Bacteroides, one of the numerically predominant genera of bacteria in the human colon and female vaginal tract, is also a common cause of internal abscesses and bacteremia in people with colon cancer or abdominal trauma. Enterotoxin producing strains of B. fragilis cause diarrheal infections,especially in people with AIDS. Resistance to two of the drugs of choice for treating Bacteroides infections, clindamycin and beta-lactam antibiotics has begun to appear and is beginning to complicate treatment of Bacteroides infections. Virtually all clinical isolates are now resistant to tetracycline whereas tetracycline resistance was once uncommon. The genes encoding resistance to tetracycline, clindamycin and beta-lactam antibiotics are being transferred by a family of large conjugative chromosomal elements (Tc r elements). Tc r elements not only transfer themselves but also mobilize co-resident plasmids and excise, circularize and transfer smaller chromosomal elements called NBUs. Resistance genes are being spread by all three routes. An unusual feature of the Tc r elements is that self-transfer, plasmid mobilization and NBU excision are stimulated by tetracycline. This feature raises questions about whether the widespread use of tetracycline for nonclinical applications like increasing feed efficiency of livestock animal's is contributing to resistance spread. Tetracycline stimulation of transfer is mediated by two transcriptional activators, RteB and RteC. The Tc r elements integrate site-specifically by a mechanism that is unlike any previously studied integration mechanism. The Tc r elements are quite large (> 70 kbp). To facilitate their study, a miniature form of the element will be constructed. The genes involved in excision and integration of the element will be sequenced and characterized and it will be determined if they are regulated by RteB/RteC. The ends of the element and target site will be mutagenized to determine what bases are essential for integration and excision. The region carrying genes that form the mating pore and initiate transfer of the circular transfer intermediate have been located in 15 kbp region. Transposon mutagenesis will be used to identify essential genes in this region. These genes will be sequenced, characterized and checked for regulation by RteB/RteC. Sequence analysis of NBU integratIon and excision indicated that these elements might integrate similarly to phage lambda, but the partial sequence of a gene that may be the integrase shows no similarity to members' of the lambda integrase family. The sequencing of the 5 kbp NBU1 integration/excision region will be completed, and genes in this region essential for excision and integration will be identified. Regulation of these genes by RteB will be determined. The ends and target site of NBU1 will be mutagenized to determine what bases are essential for integration and excision.
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