Positive regulators of Bacteroides CTnDOT excision and transfer
Tetracycline stimulates both excision and transfer of a Bacteroides Conjugative transposon CTnDOT. It was shown previously that a gene, rteC, is necessary for tetracycline-stimulated transcriptional regulation of the xis2c 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, I sought structural homologs of RteC. A three-dimensional structure prediction by Robetta suggested that RteC might have two domains, and 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. I made alanine substitutions within the putative DNA binding helix 3 region of RteC. Assays of xis2c::uidA activation by alanine mutants indicated that residues 174, 175, 178, 180, and 184 in helix 3 might contact the upstream region of PE. The same alanine RteC mutants were used to test the effect of mutations on binding, and all mutants lost binding to PE region. The upstream region of xis2c contained two inverted repeat half-sites. Mutational analysis of these half-sites showed that both half-sites are important for activity. In vivo GUS assays were also done to test whether these half-sites are important for binding. Disrupted inverted repeat sequences resulted in loss of binding to PE region. Thus, I have identified the DNA binding portion of RteC and DNA site to which it binds. The tetracycline regulated excision operon, containing xis2c, xis2d, and exc of the Bacteroides conjugative transposon CTnDOT has been shown to mediate not only the excision of CTnDOT and but also the regulation of transcription of a large operon containing genes required for transfer of the CTnDOT transfer intermediate. In this study, the importance of two excision proteins (Xis2c and Xis2d) was examined to determine their roles in regulating the transcription level of the transfer operon. In-frame deletions in xis2c and xis2d, and deletion of exc reduced the in vivo transcription induction of the operon by 58%, 97%, and 16% respectively. Xis2c and Xis2d were shown to bind to the 450-bp DNA fragment immediately upstream of the first gene in the transfer operon but Exc did not exhibit any gel shift activity. However, Exc appeared to bind cooperatively when added to Xis2d and caused a super shift of the fragment. I obtained a footprint of Xis2d covering a 50-bp region immediately upstream of the promoter of the transfer operon. Mutations within this region had varying effects on the transcriptional activity in vivo and resulted in a loss of Xis2d binding. The same mutations also affect a crude preparation of Xis2c in its ability to bind DNA. My results confirmed the importance of Xis2c, Xis2d, and Exc in the transcriptional regulation of the transfer genes.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign