Mechanism of CTX bacteriophage integration

描述(申请人提供)：这项建议的目标是了解CTXphi整合的机制及其与染色体二聚体分辨的关系。CTXphi_是一种丝状噬菌体，携带霍乱毒素的基因，霍乱毒素是霍乱弧菌的主要毒力因子。CTXphi将位点特异性整合到霍乱弧菌的染色体中，从而使非致病性霍乱弧菌产生毒素。与其他特有的噬菌体和病毒不同，CTXphi依赖于染色体编码的重组酶XerC和XerD来整合其DNA。整合发生在染色体中点附近，靠近d/f位点，这是XerC和XerD底物，用于分辨染色体二聚体。因此，这项建议的具体目标包括：1)确定CTXphi整合和重组所需的DNA序列；2)鉴定XerC和XerD在体外的重组；111)通过遗传筛选鉴定CTXphi整合所需的额外染色体基因；以及IV)鉴定噬菌体编码的rstB基因及其对CTXphi整合离子的影响。这些研究结果将拓宽我们对CTXphi生物学和霍乱弧菌致病性进化的理解，并将阐明一种新的噬菌体整合方法的机制。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand the mechanism of CTXphi integration and its relationship to chromosome dimer resolution. CTXphi_ is a filamentous bacteriophage that carries the genes for cholera toxin, the primary virulence factor of Vibrio cholerae. CTXphi integrates site-specifically into the chromosome of v. cholerae and thereby renders non-pathogenic isolates toxigenic. Unlike other characterized phages and viruses, CTXphi depends on chromosome-encoded recombinases, XerC and XerD, to integrate its DNA. Integration occurs near the chromosome mid-point in close proximity to the d/f site, which is the XerC and XerD substrate for resolution of chromosome dimers. Thus, the specific aims of this proposal include: I) definition of DNA sequences required for CTXphi integration and recombination at d/f, II) characterization of recombination by XerC and XerD in vitro; 111) identification, via a genetic screen, of additional chromosomal genes required for CTXd_ integration; and IV) characterization of the phage encoded rstB gene and examination of its effect on CTXphi integral ion. Results from these studies will broaden our understanding of CTXphi biology and the evolution of V. cholerae pathogenicity and will clarify the mechanism of a novel means of phage integration.