Construction of chimaeric promoter regions by exchange of the upstream regulatory sequences from fdhF and nif genes.

Abstract

Hybrid 5' regulatory regions were constructed in which the upstream activator sequence (UAS) and promoter of various nif genes were exchanged with the upstream regulatory sequence (URS) of the fdhF gene from Escherichia coli. They were analysed for their regulatory response under different growth conditions with the aid of fdhF'-'lacZ or nif'-'lacZ fusions. Placement of the UAS from the Bradyrhizobium japonicum nifH gene in front of the spacer (DNA region between URS and promoter) plus promoter from fdhF renders fdhF expression activatable by the Klebsiella pneumoniae NIFA protein, both under aerobic and anaerobic conditions. This excludes the possibility that the spacer of the fdhF5' flanking region contains a site recognized by a putative oxygen- or nitrate-responsive repressor. There was also considerable activation by NIFA of fdhF expression in a construct lacking the nifH UAS but containing the fdhF spacer plus promoter. Further experimental evidence suggests that this reflects a direct interaction between NIFA and RNA polymerase at the ntrA-dependent promoter. A second set of hybrid constructs in which the URS from fdhF (E. coli) was placed in front of the nifD spacer plus promoter from B. japonicum or in front of the K. pneumoniae nifH, nifU, nifB spacers and promoters, delivered inactive constructs in the case of the nifD, nifU and nifB genes. However, a nifH'-'lacZ fusion preceded by its own spacer and promoter plus the foreign fdhF URS displayed all the regulatory characteristics of fdhF expression, i.e. anaerobic induction with formate and repression by oxygen and nitrate. Although it is not known why only one out of the four nif promoters could be activated by the fdhF URS, this result nevertheless demonstrates that the various regulatory stimuli affecting expression of fdhF in E. coli have their target at the upstream regulatory sequence.