TRANSPOSITIONAL MUTAGENESIS AND MUTACINS IN S MUTANS

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Dental caries is an infectious and transmissible disease and the mutans streptococci have been implicated as major causative agents. Dental caries still affects a large segment of the population, both in the United States and world wide. Some strains of Streptococcus mutans produce an antibacterial substance called mutacin which kill other bacteria of the same or closely related species. The production of mutacin is one characteristic of S. mutans which may contribute to its ability to colonize and be sustained in the oral cavity. For this reason, mutacin may be considered a virulence factor. Because of their unique inhibitory properties, knowledge about mutacin may be applied to the eventual control of the pathogenesis of mutans streptococcal infections. Unfortunately, very little is known about its regulation or under what conditions it is made. Because these substances are made in small quantities and only under certain defined conditions, past attempts to isolate mutacin biochemically have been unsuccessful. We have successfully purified the mutacin polypeptide and determined its amino acid composition and N-terminal sequence. Combined with transpositional mutagenesis, the structural gene for mutacin has been definitively located on a cloned segment of DNA from the chromosome of S. mutans UA96. Accordingly, this proposal entails the continued genetic characterization of the mutacin locus using a variety of proven genetic techniques. The objective here is to understand, at the molecular level, how mutacin is produced and processed. Its small size coupled with its ribosomal synthesis permits a wide range of genetic manipulation. One such method is called site-directed mutagenesis which allows single base-pair changes in the structural gene which, in turn, changes the amino acid composition of the mutacin polypeptide. A change in peptide composition could cause changes in the properties of mutacin leading to enhanced spectrum of activity or physical properties. Because mutacin closely resembles the important group of peptide antibiotics called lantibiotics which are produced by other gram-positive bacteria, the potential for developing mutacin into a therapeutic agent holds great promise. In addition, because the mutacin gene locus is likely clustered as a single unit, it should be possible to transfer the entire gene locus into other bacteria such as non- cariogenic effector strains, e.g., S. sanguis or an avirulent S. mutans strain. This constitutes the basis of replacement therapy. Expression of mutacin could either assist the colonization of the engineered strain into an existing ecological milieu or prevent the colonization of invading S. mutans into the oral cavity. There are several long-term health-related interests for the study of mutacin production. First, a means could be developed to enhance the large scale production of mutacin for therapeutic application. Second, knowledge of the control or regulation of this gene will give us insight into one of the virulence factors of this important organism. Lastly and importantly, mutacin exhibits a wide range of antibacterial activity, including a pronounced bactericidal action against the human pathogens S. pyogenes and S. pneumoniae. In a growing climate of increased drug resistance among human pathogens, alternative antimicrobials such as mutacin may have life- saving value as a new antibiotic.