Localization of upstream sequence elements required for nitrate and anaerobic induction of fdn (formate dehydrogenase-N) operon expression in Escherichia coli K-12.

Abstract

Two transcriptional activators, the FNR and NARL proteins, are required for induction of the fdnGHI operon, encoding Escherichia coli formate dehydrogenase-N. The FNR protein is required for anaerobic expression, while the NARL protein mediates nitrate induction. We used primer extension to locate the transcription initiation site 29 nucleotides upstream of the fdnG translation initiation codon. Expression assays with single-copy phi (fdnG-lacZ) gene fusions containing various deletions in the fdn 5'-regulatory region delimited three distinct cis-acting elements. One site, which is located at approximately -110, was required for nitrate induction. Two other sites share sequence similarity with the FNR protein binding site core consensus. The first site, centered at -42.5, was required for anaerobic induction. We used site-specific mutagenesis to change this putative FNR protein binding site into the CRP protein binding site core consensus. This change caused the fdn operon to be expressed aerobically, subject to CRP protein control. On the other hand, converting this putative FNR protein binding site into the FNR protein binding site core consensus resulted in elevated anaerobic induction of the fdn operon and also caused weak aerobic expression. The other putative FNR protein binding site, centered at -97.5, was not involved in anaerobic induction. It might play a negative role in fdn operon expression during anaerobic growth in the absence of nitrate.