Modulation of pPS10 Host Range by Plasmid-Encoded RepA Initiator Protein
Modulation of pPS10 Host Range by Plasmid-Encoded RepA Initiator Protein
复制标题
质粒编码的 RepA 起始蛋白对 pPS10 宿主范围的调节
DOI：
10.1128/jb.185.4.1367-1375.2003
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发表时间：
2003
影响因子：
3.2
通讯作者：
E. Fernández
中科院分区：
文献类型：
--
作者：
Beatriz Maestro;J. Sanz;R. Dı́az;E. Fernández
摘要本文报道了原发现于野生型假单胞菌中的pPS10新的REPA宿主范围突变体的分离和分析。在羟胺处理后，选择了5个质粒突变体在37℃的大肠杆菌中建立，在这个温度下不能建立野生型形式。突变位于REPA起始蛋白的不同功能区，其稳定性、拷贝数和与基本复制子序列相互作用的能力也不同。其中四株扩大了寄主范围，其中一株无法在假单胞菌中复制，因此改变了寄主范围。此外，这些突变体还提高了它们在恶臭假单胞菌和粪产碱菌等大肠杆菌以外的菌株中的复制效率。这些突变都没有显著改变野生型REPA蛋白的结构或热稳定性，但在所有情况下，凝胶过滤层析都检测到与宿主编码的DNAA蛋白的相互作用增强。在蛋白质的三维模型框架内讨论了突变对REPA蛋白功能的影响。我们对不同的REPA突变体的宿主范围效应提出了可能的解释，包括增强了REPA与特定宿主复制因子(如DNAA)的限制性相互作用。
ABSTRACT We report here the isolation and analysis of novel repA host range mutants of pPS10, a plasmid originally found in Pseudomonas savastanoi. Upon hydroxylamine treatment, five plasmid mutants were selected for their establishment in Escherichia coli at 37°C, a temperature at which the wild-type form cannot be established. The mutations were located in different functional regions of the plasmid RepA initiation protein, and the mutants differ in their stable maintenance, copy number, and ability to interact with sequences of the basic replicon. Four of them have broadened their host range, and one of them, unable to replicate in Pseudomonas, has therefore changed its host range. Moreover, the mutants also have increased their replication efficiency in strains other than E. coli such as Pseudomonas putida and Alcaligenes faecalis. None of these mutations drastically changed the structure or thermal stability of the wild-type RepA protein, but in all cases an enhanced interaction with host-encoded DnaA protein was detected by gel filtration chromatography. The effects of the mutations on the functionality of RepA protein are discussed in the framework of a three-dimensional model of the protein. We propose possible explanations for the host range effect of the different repA mutants, including the enhancement of limiting interactions of RepA with specific host replication factors such as DnaA.
质粒 R6K 编码的 pi 蛋白的自动调节缺陷突变体区分回文和非回文结合位点。
DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
York,D;Filutowicz,M
通讯作者：
Filutowicz,M
质粒 R6K 复制的负控制：复制起点分子间偶联的可能作用。
DOI：
10.1073/pnas.86.20.7942
发表时间：
1989
影响因子：
11.1
作者：
McEachern,MJ;Bott,MA;Tooker,PA;Helinski,DR
通讯作者：
Helinski,DR
质粒 RK2 复制起始蛋白 TrfA 的三个羧基末端氨基酸对于革兰氏阴性细菌中 DNA 结合和复制活性的重要性。
DOI：
--
发表时间：
1993
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Cereghino,JL;Helinski,DR
通讯作者：
Helinski,DR