Deletion loop mutagenesis of the nifL promoter from Klebsiella pneumoniae: role of the -26 to -12 region in promoter function

Abstract

Nine single C-to-T transitions were introduced into the -26 to -12 region of the Klebsiella pneumoniae nifL promoter by bisulphite mutagenesis of M 13 heteroduplexes containing a 15 nucleotide single-stranded loop. Mutant promoter fragments were inserted into translational lac fusion vectors to utilise β-galactosidase activity as a measure of promoter efficiency. Mutations in invariant nucleotides found in the consensus sequence for nif promoters gave a strong ‘down’ promoter phenotype with respect to transcriptional activation. Mutations in semi-conserved residues had a much weaker down phenotype, whereas a mutation which increased homology to the consensus sequence enhanced promoter strength. One mutant showed increased activation by ntrC and decreased activation by nifA.