Mycobacterium tuberculosis accounts for more deaths worldwide than any other infectious agent. Recent increases in the number of cases have been associated with the spread of the AIDS virus and the appearance of drug-resistant strains of M. tuberculosis. The development of new treatments for mycobacteria requires an understanding of the biology of these bacteria and the ability to manipulate their genomes to determine the genetic basis of pathogenesis and drug resistance. In M. tuberculosis, studies have been hindered by inefficient methods for the introduction of DNA into and between strains, and by the low frequency of targeted recombination. In particular, the ability to make site-directed changes within the M. tuberculosis genome is crucial for understanding the role of particular genes in virulence. We propose to characterize a novel conjugation system that can transfer chromosomal DNA between strains of Mycobacterium smegmatis and use it as a tool for the analysis and transfer of DNA between mycobacteria. This naturally occurring system could offer an efficient method for the introduction of DNA into the slow-growing pathogenic mycobacteria. Furthermore, by examining conjugation between different species of mycobacteria we will assess its promiscuity and possible role in the spread of drug resistance. Finally, we have identified three M. tuberculosis genes that are highly homologous to those encoding the RecBCD pathway of recombination in Escherichia coli, which is the major pathway for conjugal recombination and double-strand break repair. We intend to characterize these genes to facilitate the development of a defined homologous recombination system in M. tuberculosis. These goals will be achieved by: 1. Cloning and characterizing the cis-acting sequences necessary for conjugal DNA transfer. 2. Identifying the trans-acting protein functions necessary for transfer. 3. Monitoring conjugal transfer from M. smegmatis into other mycobacterial species including the slow-growing pathogenic species. 4. Developing a defined allele-exchange system by characterizing the RecBCD pathway of M. tuberculosis

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Sizemore, Christine F
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Wadsworth Center
United States
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Derbyshire, Keith M; Gray, Todd A (2014) Distributive Conjugal Transfer: New Insights into Horizontal Gene Transfer and Genetic Exchange in Mycobacteria. Microbiol Spectr 2:
Gray, Todd A; Palumbo, Michael J; Derbyshire, Keith M (2013) Draft Genome Sequence of MKD8, a Conjugal Recipient Mycobacterium smegmatis Strain. Genome Announc 1:e0014813
Gray, Todd A; Krywy, Janet A; Harold, Jessica et al. (2013) Distributive conjugal transfer in mycobacteria generates progeny with meiotic-like genome-wide mosaicism, allowing mapping of a mating identity locus. PLoS Biol 11:e1001602
Wirth, Samantha E; Krywy, Janet A; Aldridge, Bree B et al. (2012) Polar assembly and scaffolding proteins of the virulence-associated ESX-1 secretory apparatus in mycobacteria. Mol Microbiol 83:654-64
Nguyen, Kiet T; Piastro, Kristina; Gray, Todd A et al. (2010) Mycobacterial biofilms facilitate horizontal DNA transfer between strains of Mycobacterium smegmatis. J Bacteriol 192:5134-42
Callahan, Brian; Nguyen, Kiet; Collins, Alissa et al. (2010) Conservation of structure and protein-protein interactions mediated by the secreted mycobacterial proteins EsxA, EsxB, and EspA. J Bacteriol 192:326-35
Nguyen, Kiet T; Piastro, Kristina; Derbyshire, Keith M (2009) LpqM, a mycobacterial lipoprotein-metalloproteinase, is required for conjugal DNA transfer in Mycobacterium smegmatis. J Bacteriol 191:2721-7
Bitter, Wilbert; Houben, Edith N G; Bottai, Daria et al. (2009) Systematic genetic nomenclature for type VII secretion systems. PLoS Pathog 5:e1000507
Cooper, Andrea M; Kaplan, Gilla; Derbyshire, Keith (2009) TB day summit in upstate New York: key issues to address. Eur J Immunol 39:1443-7
Coros, Abbie; Callahan, Brian; Battaglioli, Eric et al. (2008) The specialized secretory apparatus ESX-1 is essential for DNA transfer in Mycobacterium smegmatis. Mol Microbiol 69:794-808

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