Analysis Of The Recipient In Conjugal Plasmid Transfer

在偶联质粒转移过程中，质粒DNA穿过供体细胞膜和细胞壁系统的运动是通过IV型转运系统的活动完成的。农杆菌的VirB复合体是典型的膜定位型IV型转运复合体。VirB复合体除了在植物转化中发挥作用外，还可以支持质粒RSF1010在农杆菌之间的偶联转移。该项目的目标是根据一个意想不到的发现而制定的：在受体农杆菌菌株中表达特定的VirB蛋白亚群，可使VirB介导的RSF1010偶联转移的频率增加三个数量级以上。这很有趣，因为IV型转运底物穿过受体细胞包膜的机制尚不清楚。这项工作的具体目的是鉴定肿瘤杆菌中的基因，这些基因与VirB蛋白一起，需要增加作为配偶受体的能力。此外，将对突变受体进行细胞和分子分析，重点是分析交配对形成的新策略，以及检查受体表型中已鉴定蛋白的表达、定位和作用的实验。就该研究的更广泛影响而言，质粒偶联转移是抗生素耐药性在从土壤到医院重症监护病房等各种环境中传播的最普遍手段。然而，人们对受体细胞的作用知之甚少，特别是在需要固体表面才能成功交配的系统中。确定接受者如何影响转移效率对微生物生态学和种群生物学以及更以医学为导向的抗生素耐药性问题具有影响。

During conjugal plasmid transfer the movement of plasmid DNA across the donor cell membrane and wall systems is accomplished through the activities of Type IV transport systems. The VirB complex of Agrobacterium tumefaciens is the prototypical membrane localized Type IV transport complex. In addition to its role in plant transformation, the VirB complex can support the conjugal transfer of plasmid RSF1010 between agrobacteria. The goals of this project were developed in response to an unexpected finding: expression of specific subsets of the VirB proteins in a recipient Agrobacterium strain increases the frequency of virB-mediated conjugal RSF1010 transfer into it by over three orders of magnitude. This is intriguing because the mechanism by which Type IV transport substrates cross the recipient cell envelope are not clear. The specific aims of the work are to identify genes in A. tumefaciens that are, along with the VirB proteins, required for the increase in the capacity to serve as a conjugal recipient. Additionally, cellular and molecular analysis of the mutant recipients will be carried out, focusing on novel strategies to analyze mating pair formation as well as experiments to examine the expression, localization and role of the identified proteins in the recipient phenotype. In relation to the broader impact of the research, plasmid conjugal transfer is the most prevalent means of antibiotic resistance spread in a wide variety of environments ranging from soil to hospital intensive care units. Yet the role of the recipient cell is poorly understood, particularly in systems that require solid surfaces for successful mating. Defining how the recipient affects the efficiency of transfer has implications for microbial ecology and population biology as well as the more medically oriented problem of antibiotic resistance.