Activities of Bacteroides Conjugative Transposons: CTnGERM1 and CTnDOT

Abstract

CTnDOT is a conjugative transposon (CTn) that is found in many Bacteroides strains. Transfer of CTnDOT is stimulated 100--1000 fold if the cells are exposed to tetracycline (Tc). An operon that contains a Tc resistance gene, tetQ, and two regulatory genes, rteA and rteB, is essential for control of excision and transfer functions. TetQ, a ribosome protection type of Tc resistance protein, actually reduced operon expression, possibly by interacting with ribosomes that are translating the tetQ message. 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 of this putative hairpin structure made GUS production as high in the absence of Tc as in Tc-stimulated cells. Compensatory mutations that restored the hairpin structure led to a return of regulated production of GUS. A 3 amino acid leader peptide which extends into this hairpin was found to be essential for Tc-induction. Abolishing the translation of this 3 as leader peptide by putting in a stop codon, or changing any amino acid without changing the hairpin structure completely abolished Tc-regulation. Thus, the tetQ-rteA-rteB operon appears to be regulated by a translational attenuation mechanism. This was further supported by results from experiments involving deletion mutants.