IDENTIFICATION OF A PALINDROMIC SEQUENCE THAT IS RESPONSIBLE FOR THE UP-REGULATION OF NAPDH-FERREDOXIN REDUCTASE IN A FERREDOXIN-I DELETIONSTRAIN OF AZOTOBACTER-VINELANDII

Azotobacter vinelandii ferredoxin I (AvFdI) is one member of a class o f 7Fe ferredoxins found in a variety of organisms that are all capable of aerobic growth. Disruption of the fdxA gene, which encodes AvFdI, leads to overexpression of its redox partner, NADPH-ferredoxin reducta se (FPR), In this study the mechanism of FdI-mediated regulation of FP R was investigated, Northern analysis has shown that regulation is at the level of fpr transcription, the start site for transcription has b een identified, and it is preceded by a canonical sigma 70-type bacter ial promoter, Gel mobility shift assays show that there is a putative regulatory protein in A, vinelandii that binds specifically upstream o f the -35 region, That protein is not AvFdI, A palindromic sequence wa s identified as a putative binding site, and randomization of that seq uence completely eliminates binding of the putative regulatory protein , A luciferase reporter gene was placed under control of the A. vinela ndii fpr promoter and introduced into wild type and FdI(-) strains of A, vinelandii, Luciferase activity was enhanced 7-fold in the FdI(-) m utant relative to the wild type, Alteration of the palindromic sequenc e reduced the luciferase levels in the FdI(-) strain to those of the w ild type, demonstrating that FdI regulates FPR through the palindrome and that the reaction is an activation rather than a repression, The i dentified palindrome is similar to 50% identical to the SoxS binding s ite upstream of Escherichia coli fpr, suggesting that A, vinelandii ma y have a SoxS-like regulatory system and that the function of FdI migh t be to specifically inactivate that system.