Tetracycline-Associated Regulation of the Transfer Genes of the Bacteroides Conjugative Transposon CTnDOT

Abstract

125 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.Many human colonic Bacteroides spp. harbor a conjugative transposon (CTn) called CTnDOT. CTnDOT carries two antibiotic resistance genes tetQ and ermF. A distinctive feature of CTnDOT is that both its excision and transfer are stimulated by tetracycline. The regulation of excision has been described previously. I provide here the first characterization of the organization and regulation of the CTnDOT transfer (tra) genes. RT-PCR analysis of the region containing the tra genes showed that these genes are transcriptionally coupled in an operon and that expression of this operon is regulated at the transcriptional level. The transcription initiation site of the operon was located 290 bp upstream of the start codon of the first tra gene, traA.A previous study had shown that the excision proteins not only to participate with the integrase to catalyze the excision reaction but are also required to regulate transfer. Expression of the excision gene operon is controlled by a regulatory protein RteC. To determine whether the excision proteins affect tra gene expression directly or act indirectly through RteC, the excision gene operon was placed under control of a heterologous promoter, thereby removing RteC from the regulatory pathway. This construct, in the absence of rteC, was able to support enhanced expression of the tra operon. Thus, the excision proteins act directly to up-regulate tra operon expression and increase the transfer frequency of CTnDOT.Although the entire orf2c operon is responsible for an increase in transcription of tra genes, transfer of CTnDOT was inhibited by a DNA segment that contained the 3' end of one of the excision genes (exc). This segment contained a small open reading frame, rteR. Previous work in our lab found that RteR is acting as a small RNA rather than as a protein. Primer extension analysis showed that rteR was transcribed in the same direction as exc. Unlike exc, rteR was expressed constitutively, not regulated by tetracycline. My work shows that RteR did not affect stability of the tra mRNA, nor did it have a detectable effect on the expression of a traA:: uidA translational fusion. If the effect of RteR was measured instead by RT-qPCR, however, it mediated a 5-fold decrease in transcription of tra genes. In this respect, the effect of RteR differs from that of most other small RNAs, which act at the post-transcriptional level. Taken together, my results show that the regulation of CTnDOT transfer is complex and involves both activator proteins, which also participate in the excision process, and an inhibitory small RNA that is encoded within the excision operon. This regulatory cascade, which links excision to regulation of transfer gene expression may function to ensure that transfer does not occur before excision is complete.CTnDOT has transferred extensively among bacteria found in humans. The same is true of another CTn, Tn916. Both appear to be stably maintained in these human-associated bacteria, but this stable maintenance could be due at least in part to the periodic exposure to antibiotics experienced by such bacteria. I was able to gain access to samples taken from the vaginal tracts of non-human primates (baboons and mangabeys), and this provided an opportunity to ask whether antibiotic resistance genes normally associated with CTns like CTnDOT and Tn916 are similarly widespread in animals that are rarely if ever exposed to antibiotics. I found that tetM, a gene carried by Tn916, and tetQ , a gene carried by CTnDOT were detectable by PCR in many of these samples. Also, using PCR analysis, I was able to show that at least some of these genes were associated with the corresponding CTn. This finding supports the contention that CTns such as CTnDOT are stably maintained in the absence of antibiotic selection. Also, my finding that genes associated with these CTns are surprisingly widespread in the primate vaginal microbiota raises the question of whether there is another condition besides tetracycline in stimulating transfer and maintenance of the CTns.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD