Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids
Defining the Role of ATP Hydrolysis in Mitotic Segregation of Bacterial Plasmids
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DOI：
10.1371/journal.pgen.1003956
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发表时间：
2013-12-01
期刊：
影响因子：
4.5
通讯作者：
Bouet, Jean-Yves
中科院分区：
文献类型：
--
作者：
Ah-Seng, Yoan;Rech, Jerome;Bouet, Jean-Yves
虽然被认为是确保质粒稳定遗传的分离机制中的关键步骤，但通过分配ATP酶水解ATP本质上非常弱。同源着丝粒结合蛋白（CBP）与DNA一起刺激ATP酶水解ATP，并进行重新定位，激发质粒运动，显然证实了分区水解的必要性。然而，ATP结合单独改变ATP酶的构象和性质，使得难以严格区分ATP在体内的底物和辅因子作用。我们已经表明，F质粒CBP，SopB中的丝氨酸R36和R42的突变减少了SopA催化的ATP水解的刺激，而不改变SopA-SopB亲和力，这表明水解的作用可以使用对ATP具有正常构象响应的SopA来分析。在这里，我们报告说，强烈减少SopB介导的刺激ATP水解的结果只有轻微的不稳定的mini-F，虽然不稳定性，以及增加mini-F集群，是成比例的ATP酶赤字。出乎意料的是，减少的刺激也增加了类核上SopA重新定位的频率。这种增加是由于SopA在类核末端所花费的时间急剧缩短;迁移本身的平均速度没有变化。减少ATP水解也与mini-F定位的明显偏差相关，尽管时间平均位置仅适度变化。因此，通过特异性靶向SopB刺激的ATP水解，我们的研究揭示，即使在降低分裂簇的效率和质粒定位的恒定性的ATP酶水平下，SopB仍然激活SopA移动性和质粒定位，并维持接近野生型水平的质粒稳定性。
Hydrolysis of ATP by partition ATPases, although considered a key step in the segregation mechanism that assures stable inheritance of plasmids, is intrinsically very weak. The cognate centromere-binding protein (CBP), together with DNA, stimulates the ATPase to hydrolyse ATP and to undertake the relocation that incites plasmid movement, apparently confirming the need for hydrolysis in partition. However, ATP-binding alone changes ATPase conformation and properties, making it difficult to rigorously distinguish the substrate and cofactor roles of ATP in vivo. We had shown that mutation of arginines R36 and R42 in the F plasmid CBP, SopB, reduces stimulation of SopA-catalyzed ATP hydrolysis without changing SopA-SopB affinity, suggesting the role of hydrolysis could be analyzed using SopA with normal conformational responses to ATP. Here, we report that strongly reducing SopB-mediated stimulation of ATP hydrolysis results in only slight destabilization of mini-F, although the instability, as well as an increase in mini-F clustering, is proportional to the ATPase deficit. Unexpectedly, the reduced stimulation also increased the frequency of SopA relocation over the nucleoid. The increase was due to drastic shortening of the period spent by SopA at nucleoid ends; average speed of migration per se was unchanged. Reduced ATP hydrolysis was also associated with pronounced deviations in positioning of mini-F, though time-averaged positions changed only modestly. Thus, by specifically targeting SopB-stimulated ATP hydrolysis our study reveals that even at levels of ATPase which reduce the efficiency of splitting clusters and the constancy of plasmid positioning, SopB still activates SopA mobility and plasmid positioning, and sustains near wild type levels of plasmid stability.