Recombineering in Mycobacteria Using Mycobacteriophage Proteins
RECOMBINEERING IN MYCOBACTERIA USING MYCOBACTERIOPHAGE PROTEINS by Julia Catherine van Kessel B.S. Biology, Utica College of Syracuse University, 2003 Submitted to the Graduate Faculty of Arts and Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2008 UNIVERSITY OF PITTSBURGH SCHOOL OF ARTS AND SCIENCES This dissertation was presented by Julia Catherine van Kessel It was defended on July 24, 2008 and approved by Roger W. Hendrix, Ph.D., Biological Sciences, University of Pittsburgh William R. Jacobs, Jr., Ph.D., Albert Einstein College of Medicine Jeffrey G. Lawrence, Ph.D., Biological Sciences, University of Pittsburgh Valerie Oke, Ph.D., Biological Sciences, University of Pittsburgh Dissertation Advisor: Graham F. Hatfull, Ph.D., Biological Sciences, University of Pittsburgh ii Copyright © by Julia Catherine van Kessel 2008 iii RECOMBINEERING IN MYCOBACTERIA USING MYCOBACTERIOPHAGE PROTEINS Julia Catherine van Kessel, Ph.D. University of Pittsburgh, 2008 Genetic manipulations of Mycobacterium tuberculosis are complicated by its slow growth, inefficient DNA uptake, and relatively high levels of illegitimate recombination. Most methods for construction of gene replacement mutants are lengthy and complicated, and the lack of generalized transducing phages that infect M. tuberculosis prevents simple construction of isogenic mutant strains. Characterization and genomic analysis of mycobacteriophages has provided numerous molecular and genetic tools for the mycobacteria. Recently, genes encoding homologues of the Escherichia coli Rac prophage RecET proteins were revealed in the genome of mycobacteriophage Chec9c. RecE and RecT are functional analogues of the phage λ Red recombination proteins, Exo (exonuclease) and Beta (recombinase), respectively. These recombination enzymes act coordinately to promote high levels of recombination in vivo in E.
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