Mutational analysis of the critical bases involved in activation of the AreR-regulated σ54-dependent promoter in Acinetobacter sp strain ADP1

The areR gene in Acinetobacter sp. strain ADP1 regulates the expression of the areCBA genes, which determine growth on benzyl alkanoates. AreR is a member of the NtrC/XyIR family of regulatory proteins as determined by sequence homology. Seventy-nine bases upstream of the start of transcription is a region carrying two overlapping inverted repeat (IR) sequences that we predict to be the AreR binding site, also known as the upstream activator site (UAS). IR1 is a near-perfect (16 of 17 bp) repeat separated by 1 bp, and IR2 consists of 9- and 7-bp perfect repeats with a 3-bp gap, with the central bases of the two arms of the repeat separated by 44 and 22 bp. We report here a method for site-directed mutagenesis of chromosomal genes in ADP1 in which linear fragments generated by overlap extension PCR are used to transform ADP1 via its natural transformation system and recombinants are selected by a marker exchange-eviction strategy with a newly created sacB-Km cassette. This method was used to generate 38 strains with designed mutations in the putative UAS upstream of areCBA. The effects of the mutations on areCBA expression were measured by enzyme assays of benzyl alcohol dehydrogenase (AreB) and by reporter gene assays of lacZ inserted into areA. Substitutions or deletions in 1111 had more deleterious effects upon expression when they were in its central region, which overlaps the left arm of 11112, than when they were in its outer regions. By contrast, substitutions in the right arm of 1112 resulted in mutants with relatively high expression levels compared to that of the wild type. Effects of deletions in the right arm of IR2 were very dependent upon the length of the deletion, with 3-or 5-bp deletions reducing expression by >90% whereas an 11-bp deletion in the same area reduced the expression levels by only 50%, suggesting that alterations in the distance and the orientation of the UAS relative to the -24, -12 sigma(54) promoter are critical.