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Title:The role of Bacteroides Host Factor A in CTnDOT integration and Holliday junction resolution
Author(s):Ringwald, Kenneth W
Director of Research:Gardner, Jeffrey F
Doctoral Committee Chair(s):Gardner, Jeffrey F
Doctoral Committee Member(s):Imlay, James A; Kuzminov, Andrei; Orlean, Peter A B
Department / Program:Microbiology
Discipline:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Microbial Genetics
DNA-Protein Interactions
Abstract:Bacteroides are a common genus of Gram negative anaerobic bacteria within the human gut. Under normal conditions, Bacteroides spp. benefit the host by breaking down complex polysaccharides. However, when the gut is punctured, these species may escape to cause abscesses or infections within the blood. In the past, tetracycline was used as a treatment for these infections. However, Bacteroides opportunistic infections are now much more difficult and costly to treat due to the widespread presence of mobile genetic elements carrying antibiotic resistance genes. One of these mobile genetic elements, CTnDOT, moves by conjugation and site-selective recombination. It is an example of a Conjugative Transposon (CTn). These elements were later classified as Integrated Conjugative Elements (ICEs). They frequently carry resistance to antibiotics and always contain the genes to mediate their own transfer by conjugation. CTnDOT is 65 kb and carries genes for mobilization, transfer, integration, and excision in addition to resistance to tetracycline and erythromycin. Once integrated into the chromosome, ICEs such as CTnDOT are stably maintained. The integration reaction into the Bacteroides chromosome and the excision reaction necessary for transfer are catalyzed by an integrase, IntDOT. IntDOT is a member of the tyrosine recombinase family. It was previously known that a host factor was required for integration and that Escherichia coli Integration Host Factor (IHF) could substitute for the host factor in an in vitro integration assay. I purified DNA binding proteins from a Bacteroides thetaiotaomicron strain lacking known ICEs. The purified fractions were then tested in the in vitro integration assay. The active fraction contained a protein that we named BHFa for Bacteroides Host Factor A. Subsequent electrophoretic mobility shift assays and fluorescent footprinting assays revealed four BHFa binding sites within the attDOT DNA sequence of CTnDOT. Surprisingly, further experiments showed that other DNA bending proteins could effectively substitute for BHFa in the in vitro integration assay even when distantly related or entirely unrelated. The integration and excision reactions of CTnDOT and other tyrosine recombinases proceed through ordered strand exchanges. The first set of strand exchanges generates a Holliday junction (HJ) intermediate that isomerizes through an overlap region and which is resolved by a second set of strand exchanges. Most tyrosine recombinases require identical DNA sequences in the overlap region where the strand exchanges occur. However, IntDOT can resolve HJs containing mismatched bases in the overlap region in vivo. This ability implies a difference in the overall protein-DNA complex, called an intasome. In integration, the intasome involves at least two different proteins and two double stranded DNA molecules, attDOT and attB. For excision, the attL and attR DNA sites are required for the assembly of two separate excisive intasomes before being brought together to reform the circular element. As many as five proteins may participate in excision and four are required. In order to study the mechanisms of the integration and excision reaction, we constructed synthetic HJs. HJ intermediates were constructed by annealing four oligonucleotides from the different products and substrates. This creates the intermediate formed after the first set of strand exchanges. The synthetic HJs can be constructed with either identical or mismatched overlap regions. It had been previously shown that synthetic HJs containing only the IntDOT core sites could be processed to both products and substrates if the overlaps are identical in vitro. However, if the overlaps contain mismatches, the HJs are resolved back to substrates. This inability of IntDOT to process mismatched HJs to products may be due to the lack of arm-type sites (which IntDOT binds) and because other protein participants were absent. I hypothesized that BHFa (or another DNA-binding protein) and the arm-type sites are necessary to form the higher-ordered complexes called intasomes and to enable IntDOT to catalyze recombination through the mismatched bases. To test this, I have constructed larger synthetic HJs (composed of four annealed oligonucleotides) containing the arm-type sites. As with core-only HJs, they were resolved into either products or substrates when the overlap contains identical bases. I have also constructed core plus arm-type HJs with a mismatched overlap. When these HJs are incubated with both IntDOT and BHFa they are resolved to both substrates and products. Accordingly, it appears that both arm-types sites and BHFa are required to enable IntDOT to resolve a HJ with a mismatched overlap region.
Issue Date:2016-11-28
Type:Thesis
URI:http://hdl.handle.net/2142/95340
Rights Information:Copyright 2016 Kenneth Ringwald
Date Available in IDEALS:2017-03-01
Date Deposited:2016-12


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