New Insights into PhaM-PhaC-Mediated Localization of Polyhydroxybutyrate Granules in Ralstonia eutropha H16
New Insights into PhaM-PhaC-Mediated Localization of Polyhydroxybutyrate Granules in Ralstonia eutropha H16
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DOI：
10.1128/aem.00505-17
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发表时间：
2017-06-01
影响因子：
4.4
通讯作者：
Jendrossek, Dieter
中科院分区：
文献类型：
--
作者：
Bresan, Stephanie;Jendrossek, Dieter
聚羟基丁酸盐（PHB）颗粒的形成和定位受PhaM控制，PhaM与PHB合成酶（PhaC）和细菌类核相互作用。在这里，我们通过体外和体内的方法研究了PhaM中两个c端PAKKA基序的脯氨酸和赖氨酸残基对PHB颗粒与DNA结合的重要性。赖氨酸残基取代而非脯氨酸残基取代导致形成的PHB颗粒脱离类核。相反，在杆状细胞的两极区域形成了PHB颗粒簇，并且PHB颗粒在子细胞中的分布不均匀。通过荧光蛋白与PhaM和PhaC染色体锚定基因融合在不同背景下的表达，研究了PHB颗粒的形成。PhaM和PhaC的融合在野生型的类核区形成的PHB颗粒上显示出明显的共定位。在Delta phaC背景下，PhaM和催化活性不强的phaC （C319A）蛋白不能形成荧光焦点，说明正确定位需要形成PHB。此外，延时实验显示PhaC和PhaM蛋白在后期从形成的PHB颗粒中分离，导致PHB颗粒的非均匀群体。这可以解释为什么在允许PHB的条件下，单个PHB颗粒的生长在一定大小时停止。在许多原核生物中，PHB颗粒是碳和能量的储存化合物。因此，在细胞分裂过程中积累的PHB颗粒的均匀分布对于子细胞的最佳适应性是重要的。在富营养菌中，PhaM负责PHB合成酶的最大活性，在细胞的离散区域启动PHB颗粒形成，并将形成的PHB颗粒与类核结合。在这里，我们发现c端PhaM序列基序的四个赖氨酸残基对于PHB颗粒与类核的结合是必不可少的。此外，我们通过延时显微镜跟踪了PHB颗粒的形成，并提供了PHB颗粒老化的证据，这表现为先前PHB颗粒相关的PhaM和PHB合成酶的脱离。
The formation and localization of polyhydroxybutyrate (PHB) granules in Ralstonia eutropha are controlled by PhaM, which interacts both with the PHB synthase (PhaC) and with the bacterial nucleoid. Here, we studied the importance of proline and lysine residues of two C-terminal PAKKA motifs in PhaM for their importance in attaching PHB granules to DNA by in vitro and in vivo methods. Substitution of the lysine residues but not of the proline residues resulted in detachment of formed PHB granules from the nucleoid. Instead, formation of PHB granule clusters at polar regions of the rod-shaped cells and an unequal distribution of PHB granules to daughter cells were observed. The formation of PHB granules was studied by the expression of chromosomally anchored gene fusions of fluorescent proteins with PhaM and PhaC in different backgrounds. PhaM and PhaC fusions showed a distinct colocalization at formed PHB granules in the nucleoid region of the wild type. In a Delta phaC background, PhaM and the catalytically inactive PhaC(C319A) protein were not able to form fluorescent foci, indicating that correct positioning requires the formation of PHB. Furthermore, time-lapse experiments revealed that PhaC and PhaM proteins detach from formed PHB granules at later stages, resulting in a nonhomogeneous population of PHB granules. This could explain why growth of individual PHB granules stops under PHB-permissive conditions at a certain size.IMPORTANCE PHB granules are storage compounds for carbon and energy in many prokaryotes. Equal distribution of accumulated PHB granules during cell division is therefore important for optimal fitness of the daughter cells. In R. eutropha, PhaM is responsible for maximal activity of PHB synthase, for initiation of PHB granule formation at discrete regions in the cells, and for association of formed PHB granules with the nucleoid. Here we found that four lysine residues of C-terminal PhaM sequence motifs are essential for association of PHB granules with the nucleoid. Furthermore, we followed PHB granule formation by time-lapse microscopy and provide evidence for aging of PHB granules that is manifested by detachment of previously PHB granule-associated PhaM and PHB synthase.