Dissection of the core and auxiliary sequences in the vegetative replication origin of promiscuous plasmid RK2.
Dissection of the core and auxiliary sequences in the vegetative replication origin of promiscuous plasmid RK2.
复制标题
混杂质粒 RK2 营养复制起点的核心和辅助序列的解剖。
DOI：
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发表时间：
1995
影响因子：
5.6
通讯作者：
Christopher M Thomas
中科院分区：
文献类型：
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作者：
D. Shah;M. Cross;D. Porter;Christopher M Thomas
当提供质粒编码的复制蛋白TrfA和宿主编码的复制蛋白（包括DnaA）时，混杂IncP质粒RK2的营养复制起点（oriV）可以在许多革兰氏阴性细菌物种中起作用。已知有9个TrfA结合位点（iteron），还有2个DnaA结合位点，即TrfA iteron 4和5之间的框1，以及重复序列9下游的框2。这里提出的缺失分析表明，核心oriV需要DnaA盒1在大肠杆菌和恶臭假单胞菌的功能。这个DNA盒在铜绿假单胞菌中不是必需的，尽管它的缺失确实减少了该物种中的质粒拷贝数。推测的IHF结合位点位于DnaA box 1的上游，但在E.大肠杆菌似乎不改变复制效率或拷贝数控制。克隆的oriV可以干扰独立RK2复制子的维持。与oriV相关的复制抑制功能的分析表明，TrfA iteron 1和2之间的短推定orf对于复制抑制不是必需的，靶质粒和抑制剂质粒中重复序列5至9的存在不足以有效抑制，并且抑制与存在的同向重复序列的数量不直接相关。相反，结果表明，分离的重复序列1和2至4，增强复制抑制不成比例地对TrfA结合位点的数量的影响。结果是一致的想法，重复1至4，作为一个单一的副本，并作为一个不规则的三个组，加强能力的oriV区域形成复合物，抑制复制。我们认为，TrfA结合在这些iteron可能更容易形成对不同质粒上的oriV序列。
The vegetative replication origin (oriV) of promiscuous IncP plasmid RK2 can function in many Gram-negative bacterial species when supplied with the plasmid-encoded replication protein TrfA and host-encoded replication proteins including DnaA. Nine TrfA binding sites (iterons) are known, and also two DnaA binding sites, box 1, between TrfA iterons 4 and 5, and box 2, downstream of repeat 9. The deletion analysis presented here shows that the core oriV requires DnaA box 1 for function in Escherichia coli and Pseudomonas putida. This DnA box is not essential in Pseudomonas aeruginosa, although its deletion does reduce plasmid copy number in this species. A putative IHF binding site is located upstream of DnaA box 1, but IHF deficiency in E. coli seems not to alter replication efficiency or copy number control. Cloned oriV can interfere with maintenance of an independent RK2 replicon. Analysis of replication inhibition functions associated with oriV showed that a short putative orf between TrfA iterons 1 and 2 is not necessary for replication inhibition, the presence of repeats 5 to 9 in target and inhibitor plasmid are not sufficient for efficient inhibition and inhibition does not correlate directly with the number of direct repeats present. Rather, the results showed that the isolated repeats 1 and 2 to 4, potentiate replication inhibition disproportionately to their effect on the number of TrfA binding sites. The results are consistent with the idea that repeats 1 to 4, arranged as a single copy and as an irregular group of three, potentiate the ability of the oriV region to form complexes which inhibit replication. We suggest that TrfA bound at these iterons may be more susceptible to forming pairs between oriV sequences on different plasmids.