Conjugation and recombination in mycobacteria

分枝杆菌中的接合和重组

• 批准号: 7846531
• 负责人: KEITH M DERBYSHIRE
• 金额: $ 1.05万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-05 至 2009-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7846531
• 关键词: Accounting; Alleles; Attenuated; Biology; Cells; Cessation of life; Chromosomes; Cis-Acting Sequence; DNA; DNA Sequence; Derivation procedure; Development; Drug resistance; Epidemic; Generations; Genes; Genetic; Genetic Recombination; Genetic Screening; Genome; Genus Mycobacterium; Goals; Gram-Negative Bacteria; Grant; HIV; Horizontal Gene Transfer; Lateral; Lead; Mediating; Molecular; Molecular Genetics; Multi-Drug Resistance; Mutation; Mycobacterium avium; Mycobacterium bovis; Mycobacterium smegmatis; Mycobacterium tuberculosis; Pathogenesis; Pharmaceutical Preparations; Plasmids; Population; Process; Progress Reports; Proteins; Publishing; Role; Surface Antigens; System; Tuberculosis; Vaccines; Virulence; Virulent; Work; base; design; gene delivery system; genetic analysis; insight; mutant; mycobacterial; novel; novel strategies; pathogen; plasmid DNA; repaired; resistant strain; stem; tool

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis accounts for over 2 million deaths per year. Furthermore, the global burden of tuberculosis has been compounded by its deadly association with the AIDS virus and by the emergence of multi-drug resistant strains, which have increased the demand for new treatments to stem the tuberculosis/AIDS epidemic. The design of new drugs and vaccines requires an understanding of the biology of mycobacteria and the development of genetic tools to manipulate their genomes in order to determine the molecular basis of pathogenesis and drug resistance. Although both transformation and transduction have had important roles in the development of mycobacterial genetics, little is known about conjugal DNA transfer. Conjugation offers an important alternative for transferring DNA between mycobacteria and, in particular, as a gene delivery system for moving markers between strains and generating targeted mutations. During the last grant-period, we described a DNA transfer process in Mycobacterium smegmatis that is different from any conjugation system described to-date. This proposal is designed to characterize the M. smegmatis conjugation system by defining and identifying both cis-acting DNA sequences and trans-acting proteins that mediate DNA transfer. Such analyses will provide important mechanistic information about the process of DNA transfer and allow differences between donor and recipient cells to be determined. Moreover, by modifying transferable plasmids, a new allele-exchange system will be established, enabling the capture of segments of chromosomal DNA and the generation of targeted mutations by transfer-mediated recombination. The application of this system to the slow-growing mycobacterial pathogens will be a valuable new addition to current molecular approaches. In addition, plasmids have been isolated from Mycobacterium avium that encode DNA relaxases related to those required for classical conjugal transfer in gram-negative bacteria. The ability of these plasmids to transfer among slow-growing mycobacteria will be investigated to understand the role of conjugation in lateral transfer among mycobacterial populations and its possible role in the spread of drug resistance. The aims are: 1. To characterize cis-acting sequences required for DNA transfer and to develop transfer as a molecular genetic tool for the study of mycobacteria. 2. To identify and characterize trans-acting transfer functions in both donor and recipient cells. 3. To examine transfer of chromosomal and plasmid DNA among fast- and slow-growing mycobacteria.

描述（由申请人提供）：结核分枝杆菌每年造成200多万人死亡。此外，由于结核病与艾滋病毒的致命联系以及耐多药菌株的出现，增加了对新疗法的需求，以遏制结核病/艾滋病的流行，结核病给全球造成的负担更加沉重。新药物和疫苗的设计需要了解分枝杆菌的生物学和开发基因工具来操纵其基因组，以确定发病机制和耐药性的分子基础。虽然转化和转导在分枝杆菌遗传学的发展中具有重要作用，但对接合DNA转移知之甚少。缀合为在分枝杆菌之间转移DNA提供了一种重要的替代方法，特别是作为一种基因递送系统，用于在菌株之间移动标记物并产生靶向突变。在最后一个授权期间，我们描述了耻垢分枝杆菌中的DNA转移过程，该过程不同于迄今为止描述的任何接合系统。 这一建议是为了表征M。通过定义和鉴定介导DNA转移的顺式作用DNA序列和反式作用蛋白质，来研究耻垢病缀合系统。这种分析将提供有关DNA转移过程的重要机制信息，并允许确定供体和受体细胞之间的差异。此外，通过修饰可转移质粒，将建立新的等位基因交换系统，从而能够捕获染色体DNA片段并通过转移介导的重组产生靶向突变。该系统的应用，以缓慢增长的分枝杆菌病原体将是一个有价值的新的除了目前的分子方法。此外，已经从鸟分枝杆菌中分离出质粒，其编码与革兰氏阴性菌中经典接合转移所需的DNA松弛酶相关的DNA松弛酶。将研究这些质粒在生长缓慢的分枝杆菌之间转移的能力，以了解接合在分枝杆菌种群之间横向转移中的作用及其在耐药性传播中的可能作用。目标是：1.描述DNA转移所需的顺式作用序列，并开发转移作为分枝杆菌研究的分子遗传学工具。 2.鉴定和表征供体和受体细胞中的反式作用转移功能。 3.检测快速和慢速生长分枝杆菌之间染色体和质粒DNA的转移。

项目成果

Construction and application of mycobacterial reporter transposons.

分枝杆菌报告转座子的构建及应用。

• DOI: 10.1016/s0378-1119(00)00238-9
• 发表时间: 2000
• 期刊: Gene
• 影响因子: 3.5
• 作者: Machowski,EE;McAdam,RA;Derbyshire,KM;Mizrahi,V
• 通讯作者: Mizrahi,V

Unconventional conjugal DNA transfer in mycobacteria.

分枝杆菌中的非常规配偶 DNA 转移。

• DOI: 10.1038/ng1139
• 发表时间: 2003
• 期刊: Nature genetics
• 影响因子: 30.8
• 作者: Wang,Jun;Parsons,LindaM;Derbyshire,KeithM
• 通讯作者: Derbyshire,KeithM

Genetic alteration of Mycobacterium smegmatis to improve mycobacterium-mediated transfer of plasmid DNA into mammalian cells and DNA immunization.

耻垢分枝杆菌的遗传改变可改善分枝杆菌介导的质粒 DNA 转移至哺乳动物细胞和 DNA 免疫。

• DOI: 10.1128/iai.01877-06
• 发表时间: 2007
• 期刊: Infection and immunity
• 影响因子: 3.1
• 作者: Mo,Yongkai;Quanquin,NatalieM;Vecino,WilliamH;Ranganathan,UmaDevi;Tesfa,Lydia;Bourn,William;Derbyshire,KeithM;Letvin,NormanL;JacobsJr,WilliamR;Fennelly,GlennJ
• 通讯作者: Fennelly,GlennJ

Draft Genome Sequence of MKD8, a Conjugal Recipient Mycobacterium smegmatis Strain.

• DOI: 10.1128/genomea.00148-13
• 发表时间: 2013-04-25
• 期刊: Genome announcements
• 作者: Gray TA;Palumbo MJ;Derbyshire KM
• 通讯作者: Derbyshire KM

Polar assembly and scaffolding proteins of the virulence-associated ESX-1 secretory apparatus in mycobacteria.

分枝杆菌毒力相关 ESX-1 分泌装置的极性组装和支架蛋白。

• DOI: 10.1111/j.1365-2958.2011.07958.x
• 发表时间: 2012
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Wirth,SamanthaE;Krywy,JanetA;Aldridge,BreeB;Fortune,SarahM;Fernandez-Suarez,Marta;Gray,ToddA;Derbyshire,KeithM
• 通讯作者: Derbyshire,KeithM