CHARACTERIZATION OF FNR-ASTERISK MUTANT PROTEINS INDICATES 2 DISTINCT MECHANISMS FOR ALTERING OXYGEN REGULATION OF THE ESCHERICHIA-COLI TRANSCRIPTION FACTOR FNR

In order to gain insight into the mechanism by which the Escherichia coli transcription factor FNR is activated in response to anaerobiosis, we have analyzed FNR* mutant proteins which, unlike the wild-type protein, stimulate gene expression in the presence of oxygen in vivo. Cell extracts containing seven different FNR* mutant proteins were tested in vitro for the ability to bind to the FNR consensus DNA site in a gel retardation assay under aerobic conditions. At the concentration of protein tested, only extracts which contained FNR* mutant proteins with amino acid substitutions at position 154 showed significant DNA binding, The three position-154 FNR* mutant proteins could be further distinguished from the other mutant proteins by analysis of the in vivo phenotypes of FNR* proteins containing amino acid substitutions at either of two essential cysteine residues. In the presence of oxygen, FNR* mutant proteins with amino acid substitutions at position 154 were the least affected when either Cys-23 or Cys-122 was substituted for Ser. On the basis of these in vivo and in vitro analyses, FNR* mutant proteins appear to segregate into at least two classes, Thus, it appears that each class of FNR* substitutions alters the normal pathway of FNR activation in response to oxygen deprivation by a different mechanism.