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Research Paper |
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA1
Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27858, USA2
Author for correspondence: Susan E. H. West. Tel: +1 608 263 7884. Fax: +1 608 263 7884. e-mail: wests{at}svm.vetmed.wisc.edu
Vfr of Pseudomonas aeruginosa is 91% similar to the cAMP receptor protein (CRP) of Escherichia coli. Based on the high degree of sequence homology between the two proteins, the question arose whether Vfr had a global regulatory effect on gene expression for P. aeruginosa as CRP did for E. coli. This report provides two-dimensional polyacrylamide gel electrophoretic evidence that Vfr is a global regulator of gene expression in P. aeruginosa. In a vfr101::aacC1 null mutant, at least 43 protein spots were absent or decreased when compared to the proteome pattern of the parent strain. In contrast, 17 protein spots were absent or decreased in the parent strain when compared to the vfr101::aacC1 mutant. Thus, a mutation in vfr affected production of at least 60 proteins in P. aeruginosa. In addition, the question whether Vfr and CRP shared similar mechanistic characteristics was addressed. To ascertain whether Vfr, like CRP, can bind cAMP, Vfr and CRP were purified to homogeneity and their apparent dissociation constants (Kd) for binding to cAMP were determined. The Kd values were 1·6 µM for Vfr and 0·4 µM for CRP, suggesting that these proteins have a similar affinity for cAMP. Previously the authors had demonstrated that Vfr could complement a crp mutation and modulate catabolite repression in E. coli. This study presents evidence that Vfr binds to the E. coli lac promoter and that this binding requires the presence of cAMP. Finally, the possible involvement of Vfr in catabolite repression control in P. aeruginosa was investigated. It was found that succinate repressed production of mannitol dehydrogenase, glucose-6-phosphate dehydrogenase, amidase and urocanase both in the parent and in two vfr null mutants. This implied that catabolite repression control was not affected by the vfr null mutation. In support of this, the cloned vfr gene failed to complement a mutation in the P. aeruginosa crc gene. Thus, although Vfr is structurally similar to CRP, and is a global regulator of gene expression in P. aeruginosa, Vfr is not required for catabolite repression control in this bacterium.
Keywords: Vfr, catabolite repression control, global regulator, cAMP binding, cAMP receptor protein
Abbreviations: CRP, cAMP receptor protein
a Present address: Department of Microbiology and Immunology, Medical College of Virginia campus of Virginia Commonwealth University, Richmond, VA 23298-0678, USA.
b Present address: Department of Cellular and Molecular Toxicology, Abbott Laboratories, Abbott Park, IL 60064-6104, USA.
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