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Journal of Bacteriology, May 2004, p. 2548-2557, Vol. 186, No. 9
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.9.2548-2557.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Regulation of a Bacteroides Operon That Controls Excision and Transfer of the Conjugative Transposon CTnDOT

Department of Microbiology, University of Illinois, Urbana, Illinois 61801

Received 28 October 2003/ Accepted 14 January 2004

CTnDOT is a conjugative transposon (CTn) that is found in many Bacteroides strains. Transfer of CTnDOT is stimulated 100- to 1,000-fold if the cells are first exposed to tetracycline (TET). Both excision and transfer of CTnDOT are stimulated by TET. An operon that contains a TET resistance gene, tetQ, and two regulatory genes, rteA and rteB, is essential for control of excision and transfer functions. At first, it appeared that RteA and RteB, which are members of a two-component regulatory system, might be directly responsible for the TET effect. We show here, however, that neither RteA nor RteB affected expression of the operon. TetQ, a ribosome protection type of TET resistance protein, actually reduced operon expression, possibly by interacting with ribosomes that are translating the tetQ message. Fusions of tetQ with a reporter gene, uidA, were only expressed at a high level when the fusion was cloned in frame with the first six codons of tetQ. However, out of frame fusions or fusions ending at the other five codons of tetQ showed much lower expression of the uidA gene. Moreover, reverse transcription-PCR amplification of tetQ mRNA revealed that despite the fact that the uidA gene product, ß-glucuronidase (GUS), was produced only when the cells were exposed to TET, tetQ mRNA was produced in both the presence and absence of TET. Computer analysis of the region upstream of the tetQ start codon predicted that the mRNA in this region could form a complex RNA hairpin structure that would prevent access of ribosomes to the ribosome binding site. Mutations that abolished base pairing in the stem that formed the base of this putative hairpin structure made GUS production as high in the absence of TET as in TET-stimulated cells. Compensatory mutations that restored the hairpin structure led to a return of regulated production of GUS. Thus, the tetQ-rteA-rteB operon appears to be regulated by a translational attenuation mechanism.

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