Gene fusion technology has been exploited in this project to study the regulation of genes involved in the switch from aerobic to anaerobic metabolism in E.coli. Protein or operon fusions were created when the appropriate transducing phage inserted in frame with a target gene. XplacMu was found to be the most versatile vector, being capable of translocating the lac genes, minus their transcriptional and translational signals, to any site in the chromosome. The expression of p-galactosidase from these fusions will reflect both the transcriptional and translational activity of the target gene in response to a particular stimulus, in this case the presence or absence of molecular oxygen.
Insertion of XplacMu into the control sequence of an anaerobically or aerobically regulated gene was detected by its anaerobic Lac+/aerobic Lac- (or vice versa) phenotype. Secondary mutagenesis of such fusions defines those control proteins involved in the switch by screening for a Lac+/Lac+ or Lac~/Lac“ phenotype following TnlO mutagenesis.
A library of fusion strains was identified by map position and phenotype and control of the anaerobically regulated fusions was further investigated. As the Fnr protein is essential for anaerobic respiration the regulation of the fusions by this pleiotropic activator protein was monitored.
The expression of two fusion strains was regulated by another gene, designated adhC. The pleiotropic nature of this locus may be indicative of a second activator protein involved in the regulation of fermentative pathways. The cloning and subsequent genetic manipulation of this gene was simplified by constructing an adhC :: XplacHu fusion. Dual regulation by Fnr and AdhC was investigated, as was the regulation of anaerobic Lac+ fusions unaffected by either activator.
The interrelationship of the various pathways of anaerobic metabolism was considered together with putative effector metabolites of the regulatory proteins involved.