THE NDH-BINDING PROTEIN (NBP) REGULATES THE NDH GENE OF ESCHERICHIA-COLI IN RESPONSE TO GROWTH-PHASE AND IS IDENTICAL TO FIS

The ndh gene that encodes the non-proton-translocating NADH dehydrogen ase II of Escherichia coli is anaerobically repressed by FNR. However, in the absence of FNR, ndh expression is enhanced by anaerobic growth in media containing amino acids. Two potential regulatory proteins th at may be associated with this activation have previously been detecte d, Arr (amino acid response regulator) and Nbp (ndh-binding protein). Studies with the heat-stable Nbp have now shown that it is present in E. coil grown both aerobically and anaerobically in rich and minimal m edia, indicating that it is not specifically associated with the anaer obic enhancement of ndh expression. The Nbp activity of aerobic cultur es was maximal during exponential growth phase (when ndh promoter acti vity is minimal) but fell rapidly as cultures entered stationary phase and ndh expression increased. Protein purification and mutant studies have further shown that Nbp is identical to the Fis protein (factor f or inversion stimulation). Three major and two minor Nbp (Fis)-binding sites have been identified in the ndh promoter by gel retardation and DNase I footprinting. The major sites are centred at -123, -72 and +5 1, in decreasing order of binding affinity. At low concentrations, Nbp (Fis) increased transcription from the ndh promoter by up to 25%, whe reas at higher concentrations it prevented RNA polymerase (RNAP) bindi ng and open complex formation. Consequently, Nbp (Fis) can both activa te and repress transcription from the ndh promoter, The results sugges t that Nbp (Fis) serves to ensure that the energetically efficient pro ton-translocating NADH dehydrogenase I is used in preference to the no n-proton translocating NADH dehydrogenase II during periods of rapid g rowth, by repressing expression of the ndh gene.