Recent Papers

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/XylR family of regulatory proteins as determined by sequence homology. 79 bases upstream of the start of transcription is a region carrying two overlapping inverted repeat sequences that we predict to be the AreR binding site, also known as the Upstream Activator Site (UAS). IR1 is a near perfect (16/17 bp) repeat separated by 1 bp, and IR2 consists of  9 bp and 7 bp perfect repeats with a 3 bp gap, with the two arms of the repeat separated by 16 bp. We report here a method for site-directed mutagenesis of chromosomal genes in ADP in which linear fragments generated by overlap extension PCR are transformed into ADP1 via its natural transformation system and recombinants selected by a marker exchange eviction strategy with a newly created sacB-Km cassette. This 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 IR1 had a more deleterious effect upon expression when they were in its central region, which overlapped the left-hand arm of IR2, than when they were in its outer regions.  By contrast substitutions in the right hand arm of IR2 resulted in mutants with relatively high expression levels compared to the wild type. Deletions in the right hand arm of IR2 were very dependent upon the length of the deletion with 3 or 5 bp deletions reducing expression by > 90%, whereas an 11bp 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.