Conjugal Transfer of Bacteriodes Antibiotic Resistances

University Of Illinois At Urbana-Champaign, Urbana IL

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Abstract

Bacteroides species are Gram-negative obligate anaerobes that account for 20-30% of the bacterial population that normally inhabits the human colon. Some Bacteroides species can cause serious infections if they escape from the human colon during surgery. These infections can be difficult to treat because Bacteroides species are becoming increasingly resistant to antibiotics. We have shown that integrated conjugative element called conjugative transposons have been responsible for much of the spread of resistance genes among these species. This proposal focuses on a the conjugative transposon CTnDOT. Transfer of CTnDOT is triggered by exposure of the bacteria to tetracycline. Both excision from the chromosome to form a circular transfer intermediate and transfer itself are stimulated by tetracycline. In previous funding periods, we have described the first steps in the regulatory pathway that leads to enhanced transcription of the excision genes, which encode the proteins that participate with the CTnDOT integrase to produce the circular intermediate. We made the surprising discovery that these same proteins are also necessary and sufficient to increase transcription of the transfer genes. Moreover, there is a small regulatory RNA, encoded in the excision gene region, that suppresses transfer. The proposed work has three specific aims. First, we will locate the DNA binding sites of the two excision proteins that bind DNA specifically (Orf2c, Orf2d) and we will determine why a third excision protein (Exc) is also needed as part of the regulatory complex. We will also characterize the proteins with respect to structure and interactions with each other. Second, we will determine how the small RNA, RteR, acts to reduce transfer of the circular form. Third, we will determine whether the genes that encode mobilization proteins, proteins that nick the circular form and initiate transfer, are regulated similarly to the transfer genes and if so, whether the same proteins regulate both processes.