Excision, transfer, and integration of the bacteroides integrative and conjugative element CTnDOT

Hopp, Crystal Marie

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Description

Title

Excision, transfer, and integration of the bacteroides integrative and conjugative element CTnDOT

Author(s)

Hopp, Crystal Marie

Issue Date

2016-08-10

Director of Research (if dissertation) or Advisor (if thesis)

Gardner, Jeffrey F.

Doctoral Committee Chair(s)

Gardner, Jeffrey F.

Committee Member(s)

• Olsen, Gary J.
• Whitaker, Rachel J.
• Orlean, Peter A. B.

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• CTnDOT
• Bacteroides
• Integrative and Conjugative Elements (ICEs)

Abstract

Increased resistances to antibiotics is a pressing problem in the world today. When new antibiotics are introduced, resistance to that antibiotic within the microbial community soon follows. One of the many contributing factors to increased resistances to antibiotics is the spread of mobile genetic elements that encode antibiotic resistance genes. These elements are able to transfer themselves to new host cells by conjugation, which may result in the widespread dissemination of an element throughout a given microbial population. The Bacteroides sp. are known for harboring wide array of integrative and conjugative elements. One of the most well studied examples is the 65kb CTnDOT, which encodes both erythromycin and tetracycline resistances. Bacteroides are prevalent within the human gastrointestinal tract, which means that CTnDOT is capable of transferring to other resident microbes within this environment. The transfer of CTnDOT is repressed under normal conditions, but when tetracycline is present the propagation of the element is stimulated through a complex regulatory cascade. The excision operon is a key component of this regulation, and is involved at many different levels of the regulatory cascade. As the name implies, the expression of the excision operon promotes the excision of CTnDOT from the host chromosome. Once the element is excised it needs somewhere to go, which the excision operon also facilitates by increasing the transcription of both the mobilization (mob) and transfer (tra) operons. These operons encode the proteins needed to assemble the mating apparatus, and the proteins responsible for shuttling the excised element to the mating apparatus for transfer. The work presented within this dissertation focuses on the different aspects of the excision operon. First the mechanistic properties of the excision proteins at the mob and tra region were investigated. It was shown that the Xis2d protein binds the DNA between the mob and tra promoters, and that the Exc protein can be recruited in the presence of Xis2d to help initiate the transcription of the mob operon. The contribution of each of the excision genes to the in vivo excision reaction was also analyzed. It was shown that each of the genes within the excision operon play a significant role in the excision reaction. The presence of xis2c, xis2d, and exc was essential for detecting excised product, which confirms that exc is required for in vivo excision. Also, a deletion of orf3 significantly reduced the production of excised products, which is the first time this gene has shown a discernable effect in a CTnDOT assay. This wasn't the only discovery of gene functionality that was uncovered, the previously uncharacterized orf2a and orf2b genes were found to contribute to the excision reaction. The excision, transfer, and mobilization of CTnDOT ultimately lead to propagation of this element into new recipients. Once within the recipient cell, the element integrates into an attB site located within the recipient chromosome. This dissertation also provides an in vivo analysis of CTnDOT integration, where 18 alternative attB sites were identified. In summation, the work presented within this dissertation has clarified the main aspects of CTnDOT propagation, by revealing important contributions of the excision operon to the regulation of the element.

Graduation Semester

2016-12

Type of Resource

text

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Copyright and License Information

Copyright 2016 Crystal Hopp

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