Dept. of Microbiology & Immunology

University of Tennessee, Memphis

Stephen B. Melville, Ph.D.

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Office:  (901) 448-6752

Lab:  (901) 448-8254

sbmelville@utmem.edu

Office:  101D M.S.B.

Lab: 123 M.S.B.

Laboratory web page

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Research Program:

    The research in my laboratory involves the molecular pathogenesis of the bacterium Clostridium perfringens. C. perfringens is the most widely occurring pathogenic bacterium, readily found in soil samples and intestinal contents of humans and animals. It is a gram-positive, spore-forming, anaerobic bacterium that causes many diseases in domesticated animals. It is also the cause of several human diseases, including gas gangrene (clostridial myonecrosis), infantile necrotic enterocolitis, and enteritis necroticans all of which are potentially fatal. In addition, C. perfringens is a common source of food poisoning in humans, being responsible for 10% of the outbreaks of known causes in the U. S. alone. After ingestion of contaminated food (usually a meat or meat product, especially beef or poultry) containing vegetative cells, food poisoning symptoms are caused by production of a potent enterotoxin protein (CPE) made by sporulating cells in the gastro-intestinal (G-I) tract. The illness is not often fatal, but occasional deaths do occur among elderly and immunocompromised patients.

    In our earlier work, we found that CPE expression is regulated at the transcriptional level during the sporulation cycle. First, when C. perfringens strain NCTC 10240 was inoculated into sporulation medium , we detected only very low levels of cpe mRNA and no CPE protein in the exponential growth phase. However, the levels of both cpe mRNA and protein rose rapidly and in synchrony about one hour after the cells entered stationary phase, indicating that the increase in CPE expression as cells enter sporulation results from increased transcription of the cpe gene. We also constructed C. perfringens E. coli shuttle plasmids vectors that have the promoters of the cpe gene linked to the gene gusA from E. coli, which encodes the enzyme beta-glucuronidase. These vectors allow us to measure cpe transcription simply by measuring the amount of beta-glucuronidase enzyme that is produced in the cell. Using these constructs, we demonstrated that there are three independent promoters responsible for cpe transcription.

    The three promoters that we identified, called P1, P2, and P3, have DNA recognition sequences for alternative sigma factors that are very similar to those found in another sporulating bacterium, Bacillus subtilis. These alternative sigma factors are involved in controlling the synthesis of proteins involved in forming a spore. Sigma E and sigma K are active in the mother cell, while sigma F and sigma G are active in the forespore compartment. The CPE protein is made only in the mother cell and the CPE promoters are similar to sigma E and sigma K-dependent promoters; therefore our model is that these sigma factors are responsible for regulating cpe transcription during the sporulation cycle. We have cloned the genes encoding all of the sporulation sigma factors in C. perfringens. We are now overproducing the sigma E and sigma K proteins and using them in in vitro assays to test our model that they are necessary for cpe transcriptional regulation.

    My laboratory has also opened up a new area of research concerning the role of sialic acid metabolism and sialidase enzymes have in the pathogenesis of the organism. We have recently cloned and sequenced an operon encoding two genes (nanE and nanA) involved in breaking down sialic acid for use as a nutrient source. We are now investigating the role of the sialidase enzymes (NanI and NanH) in nutrient acquisition and pathogenesis of the bacterium

Walters, D. M., V. L. Stirewalt, and S. B. Melville. 1999. Cloning, sequence and transcriptional regulation of the operon encoding a putative N-acetylmannosamine-6-phosphate epimerase (nanE) and sialic acid lyase (nanA) in Clostridium perfringens. J. Bacteriol. 181:4526-4532.

Zhao, Y. and S. B. Melville. 1998. Identification and characterization of sporulation-dependent promoters upstream of the enterotoxin gene (cpe) of Clostridium perfringens. J. Bacteriol. 180:136-142.

Melville, S. B., R. Collie, and B. A. McClane. 1997. Regulation of enterotoxin production in Clostridium perfringens, p. 471-487. In J. R. Rood, G. Songer, B. McClane, and R. Titball (ed.) Molecular biology and pathogenesis of the Clostridia. Academic Press (London).

Melville, S. B., R. Labbe, and A. L. Sonenshein. 1994. Expression from the Clostridium perfringens cpe promoter in C. perfringens and Bacillus subtilis. Infect. Immun. 62:5550-5558.