Jasni, A; (2013) Investigation into the regulation and transfer of conjugative transposons of the Tn916-like family. Doctoral thesis , UCL (University College London).

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Abstract

The Tn916-like family of conjugative transposons are broad host range mobile genetic elements and are clinically important as they are one of the major vectors responsible for the spread of antibiotic resistance among bacterial pathogens. This study was designed to investigate the behaviour of the conjugative transposons Tn916 and Tn5397, focusing on the transcriptional regulation and transfer. The proposed regulatory system of Tn916 involves transcriptional attenuation upstream of tet(M) and is regulated by tetracycline. The translation of orf12 is central to this regulatory mechanism as it is the translating ribosome upon orf12 RNA that is hypothesised to destroy, or prevent the formation of the transcriptional terminators. This hypothesis was tested using a Bacillus subtilis construct, with a 2 bp mutation disrupting the start codon of orf12. This construct is expected to result in the transcriptional terminators being permanently formed as the ribosome will no longer translate the orf12 and destroy them. Results indicate a lower transcription of tet(M) and downstream genes which was supported by the slower growth rate of the B. subtilis mutant compared to the wild type upon challenged with tetracycline. When tetracycline is present, a reduced fitness of this mutant was observed compared to the wild type. However, the transfer frequency of the B. subtilis mutant was similar to that of the wild type. The transcription of Tn916 and Tn5397 was investigated by quantifying the expression level using reporter assays, where the Ptet(M) promoter and the open reading frames upstream of tet(M) were cloned upstream of a ß-glucuronidase gene. In the presence of tetracycline, Tn916 wild type construct was upregulated whereas the Tn5397 wild type construct showed a constant expression level. Disruption of the start codon of orf12 (Tn916) and orf26 (Tn5397) has also led to a constant expression level of ß-glucuronidase. The termination efficiency of the Tn916 terminators was estimated using promoter assays and a published algorithm. Results suggest that the large terminator is more efficient [47% (± 18)] than the small terminator [23% (± 15)], which was supported by the algorithm analysis for Tn916. Finally, reciprocal gene transfer of Tn5397 between Clostridium difficile and Enterococcus faecalis was demonstrated. The transfer frequency [± standard deviation (SD)] detected was 8.85 x 10-8 (± 2.14 x 10-7) per recipient. Tn5397 integrates into the genome of E. faecalis at a single site that is within an orf encoding the phosphotransferase (PTS) IIA component. Comparative growth curves showed that the acquisition of Tn5397 has a very small effect on the growth of E. faecalis. This work has extended the current knowledge of the regulation and transfer of conjugative transposons of the Tn916-like family. It has provided a better understanding about the mechanism of transcriptional regulation of these elements.

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