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Mechanism of conjugal transfer in IncI plasmids

IncI质粒中的接合转移机制

基本信息

批准号：
17570007
负责人：
KOMANO Teruya
金额：
$ 2.18万
依托单位：
Tokyo Metropolitan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570007/
关键词：
Genetics Bacteria IncI plasmid R64 Conjugal transfer Shufflon

项目摘要

The 54-kb transfer region of IncI1 plasmid R64 contains 49 genes, in which 24 genes are essential for matings in general. Additional 12 pil genes encoding type IV pili are required only for liquid matings to reveal the relationship between the structure and function of PilU. Multiple DNA inversions of R64 shufflon select one of seven PilV adhesins with different C-terminal segments.1) Forty-two mutants of R64 pilU gene encoding prepilin peptidase were isolated. The PilU protein was shown to belong to the aspartic acid peptidase family.2) TraC, a putative positive regulator for transfer gene expression, was shown to inhibit shufflon inversions by interacting with a C-terminal segment of Rci, the shufflon-specific recombinase.3) The sfx recombination sequences of R64 shufflon have been shown to be asymmetric. Frequencies of seven pilV genes were not equal in equilibrium. When in vivo inversion of an artificial shufflon segment carrying two sfx sequence with different left and right arms at both ends was induced by concomitant presence of rci plasmid, the frequencies of shufflon segments with normal or reverse orientation were not equal after inversions reached equilibrium. Theses results indicate that the asymmetry of the sfx sequences results in the unequal frequency of the seven pilV genes in equilibrium in R64 shufflon.4) DNA transfer in R64 conjugation is initiated by the introduction of a site-and strand-specific nick at the oriT site by NikAB relaxase. NMR analysis indicated that two molecules of NikA N-terminal segment (NikA1-51) form a ribbon-helix-helix fold. SELEX analysis indicated that NikA binds to ACGGTA sequence present in the repeat A sequence of R64 oriT.

IncI1质粒R64的54-kb转移区包含49个基因，其中24个基因对于一般交配是必需的。仅在液体交配时才需要编码 IV 型菌毛的额外 12 个 pil 基因，以揭示 PilU 的结构和功能之间的关系。 R64 shufflon的多重DNA倒转选择了具有不同C末端片段的7种PilV粘附素之一。1)分离了42个编码prepilin肽酶的R64 pilU基因突变体。 PilU 蛋白被证明属于天冬氨酸肽酶家族。2) TraC（一种假定的转移基因表达正调节因子）被证明可以通过与 Rci（shufflon 特异性重组酶）的 C 末端片段相互作用来抑制 shufflon 倒位。3) R64 shufflon 的 sfx 重组序列被证明是不对称的。七个 pilV 基因的频率在平衡状态下不相等。当rci质粒的同时存在诱导两端带有不同左臂和右臂的两个sfx序列的人工shufflon片段体内倒位时，倒位达到平衡后，具有正常或反向方向的shufflon片段的频率不相等。这些结果表明，sfx序列的不对称性导致R64 shufflon中平衡状态下的7个pilV基因的频率不等。4) R64缀合中的DNA转移是通过NikAB松弛酶在oriT位点引入位点和链特异性切口来启动的。 NMR分析表明NikA N端片段(NikA1-51)的两个分子形成带状-螺旋-螺旋折叠。 SELEX 分析表明 NikA 与 R64 oriT 重复 A 序列中存在的 ACGGTA 序列结合。