Recombineering in mycobacteria using mycobacteriophage proteins
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 ? 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 ? 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 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 ? 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.
Advisor:William Jacobs, Jr.; Roger Hendrix; Jeffrey Lawrence; Graham Hatfull; Valerie Oke
School:University of Pittsburgh
School Location:USA - Pennsylvania
Source Type:Master's Thesis
Date of Publication:11/03/2008