Roberts, Adam Paul; (2002) Investigation into the molecular genetics of the conjugative transposon, Tn5397. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Tn5397 has been identified as a conjugative transposon found in the Gram-positive human pathogen Clostridium difficile and is related to the well-characterised enterococcal conjugative transposon Tn916. However Tn5397 differs from Tn916 in two crucial ways in that the ends of the two elements are completely different, with the gene tndX replacing the int and xis genes ofTn5397, and Tn5397 contains a group II intron. The aim of this project was to determine the mechanism of excision and insertion, regulation, and possible role of the group II intron on the mobility of Tn5397. Tn5397 was sequenced in its entirety. This data showed the presence of 21 open reading frames organised into four distinct 'modules'. The excision module comprises the gene tndX that was shown, by mutation studies, to be required for excision and circularisation of the element in a Bacillus subtilis host. The conjugation and antibiotic resistance modules are related to those of Tn916. The regulation module was also related to Tn916 but contains a deletion of 84 bp that was predicted to alter the mechanism of regulation. A mechanism of insertion and excision is proposed. Excision is mediated by TndX which catalyses 2 base-pair staggered endonucleolytic cleavage at a GA dinucleotide located at each end of the element, which is then excised as a circular molecule. Integration is essentially the reverse of this process. Up-regulation of transcription of the tetracycline resistance gene, tet(M) and downstream genes was shown to occur in the presence of tetracycline. A model for this regulation is presented that involves both transcriptional attenuation and translational control mediated by ribosome binding site occlusion. The group II intron present within Tn5397 was shown to splice from its host gene in vivo. A mutant that was unable to splice however was shown to have no detectable effect on either excision or insertion or conjugative transfer of Tn5397. Finally the host range of the element was investigated in an oral biofilm environment. Tn5397 was shown to transfer from a B. subtilis donor to a streptococcal host even though the donor could not be isolated from the biofilm after 6 hours. This work has extended the current knowledge of the genetics of conjugative transposons of the Tn916-like family. It has provided valuable insights into the evolution of these transposons and shows that different types of elements probably undergo recombination reactions resulting in new genetic elements.

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