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Investigation of the environmental effect on the Regulation and Transfer of Conjugative Transposon Tn916

Al Harbi, Deena Saleh; (2020) Investigation of the environmental effect on the Regulation and Transfer of Conjugative Transposon Tn916. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Antibiotic resistance is a major global concern, as it has spread rapidly and prevents the treatment of infectious diseases. One of the main reasons for this is the acquisition of resistance genes carried on mobile genetic elements (MGEs). There are many different types of MGEs, including transposons, plasmids, insertion sequences, pathogenicity islands, bacteriophages and gene cassettes. Tn916 is a conjugative transposon that contains the tetracycline-resistance gene tet(M). This transposon transfers to a broad range of bacteria. In this study, the research aimed to determine the behaviour of Tn916 in the human oral cavity, which is the portal to the digestive system. Bacillus subtilis (BS34A) has been used as a Tn916 donor, and six Streptococcus spp. (S. oralis, S. pyogenes, S. salivarius, S. mutans, S. sanguinis and S. parasanguinis) and Enterococcus faecalis as recipients. The donor B. subtilis is considered as a transient bacterium within the oral cavity, normally inhabiting soil, and the recipients are all considered normal oral and gastrointestinal bacteria. Filter-mating assays were carried out in pairs between donor–recipients under conditions that mimic those found in the mouth. It was observed that B. subtilis was able to transfer Tn916 into S. oralis, S. pyogenes and E. faecalis under anaerobic conditions at frequencies ranging from 10‾⁹ to 10‾⁷ transconjugants per recipient within 1 min on solid and liquid media. In contrast, no transfer was observed aerobically or when mating with S. salivarius, S. mutans, S. sanguinis and S. parasanguinis as recipients. However, after a mutant strain of B. subtilis onTn916 (Δorf12 terminator of Tn916) was developed, transfer of Tn916 occurred from B. subtilis into S. oralis, S. pyogenes and E. faecalis aerobically at frequencies ranging from10‾⁹ to 10‾⁸ transconjugants per recipient and anaerobically only into S. pyogenes. Using qPCR, the copy number of circular Tn916 in different conditions (aerobic and anaerobic) with and without a recipient (E. faecalis JH2-2) was determined. There were10⁷copies/μl in the absence of a recipient in both conditions. However, in the presence of the recipient, the copy numbers increased significantly to 10⁸ copies/μl anaerobically compared to 10⁷ copies/μl aerobically. Furthermore, the role of the orf12 terminator in aerobic and anaerobic conditions was determined by quantifying the expression level using an enzymatic reporter assay. The transcription level from ptet(M) is controlled by the terminator on orf12, and it was almost the same in both conditions. However, the transcription level was significantly increased when orf12 terminator mutated and the condition was switched from aerobic to anaerobic. In conclusion, transient bacteria in the oral cavity may have ample opportunities to disseminate their DNA to the oral microbiota.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Investigation of the environmental effect on the Regulation and Transfer of Conjugative Transposon Tn916
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10091452
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