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Genetics and Molecular Biology |
Department of Genetics, Institute of Molecular and Cell Biology, Estonian Biocentre and Tartu University, 51010 Tartu, Estonia1
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois, USA2
Author for correspondence: Maia Kivisaar. Tel: +372 7 375 015. Fax: +372 7 420 286. e-mail: maiak{at}ebc.ee
The promoter of the plasmid-borne pheBA genes encoding enzymes for phenol degradation resembles the catBCA promoter and is activated by CatR, the regulator of the chromosomally encoded catechol-degradative catBCA genes in Pseudomonas putida. In this study, site-directed mutagenesis of the pheBA promoter region was performed. The interrupted inverted repeat sequence of the CatR recognition binding site (RBS) of the pheBA promoter is highly homologous to that of the catBCA promoter. However, the RBS was shown not to be the sole important feature for high-affinity binding of CatR to this site. Mutagenesis of the activation binding site (ABS) of CatR, which overlaps the -35 hexamer sequence TTGGAT of the promoter, revealed that the two G nucleotides in this sequence are important for promoter activity but not for CatR binding. All other substitutions made in the ABS negatively affected both the promoter activity and CatR binding. The spacer sequence of the pheBA and catBCA promoters between the -10 and -35 hexamers is 19 bp, which is longer than optimal. However, reducing the spacer region of the pheBA promoter was not sufficient for CatR-independent promoter activation. An internal binding site (IBS) for CatR is located downstream of the transcriptional start site of the catBCA genes and it negatively regulates the operon. A similar IBS was identified in the case of the pheBA operon and tested for its functionality. The results indicate a conservation of CatR-mediated regulation mechanisms between the pheBA promoter and the catBCA promoter. This universal mechanism of CatR-mediated transcriptional activation could be of great importance in enabling catechol-degrading bacteria to expand their substrate range via horizontal transfer of the phenol degradative genes.
Keywords: Pseudomonas putida, transcription activation, CatR, pheBA and catBCA operons, evolution of catabolic pathways
Abbreviations: ABS, activation binding site; CCM, cis,cis-muconate; ß-Gal, ß-galactosidase; IBS, internal binding site; RBS, recognition binding site
a Present address: Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA.
This article has been cited by other articles:
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  M. Putrins, A. Tover, R. Tegova, U. Saks, and M. Kivisaar Study of factors which negatively affect expression of the phenol degradation operon pheBA in Pseudomonas putida Microbiology, June 1, 2007; 153(6): 1860 - 1871. [Abstract] [Full Text] [PDF]
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 B. M. Bundy, L. S. Collier, T. R. Hoover, and E. L. Neidle Synergistic transcriptional activation by one regulatory protein in response to two metabolites PNAS, May 28, 2002; 99(11): 7693 - 7698. [Abstract] [Full Text] [PDF]
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A. Tover, E.-L. Ojangu, and M. Kivisaar Growth medium composition-determined regulatory mechanisms are superimposed on CatR-mediated transcription from the pheBA and catBCA promoters in Pseudomonas putida Microbiology, August 1, 2001; 147(8): 2149 - 2156. [Abstract] [Full Text] [PDF]
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