Bacteroides species are the most important anaerobic bacteria causing human disease in the United States, and are also the most numerous gram-negative organisms in the gastrointestinal tract where they promote normal physiologic function. Bacteroides can acquire resistance to all major drugs. Genetic analysis of Bacteroides is essential to understand both the normal physiology of these organisms and the development of drug resistance. The objectives of this project continue to be the molecular characterization of genetic mechanisms of antibiotic resistance transfer in Bacteroides and the development of methods for the genetic manipulation of these organisms. Considerable progress in Bacteroides genetics has been made: E.coli/Bacteroides shuttle vector systems allow cloning and manipulation of Bacteroides genes; both plasmid and non-plasmid conjugation systems have been isolated, and Bacteroides genes for clindamycin (Cl-R) and tetracycline (Tc-R) resistance have been cloned. Major questions in Bacteroides genetics involve: 1) the molecular structure and function of the anaerobic Tc-R locus which is widespread in Bacteroides and is a very useful genetic marker, and 2) the mechanism underlying the novel, non-plasmid-mediated conjugation systems that are widespread in Bacteroides.
The aims of this proposal are: 1) to map and sequence the Bacteroides Tc-R gene that has been cloned but is not expressed in E. coli. These studies will use standard deletion analysis and sequencing technology as well as the shuttle-transfer system between E. coli and Bacteroides mediated by IncP plasmids. 2) to use the cloned Tc-R and Cl-R genes in cosmid and plasmid libraries to elucidate the mechanism of the non-plasmid mediated conjugation systems of Bacteroides. Current evidence suggests that these systems may be """"""""conjugative transposons"""""""", but the mechanism of transfer is not understood. The size of these transfer elements will be mapped on overlapping cosmids, and the junctions between the elements and the insertion sites in the chromosome will be located and sequenced to determine the mechanisms of the recombination events. The genes and cis-acting DNA sequences required for transfer of the elements will be characterized, and a new site-specific recombination determinant located on the transfer elements will be cloned and sequenced. These studies will elucidate the basic mechanism of a new type of conjugal transfer system and provide valuable tools for further genetic studies in Bacteroides.
