Environmental gene regulation by DNA supercoiling in Borrelia


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University of Montana
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1999
Environmental gene regulation by DNA supercoiling in Borrelia burgdorferi
Janet Alverson The University of Montana

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Recommended Citation Alverson, Janet, "Environmental gene regulation by DNA supercoiling in Borrelia burgdorferi" (1999). Graduate Student Theses, Dissertations, & Professional Papers. 10585. https://scholarworks.umt.edu/etd/10585
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Environmental Gene Regulation by DNA Supercoiling in Borrelia burgdorferi
Janet Alverson DVM Virginia-Maryland Regional College o f Veterinary Medicine, 1987
B.S. University of Maryland, 1983 Presented in partial fulfillment o f the requirements for the degree of
Doctor o f Philosophy The University of Montana
1999
Approved by: Chairperson
Dean, Graduate School
Date
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Alverson, Janet, DVM, Ph.D., December 1999

Microbiology

Environmental Gene Regulation by DNA Supercoiling in Borrelia burgdorferi (97 pp.)

Director: D.Scott Samuels

The synthesis of the outer surface proteins OspC and OspA increased and decreased, respectively, when cultures o f Borrelia burgdorferi were shifted from 23 °C, which
models the temperature o f the tick vector, to 35°C, which approximates the mammalian host body temperature. The changes in protein synthesis correlated with changes in mRNA transcript levels showing that this regulation occurs at the gene level. This change in outer surface protein production is due to changes in DNA supercoiling with DNA from B. burgdorferi cultures grown at 35°C being less supercoiled than that o f cultures grown at 23°C. OspC synthesis increased and OspA synthesis decreased when DNA supercoiling was artificially relaxed by treating cultures with coumermycin Ai, a DNA gyrase inhibitor. In addition, OspC synthesis was higher in DNA gyrase mutants than wild type cells and DNA from the gyrase mutants was more relaxed. Promoter studies using a chloramphenicol acetyltransferase (CAT) reporter system in Escherichia coli and B. burgdorferi cells showed that ospC is regulated by DNA supercoiling in both bacteria while ospA is responsive to DNA supercoiling in B. burgdorferi, but not in E. coli. This suggests the presence o f an ospA repressor in B. burgdorferi. These results, taken together, suggest that B. burgdorferi senses environmental changes in temperature by altering the level o f DNA supercoiling, which then affects the program o f gene expression. This implies that DNA supercoiling acts as a signal transducer for environmental regulation of outer surface protein synthesis. The gyrB promoter was also shown to be homeostatically regulated by DNA supercoiling while other B. burgdorferi promoters were shown to be non-responsive to DNA supercoiling. The synthesis of heat shock protein, GroEL is up-regulated in gyrB mutants. This suggests that expression of groEL and gyrB, both genes carried on linear DNA molecules, respond to changes in DNA supercoiling in vivo.

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Table of Contents
Chapter 1. Introduction
1.1 Lyme disease 1.2 Environmental adaptation o f bacteria 1.3 DNA supercoiling 1.4 DNA gyrase and gyrase inhibitors 1.5 Heat shock proteins 1.6 Specific aims 2. Methods 2.1 Chapter 3 methods 2.2 Chapter 4 methods 2.3 Chapter 5 methods 2.4 Chapter 6 methods 3. Temperature shift affects gene expression and relaxes DNA supercoiling 4. The effect o f pharmacological and genetic manipulation of DNA supercoiling on gene expression and protein synthesis 5. Regulation o f promoter activity by DNA supercoiling 6. Over-expression o f groEL in coumermycin-resistant gyrB mutants 7. Discussion 8. References

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Table 1. 2. 3.

List of Tables Title Oligonucleotides Plasmids MIC o f chloramphenicol in E. coli

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Environmental gene regulation by DNA supercoiling in Borrelia