Mutational Analysis of the Critical Bases Involved in Activation of the AreR-Regulated σ 54 -Dependent Promoter in Acinetobacter sp. Strain ADP1
Open Access
- 1 September 2003
- journal article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 69 (9), 5627-5635
- https://doi.org/10.1128/aem.69.9.5627-5635.2003
Abstract
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. 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 IR1 had more deleterious effects upon expression when they were in its central region, which overlaps the left arm of IR2, than when they were in its outer regions. By contrast, substitutions in the right arm of IR2 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 σ 54 promoter are critical.Keywords
This publication has 36 references indexed in Scilit:
- In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated ς 54 -Dependent Po PromoterJournal of Bacteriology, 2001
- areCBA Is an Operon in Acinetobacter sp. Strain ADP1 and Is Controlled by AreR, a ς 54 -Dependent RegulatorJournal of Bacteriology, 2001
- Bacteria Are Not What They Eat: That Is Why They Are So DiverseJournal of Bacteriology, 2000
- THE β-KETOADIPATE PATHWAY AND THE BIOLOGY OF SELF-IDENTITYAnnual Review of Microbiology, 1996
- Activation of Transcription at σ54-dependent Promoters on Linear Templates Requires Intrinsic or Induced Bending of the DNAJournal of Molecular Biology, 1996
- ATP Binding to the σ54-Dependent Activator XylRTriggers a Protein Multimerization Cycle Catalyzed by UAS DNACell, 1996
- A wide-host-range suicide vector for improving reverse genetics in Gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocoliticaGene, 1991
- The integration host factor stimulates interaction of RNA polymerase with NIFA, the transcriptional activator for nitrogen fixation operonsCell, 1990
- Site-directed mutagenesis by overlap extension using the polymerase chain reactionGene, 1989
- The Growth of Micro-organisms in Relation to their Energy SupplyMicrobiology, 1960