Sciochetti Stephen A, Lane Todd, Ohta Noriko, Newton Austin
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
J Bacteriol. 2002 Nov;184(21):6037-49. doi: 10.1128/JB.184.21.6037-6049.2002.
The Caulobacter crescentus sensor kinase DivJ is required for an early cell division step and localizes at the base of the newly formed stalk during the G1-to-S-phase transition when the protein is synthesized. To identify sequences within DivJ that are required for polar localization, we examined the ability of mutagenized DivJ sequences to direct localization of the green fluorescent protein. The effects of overlapping C-terminal deletions of DivJ established that the N-terminal 326 residues, which do not contain the kinase catalytic domain, are sufficient for polar localization of the fusion protein. Internal deletions mapped a shorter sequence between residues 251 and 312 of the cytoplasmic linker that are required for efficient localization of this sensor kinase. PleC kinase mutants, which are blocked in the swarmer-to-stalked-cell transition and form flagellated, nonmotile cells, also fail to localize DivJ. To dissect the cellular factors involved in establishing subcellular polarity, we have examined DivJ localization in a pleC mutant suppressed by the sokA301 allele of ctrA and in a pleD mutant, both of which display a supermotile, stalkless phenotype. The observation that these Mot(+) strains localize DivJ to a single cell pole indicate that localization may be closely coupled to the gain of motility and that normal stalk formation is not required. We have also observed, however, that filamentous parC mutant cells, which are defective in DNA segregation and the completion of cell separation, are motile and still fail to localize DivJ to the new cell pole. These results suggest that formation of new sites for DivJ localization depends on events associated with the completion of cell separation as well as the gain of motility. Analysis of PleC and PleD mutants also provides insights into the function of the His-Asp proteins in cell cycle regulation. Thus, the ability of the sokA301 allele of ctrA to bypass the nonmotile phenotype of the pleC null mutation provides evidence that the PleC kinase controls cell motility by initiating a signal transduction pathway regulating activity of the global response regulator CtrA. Analysis of the pleD mutant cell cycle demonstrates that disruption of the swarmer-to-stalked-cell developmental sequence does not affect the asymmetric organization of the Caulobacter cell cycle.
新月柄杆菌传感器激酶DivJ是细胞早期分裂步骤所必需的，在G1期到S期转换期间，当该蛋白合成时，它定位于新形成的柄的基部。为了确定DivJ中极性定位所需的序列，我们检测了诱变后的DivJ序列指导绿色荧光蛋白定位的能力。DivJ重叠C末端缺失的影响表明，不包含激酶催化结构域的N末端326个残基足以使融合蛋白进行极性定位。内部缺失确定了细胞质连接子第251至312位残基之间一段较短的序列，该序列是该传感器激酶有效定位所必需的。PleC激酶突变体在游动细胞到柄细胞的转变过程中受阻，形成有鞭毛的、不能运动的细胞，也不能使DivJ定位。为了剖析参与建立亚细胞极性的细胞因子，我们检测了在被ctrA的sokA301等位基因抑制的pleC突变体和pleD突变体中的DivJ定位，这两种突变体均表现出超运动性、无柄表型。这些Mot(+)菌株将DivJ定位于单个细胞极的观察结果表明，定位可能与运动性的获得密切相关，并且正常的柄形成不是必需的。然而，我们还观察到，丝状parC突变体细胞在DNA分离和细胞分离完成方面存在缺陷，它们具有运动性，但仍然不能将DivJ定位于新的细胞极。这些结果表明，DivJ定位新位点的形成取决于与细胞分离完成以及运动性获得相关的事件。对PleC和PleD突变体的分析也为组氨酸-天冬氨酸蛋白在细胞周期调控中的功能提供了见解。因此，ctrA的sokA301等位基因绕过pleC无效突变的非运动表型的能力提供了证据，表明PleC激酶通过启动调节全局反应调节因子CtrA活性的信号转导途径来控制细胞运动性。对pleD突变体细胞周期的分析表明，游动细胞到柄细胞发育序列的破坏并不影响新月柄杆菌细胞周期的不对称组织。
