Characterization of the Bacteroides CTnDOT regulatory protein RteC

Excision of the Bacteroides conjugative transposon CTnDOT is stimulated by tetracycline. It was shown previously that a gene, rteC, is necessary for tetracycline-stimulated transcriptional regulation of the orf2c operon, which contains the excision genes. The protein encoded by this gene, RteC, did not have primary amino acid sequence homology to any known proteins in the databases. Accordingly, we sought structural homologs of RteC. A three-dimensional structure prediction by Robetta suggested that RteC might have two domains and that the C-terminal domain might have a winged helix motif. Based on the Robetta prediction, the human transcriptional factors E2F-4 and DP2 were identified as the most likely structural homologs of RteC. We made alanine substitutions within the putative DNA binding helix 3 region of RteC. Assays of orf2c::uidA activation by alanine mutants indicated that residues 174, 175, 178, 180, and 184 in helix 3 might contact the upstream region of P(E). The upstream region of orf2c contained two inverted-repeat half sites. Mutational analysis of these half sites showed that both half sites are important for activity. Thus, we have identified the DNA binding portion of RteC and the DNA site to which it binds.