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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Different phenotypes of Walker-like A box mutants of ParA homolog IncC of broad-host-range IncP plasmids
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Different phenotypes of Walker-like A box mutants of ParA homolog IncC of broad-host-range IncP plasmids

机译：广泛宿主范围的IncP质粒的ParA同系物IncC的Walker样A盒突变体的不同表型

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The promiscuous IncPα plasmids RK2 and R995 encode a broad-host-range partition system, whose essential components include the incC and korB genes and a DNA site (O B) to which the korB product binds. IncC2, the smaller of the two incC products, is sufficient for stabilization of R995ΔincC. It is a member of the type Ia ParA family of partition ATPases. To better understand the role of ATP in partition, we constructed three alanine-substitution mutants of IncC2. Each mutation changed a different residue of the Walker-like ATP-binding and hydrolysis motif, including a lysine (K10) conserved solely among members of the ParA and MinD families. All three IncC2 mutants were defective in plasmid partition, but they differed from one another in other respects. The IncC2 T16A mutant, predicted to be defective in Mg 2+ coordination, was severely impaired in all activities tested. IncC2 K10A, predicted to be defective in ATP hydrolysis, mediated enhanced incompatibility with R995 derivatives. IncC2 K15A, predicted to be defective in ATP binding, exhibited two distinct incompatibility properties depending on the genotype of the target plasmid. When in trans to plasmids carrying a complementable incC deletion, IncC2 K15A caused dramatic plasmid loss, even at low levels of expression. In trans to wild-type R995 or to R995ΔincC carrying a functional P1 partition system, IncC2 K15A-mediated incompatibility was significantly less than that caused by wild-type IncC2. All three Walker-like A box mutants were also defective for the host toxicity that normally results from co-overexpression of incC and korB. The phenotypes of the mutants support a model in which nucleotide hydrolysis is required for separation of paired plasmid complexes and possible interaction with a host factor.
机译：混杂的IncPα质粒RK2和R995编码一个广泛的宿主范围的分区系统，其主要成分包括incC和korB基因以及与korB产物结合的DNA位点（OB）。两个incC产品中较小的IncC2足以稳定R995ΔincC。它是Ia ParA型分区ATPase家族的成员。为了更好地了解ATP在分区中的作用，我们构建了三个IncC2的丙氨酸替代突变体。每个突变都改变了Walker样ATP结合和水解基序的不同残基，包括仅在ParA和MinD家族成员中保守的赖氨酸（K10）。所有三个IncC2突变体在质粒分配上均存在缺陷，但在其他方面却彼此不同。预计在Mg 2+配体中存在缺陷的IncC2 T16A突变体在所有测试的活动中均受到严重损害。预计在ATP水解中存在缺陷的IncC2 K10A介导了与R995衍生物增强的不相容性。预期在ATP结合方面有缺陷的IncC2 K15A表现出两种不同的不相容性，具体取决于靶质粒的基因型。当与携带互补的incC缺失的质粒反式表达时，即使在低水平的表达下，IncC2 K15A也引起了显着的质粒损失。在携带功能性P1分隔系统的野生型R995或R995ΔincC的反式中，IncC2 K15A介导的不相容性明显小于野生型IncC2引起的不相容性。所有三个Walker-like A box突变体也都缺乏宿主毒性，而宿主毒性通常是由incC和korB共同过量表达引起的。突变体的表型支持一个模型，其中分离成对的质粒复合物以及可能与宿主因子相互作用需要核苷酸水解。

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