro . The [2Fe-2S] centers are thought to be anchored by a C-terminal cluster of four cysteine residues in SoxR. Here we analyze mutant SoxR derivatives with individual cysteines replaced by alanine residues (Cys-->Ala). The mutant proteins in cell-free extracts bound the soxS promoter with wild-type affinity, but upon purification lacked Fe or detectable transcriptional activity for soxS in vitro . Electron paramagnetic resonance measurements in vivo indicated that the Cys-->Ala proteins lacked the [2Fe-2S] centers seen for wild-type SoxR. The Cys-->Ala mutant proteins failed to activate soxS expression in vivo in response to paraquat, a superoxide- generating agent. However, when expressed to approximately 5% of the cell protein, the Cys-->Ala derivatives increased basal soxS transcription 2-4-fold. Overexpression of the Cys119-->Ala mutant protein strongly interfered with soxS activation by wild-type SoxR in response to paraquat. These studies demonstrate the essential role of the [2Fe-2S] centers for SoxR activation in vivo ; the data may also indicate oxidant-independent mechanisms of transcriptional activation by SoxR.

1. Membrane fractions were obtained from homogenates of olfactory rosettes from Atlantic salmon (Salmo salar) or from isolated olfactory cilia and homogenates of deciliated olfactory rosettes. 2. Specific binding of L-[3H]alanine was saturable, high-affinity, and effectively inhibited by L-threonine, L-serine and L-alanine but not by L-lysine or L-glutamic acid. Comparable results were obtained with L-[3H]serine except for the presence of a second, lower affinity, binding site for L-alanine but not L-serine. 3. Specific binding of L-[3H]alanine was inhibited by low concentrations of mercury ion, acidic pH, and high concentrations of cadmium, copper or zinc ions. Aluminum had no effect. 4. Specific binding sites for L-alanine were present in membranes from isolated cilia at a level 2-fold that of membranes prepared from the deciliated rosette. 5. Ouabain sensitive Na+, K(+)-ATPase activity was also determined in cilia preparations. This enzyme was present in cilia at a level approximately 3-fold that of membranes prepared from the deciliated rosette. 6. The results are consistent with the presence of an olfactory alanine receptor in S. salar with binding characteristics similar to those of a variety of other fish species and with a localization on olfactory cilia as well as non-ciliated receptor cell membranes.