Molecular genetic analysis of an FNR-dependent anaerobically inducible Escherichia coli promoter

From the effects of 13 deletions and three linker-scanner mutations at the Escherichia coli nirB promoter we have located sequences necessary for FNR-dependent induction of activity by anaerobiosis and further nitrite-dependent stimulation of expression. We describe a nirB promoter derivative that allows the cloning of 'cassettes' carrying different FNR-binding sequences and experiments in which a number of point mutations were introduced into these sequences. FNR-dependent stimulation of expression from the nirB promoter is critically dependent on the location of the FNR-binding site, and deletion or insertion of one base pair is sufficient to disrupt promoter function. We have transferred a number of cassette FNR-binding sequences from the nirB promoter to the unrelated melR promoter. The insertion of FNR-binding sequences at the melR promoter is sufficient to confer fnr-dependency on expression. However expression from these hybrid promoters is not as efficiently repressed during aerobic growth, suggesting that the function of bound FNR is dependent on the sequence context of the FNR-binding sequence.     

Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa.

A mutant of Pseudomonas aeruginosa was characterized which could not grow anaerobically with nitrate as the terminal electron acceptor or with arginine as the sole energy source. In this anr mutant, nitrate reductase and arginine deiminase were not induced by oxygen limitation. The anr mutation was mapped in the 60-min region of the P. aeruginosa chromosome. A 1.3-kb chromosomal fragment from P. aeruginosa complemented the anr mutation and also restored anaerobic growth of an Escherichia coli fnr deletion mutant on nitrate medium, indicating that the 1.3-kb fragment specifies an FNR-like regulatory protein. The arcDABC operon, which encodes the arginine deiminase pathway enzymes of P. aeruginosa, was rendered virtually noninducible by a deletion or an insertion in the -40 region of the arc promoter. This -40 sequence (TTGAC....ATCAG) strongly resembled the consensus FNR-binding site (TTGAT....ATCAA) of E. coli. The cloned arc operon was expressed at low levels in E. coli; nevertheless, some FNR-dependent anaerobic induction could be observed. An FNR-dependent E. coli promoter containing the consensus FNR-binding site was expressed well in P. aeruginosa and was regulated by oxygen limitation. These findings suggest that P. aeruginosa and E. coli have similar mechanisms of anaerobic control.