This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jones, R. M. Articles by Williams, P. A. Search for Related Content PubMed PubMed Citation Articles by Jones, R. M. Articles by Williams, P. A. Agricola Articles by Jones, R. M. Articles by Williams, P. A.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, September 2003, p. 5627-5635, Vol. 69, No. 9
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.9.5627-5635.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Mutational Analysis of the Critical Bases Involved in Activation of the AreR-Regulated ⁵⁴-Dependent Promoter in Acinetobacter sp. Strain ADP1

School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd LL57 2UW, Wales, United Kingdom

Received 26 March 2003/ Accepted 2 July 2003

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 ⁵⁴ promoter are critical.

This article has been cited by other articles:

• Gore, J. M., Ran, F. A., Ornston, L. N. (2006). Deletion Mutations Caused by DNA Strand Slippage in Acinetobacter baylyi. Appl. Environ. Microbiol. 72: 5239-5245 [Abstract] [Full Text]  
• Buchan, A., Ornston, L. N. (2005). When Coupled to Natural Transformation in Acinetobacter sp. Strain ADP1, PCR Mutagenesis Is Made Less Random by Mismatch Repair. Appl. Environ. Microbiol. 71: 7610-7612 [Abstract] [Full Text]  
• Metzgar, D., Bacher, J. M., Pezo, V., Reader, J., Doring, V., Schimmel, P., Marliere, P., de Crecy-Lagard, V. (2004). Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering. Nucleic Acids Res 32: 5780-5790 [Abstract] [Full Text]  
• Parke, D., Ornston, L. N. (2004). Toxicity Caused by Hydroxycinnamoyl-Coenzyme A Thioester Accumulation in Mutants of Acinetobacter sp. Strain ADP1. Appl. Environ. Microbiol. 70: 2974-2983 [Abstract] [Full Text]