George P. Munson, Ph.D.

Assistant Professor of Microbiology and Immunology
Room 3038, Rosenstiel Medical Sciences Building
1600 NW 10th Avenue
Telephone: 305- 243-5317
Fax: 305- 243-4623
Email: gmunson@miami.edu

Research Interests:

Microbial Pathogenesis, Transcriptional Regulation.

Entertoxigenic E. coli (ETEC) cause diarrheal disease resulting in an estimated 700 thousand deaths annually. The majority of mortality cases are infants and young children in developing countries. Presently there are no readily available vaccines to protect humans against this pathogen. Domesticated animals may also be infected by ETEC resulting in significant agricultural and economic impact. To better understand the pathology of ETEC infections, my laboratory is characterizing Rns, a 32 kDa member of the AraC superfamily, that is required for the expression of known or suspected virulence genes. Our goals are to understand how Rns functions as a transcriptional regulator and to identify and characterize the genes that it regulates. Rns homologs have been identified in other strains of ETEC, enteroaggregative E. coli, enteropathogenic E. coli, Shigella flexneri, Yersinia sp., Vibrio cholerae and many other bacterial pathogens. Some of these homologs are functionally interchangeable with Rns and like Rns, they may play pivotal roles during the course of an infection. Thus Rns is the prototype for a group of virulence regulators and our research has implications beyond any one particular strain of ETEC.

Specific areas of interest include:

I. Rns as a model for a new class of transcriptional activators

Rns positively regulates its own expression through an arrangement of binding sites that is unprecedented for a prokaryotic activator. While the vast majority of activators bind within a small region just upstream of the –35 hexamer, Rns has no upstream promoter–proximal binding site. Rather Rns has one binding site centered 220 bp upstream of the transcription start site. Despite the distance of this site from the transcription complex, it is required for Rns to initiate transcription from its promoter, Prns. Even more unusual, Rns has three binding sites downstream of the transcription start site and one of these is required for positive autoregulation of Prns. At present, this makes Rns one of only three activators that have been shown to utilize a downstream binding site. However this phenomena may be more common than is currently suggested by the literature because my studies have shown that several other virulence activators within the AraC family are also capable of utilizing a downstream binding site. Because this mechanism of activation may be important for the virulence of many bacterial pathogens, we are studying it in greater detail. In particular we would like to understand how Rns communicates with the transcription complex and which steps of transcription are affected by Rns binding to sites downstream of the transcription start site.

II. Structural and functional analysis of Rns

Like most other AraC family members Rns has two predicted helix-turn-helix motifs within its carboxy terminus which is thought to constitute the regulator’s DNA binding domain. Currently there are two crystallographic structures of AraC family members bound to DNA and each presents a different model of DNA binding. The structure of MarA reveals that both its helix-turn-helix motifs make contacts in the major groove of the DNA helix. However Rob places only one recognition helix in the major groove. We would like to determine which model of DNA binding is applicable to Rns through a variety of techniques including alanine scanning mutagenesis and loss of contact analysis.

Also of interest is the function of the amino terminal domain of the Rns. This domain may be required for dimerization, transcriptional activation, ligand binding, or any combination of the three.

III. Identification of genes within the Rns regulon

Recently we have shown that an MBP–Rns fusion protein can be used to isolate and clone DNA fragments carrying Rns binding sites from total genomic DNA of ETEC strains. This research has led to the identification of other genes that are regulated by Rns. One of the more interesting findings from this study is that Rns regulates the expression of some genes that are common to ETEC and E. coli K-12 even though K-12 strains do not carry rns. Although this method has been successful, it has not yet been taken to completion. We plan to continue this biochemical approach to identify additional genes within the Rns regulon. An alternative approach is the application of information theory to identify potential Rns binding sites within genomic databases. The accuracy of these predictions should be assisted by my labs analysis of Rns interactions with DNA. Longer term investigations will address the function of Rns regulated genes and their role in ETEC pathogenesis. It is possible that the products of some of these genes could be the targets for preventive therapies against ETEC and other bacterial infections.

Selected Publications:

Basturea GN, Bodero MD, Moreno ME, Munson GP. 2008. "Residues near the amino terminus of Rns are essential for positive autoregulation and DNA binding." Journal of Bacteriology 190(7):2279-85.

Pilonieta MC, Munson GP. 2008. "The chaperone IpgC copurifies with the virulence regulator MxiE." Journal of Bacteriology 190(6):2249-51.

Pilonieta MC, Bodero MD, Munson GP. 2007. "CfaD-dependent expression of a novel extracytoplasmic protein from enterotoxigenic Escherichia coli." Journal of Bacteriology 189(14):5060-7.

Bodero MD, Pilonieta MC, Munson GP. 2007. "Repression of the inner membrane lipoprotein NlpA by Rns in enterotoxigenic Escherichia coli." Journal of Bacteriology 189(5):1627-32.

Munson, George P., Lisa G. Holcomb, Heather L. Alexander, and June R. Scott. 2002. "In vitro identification of Rns-regulated genes," Journal of Bacteriology 184(4):1196-1199.

Munson, George P., Lisa G. Holcomb, and June R. Scott. 2001. “Novel group of virulence activators within the AraC family that are not restricted to upstream binding sites,” Infection and Immunity 69(1):186-193.

Munson, George P., and June R. Scott. 2000. “Rns, a virulence regulator within the AraC family, requires binding sites upstream and downstream of its own promoter to function as an activator,” Molecular Microbiology 36(6):1391–1402.

Sakellaris, Harry, George P. Munson and June R. Scott. 1999. “A conserved residue in the tip proteins of CS1 and CFA/I pili of enterotoxigenic Escherichia coli that is essential for adherence,” Proceedings of the National Academy of Sciences USA 96(22):12828–12832.

Munson, George P., and June R. Scott. 1999. “Binding site recognition by Rns, a virulence regulator in the AraC family,” Journal of Bacteriology 181(7):2110-2117.