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Recombineering in Mycobacteria Using Mycobacteriophage Proteins

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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. coli and related bacteria using short regions of homology, facilitating the development of a powerful genetic technique called ‘recombineering.’ Biochemical characterization of Che9c gp60 and gp61 demonstrated that they possess exonuclease and DNA binding activities, respectively, similar to RecET and λ Exo/Beta. Expression of gp60/gp61 in M. smegmatis and M. tuberculosis substantially increases homologous recombination such that 90% of recovered colonies are the desired gene replacement mutants. Further development of this system demonstrated that Che9c gp61 iv facilitates introduction of selectable and non-selectable point mutations on mycobacterial genomes at high frequencies using short (<50 nt) ssDNA substrates. The mycobacterial recombineering system provides a simple and efficient method for mutagenesis with minimal DNA manipulation. While it is clear that similar phage-encoded recombinase homologues are rare, they can be readily identified by genomic studies and by in vivo characterization. Several putative recombination systems have been identified in mycobacteriophages Halo, BPs, and Giles, and recombineering of drug-resistance point mutations provides an easy assay for recombinase activity. Analysis of recombinases from various phages – including λ Beta and E. coli RecT – indicates that these proteins function best in their native bacteria. The mycobacteriophage-encoded proteins exhibited varying levels of activity, suggesting that analysis of multiple proteins is required to achieve optimal recombination frequencies. The apparent species-specific nature of these recombinases suggests the recombineering technology could likely be extended to any bacterial system through characterization of host-specific bacteriophages. v TABLE OF CONTENTS PREFACE.................................................................................................................................xviii 1.0 INTRODUCTION........................................................................................................ 1 1.1 GENETICS AND RECOMBINATION IN MYCOBACTERIA .................... 2 1.1.1 Barriers to genetics in M. tuberculosis ........................................................ 2 1.1.2 Genetics in other mycobacteria ................................................................... 5 1.1.3 Recombination in mycobacteria .................................................................. 7 1.1.3.1 Gene replacement by homologous recombination in M. smegmatis.8 1.1.3.2 Evidence of illegitimate recombination in M. tuberculosis.............. 11 1.1.3.3 The recombination genes of M. tuberculosis..................................... 12 1.1.3.4 The debate over homologous and illegitimate recombination in mycobacteria ...................................................................................................... 14 1.1.4 Mycobacteriophage-derived genetic tools................................................. 16 1.1.5 Genetic techniques for allelic replacement............................................... 18 1.1.5.1 AES structural modifications ............................................................ 21 1.1.5.2 Treatment of the AES......................................................................... 22 1.1.5.3 Plasmid delivery of the AES .............................................................. 23 1.1.5.4 The counter-selection strategy........................................................... 24 1.1.5.5 Specialized transduction .................................................................... 27 vi 1.2 SINGLE STRAND ANNEALING PROTEINS.............................................. 31 1.2.1 Single strand annealing protein families .................................................. 34 1.2.2 The Red recombination proteins............................................................ 36 1.2.3 The Rac prophage RecET recombination proteins ................................. 38 1.2.4 The P22 Erf, Arf, and Abc recombination proteins ................................ 40 1.2.5 SSAP mechanisms of recombination in vivo: single strand annealing versus strand exchange.............................................................................................. 41 1.3 RECOMBINEERING IN ESCHERICHIA COLI........................................... 42 1.3.1 Recombineering systems: λ Red and RecET............................................ 43 1.3.2 The recombineering strategy for mutagenesis ......................................... 45 1.3.2.1 Recombineering with dsDNA substrates.......................................... 46 1.3.2.2 Recombineering with ssDNA substrates........................................... 48 1.4 SPECIFIC AIMS OF THIS STUDY................................................................ 55 1.4.1 Specific Aim 1: Bioinformatic and biochemical analysis of mycobacteriophage Che9c-encoded RecET homologues. ...................................... 56 1.4.2 Specific Aim 2: Development of a mycobacterial recombineering system using mycobacteriophage Che9c-encoded recombination proteins. ..................... 56 1.4.3 Specific Aim 3: Identification of additional mycobacteriophage-encoded recombination systems............................................................................................... 57 2.0 MYCOBACTERIOPHAGE CHE9C ENCODES RECE AND RECT HOMOLOGUES......................................................................................................................... 58 2.1 INTRODUCTION ............................................................................................. 58 vii 2.2 BIOINFORMATIC ANALYSES OF MYCOBACTERIOPHAGES REVEALS A PUTATIVE RECOMBINATION SYSTEM............................................ 61 2.3 PURIFICATION OF CHE9C GP60 AND GP61 PROTEINS ...................... 65 2.4 CHE9C GP60 IS AN EXONUCLEASE .......................................................... 67 2.5 CHE9C GP61 BINDS SSDNA AND DSDNA ................................................. 69 2.6 CONCLUSIONS................................................................................................ 74 3.0 DEVELOPMENT OF THE MYCOBACTERIAL RECOMBINEERING SYSTEM. ..................................................................................................................................... 76 3.1 INTRODUCTION ............................................................................................. 76 3.2 EXPRESSION OF CHE9C RECOMBINATION GENES IN VIVO ........... 78 3.3 ALLELIC REPLACEMENT MUTAGENESIS............................................. 85 3.3.1 Che9c gp60 and gp61 promote homologous recombination in vivo ....... 85 3.3.2 Recombineering requires both Che9c gp60 and gp61............................. 87 3.3.3 Recombineering of the M. smegmatis groEL1 gene.................................. 88 3.3.4 Recombineering frequencies are limited by DNA uptake efficiency...... 91 3.3.5 Recombineering of other M. smegmatis genes.......................................... 92 3.3.6 Recombineering of the M. tuberculosis groEL1 gene............................... 93 3.3.7 Recombineering efficiently targets replicating plasmids. ....................... 96 3.4 POINT MUTAGENESIS .................................................................................. 98 3.4.1 ssDNA recombineering of replicating plasmids requires only Che9c gp61…… ..................................................................................................................... 98 3.4.2 Introducing point mutations in the M. smegmatis chromosome by ssDNA recombineering........................................................................................................
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