Characterization of the Pseudomonas aeruginosa vfr gene and its role in the regulation of exotoxin A production

My doctoral research was carried out in the laboratory of Dr. Susan West at the University of Wisconsin at Madison.  I was in the Program in Cellular and Molecular Biology.  The title of my dissertation was “Characterization of the Pseudomonas aeruginosa vfr gene and its role in the regulation of exotoxin A production”.

My dissertation research focused on characterizing the vfr gene and its role in the regulation of exotoxin A (ETA) production in the bacterium Pseudomonas aeruginosa. Vfr was initially identified due to its pleotrophic effect of the expression of several virulence factors or their regulators. Vfr is 67% identical and 91% similar to Escherichia coli CRP, a global regulator of gene expression, and the cloned vfr gene complemented an E. coli crp deletion mutant. In P. aeruginosa, Vfr activated or repressed the production of over 60 proteins.

Pseudomonasaeruginosa vfr was characterized based on its similarities to and differences from E. coli crp. I identified an open reading frame (ORF) divergent to vfr that was 54% identical and 87% similar to an uncharacterized ORF divergent to crp. In E. coli, CRP-dependent expression from the divergent ORF promoter represses the expression of crp because the two promoters overlap. However, in P. aeruginosa, these promoters did not overlap and activation of the divergent ORF promoter was not Vfr-dependent. Thus, the regulatory mechanisms that control vfr expression are different from the ones that control crp expression in E. coli.

Since CRP plays a central role in the catabolite repression response, I also examined the role of Vfr in catabolite repression in E. coli and P. aeruginosa. Using genetic and biochemical analyses, I found that Vfr could replace CRP to function as a component of the E. coli cAMP-modulated catabolite repression control system. However, Vfr was not required for catabolite repression in P. aeruginosa. Thus, although Vfr and CRP are functionally similar as global transcriptional regulators, their specific roles may have evolved to accommodate the particular needs of their respective organisms which have very different natural niches and physiologies.

Finally, I characterized the role of Vfr in the regulation of Vfr-dependent gene expression. I found that Vfr bound sequences in the toxA, regA P1 (a positive regulator of toxA expression), lasRpromoters.  E. coli did not bind the regA P1 or toxA promoters, although it bound the lasR promoter. Thus, Vfr appears to have a broader DNA recognition specificity than CRP.  P. aeruginosagenes in the Vfr regulon have been divided into two groups based on the ability of Vfr and CRP to activate their expression. Only Vfr is capable of activating Class A genes, while CRP can replace Vfr to activate Class B genes. Based on these observations, I constructed chimeric Vfr/CRP proteins to begin localization of the unique regions of Vfr that are required for the regulation of Class A genes. Although the chimeric proteins activated lasR expression, neither the Vfr-CRP nor the CRP-Vfr proteins activated toxA and regA expression, suggesting that Vfr contains unique motifs in both the N and C terminus that are required for activation of Class A gene expression.

Papers from doctoral research:

Kanack KJ, Runyen-Janecky LJ, Ferrell EP, Suh SJ, West SE. 2006. Characterization of DNA-binding specificity and analysis of binding sites of the Pseudomonas aeruginosa global regulator, Vfr, a homologue of the Escherichia coli cAMP receptor protein.Microbiology. 2006 Dec;152(Pt 12):3485-96.

Suh SJ, Runyen-Janecky LJ, Maleniak TC, Hager P, MacGregor CH, Zielinski-Mozny NA, Phibbs PV Jr, West SE. 2002. Effect of vfr mutation on global gene expression and catabolite repression control of Pseudomonas aeruginosa. Microbiology. 2002 May;148(Pt 5):1561-9.

Runyen-Janecky, L. J., A. K. Sample, T. C. Maleniak, and S. E. H. West. 1997. A divergently transcribed open reading frame is located upstream of the Pseudomonas aeruginosa vfr gene, a homolog of Escherichia coli crp. J. Bacteriol. 179:2802-2809.

Albus, A. M., E. C. Pesci, L. J. Runyen-Janecky, S. E. H. West, and B. H. Iglewski. 1997. Vfr controls quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179:3928-3935.

West, S. E. H., A. K. Sample, and L. J. Runyen-Janecky. 1994. The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family. J. Bacteriol. 176:7532-7542.

West, S. E. H., H. P. Schweizer, C. Dall, A. K. Sample, and L. J. Runyen-Janecky. 1994. Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 128:81-86.