Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16
Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16
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DOI：
10.1128/jb.00779-12
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发表时间：
2012-11-01
影响因子：
3.2
通讯作者：
Jendrossek, Dieter
中科院分区：
文献类型：
--
作者：
Pfeiffer, Daniel;Jendrossek, Dieter
聚（3-羟基丁酸酯）（PHB）颗粒被主要由相蛋白和其他PHB颗粒相关蛋白（PGAP）组成的表面层所覆盖。相蛋白是小的两亲性蛋白质，其决定积聚的PHB颗粒的数量和大小。真养罗尔斯通氏菌已知有五种相蛋白（PhaP 1至PhaP 5）。在这项研究中，我们通过检查R.具有“相蛋白2基序”的序列的真养生物基因组。为了确定相应的蛋白质是否代表真正的PGAP，构建了与eYFP（增强型黄色荧光蛋白）的融合体。还制备了eYFP与PhaPl至PhaP 5的类似融合物以及与PHB合酶（PhaCl）、无活性PhaCl变体（PhaCl-C319 A）和PhaC 2的融合物。在野生型和PHB阴性背景中研究所有融合。发现所有PhaC变体和PhaP 1至PhaP 5与PHB颗粒共定位。此外，具有H16_B1988和H16_B2326的eYFP融合物与PHB共定位。然而，H16_B2296与eYFP的融合物不与PHB颗粒共定位，但与类核区域共定位。值得注意的是，所有融合体（除了H16_B2296）都可溶于Delta phaCl菌株。这些数据证实H16_B1988和H16_B2326而不是H16_B2296编码真正的PGAP，我们建议将其命名为PhaP 6（H16_B1988）和PhaP 7（H16_B2326）。当本地化的相蛋白进行了研究，在不同阶段的聚羟基丁酸积累，PhaP 6和PhaP 7的融合是可溶的，在第一个3小时的条件下，聚羟基丁酸允许的，虽然聚羟基丁酸颗粒出现后10分钟。在以后的时间点，融合共定位与聚羟基丁酸。值得注意的是，表达eYFP与PhaP 5、PhaP 6或PhaP 7融合的菌株的PHB颗粒主要位于细胞极附近或未来隔膜形成的区域中。在其他PGAP（PhaP 1至PhaP 4、PhaC 1、PhaC 1-C319 A和PhaC 2）中未观察到这种现象，表明某些相位蛋白可能具有额外的功能。phaP 6或phaP 7的染色体缺失对PHB颗粒的形成没有可见的影响。
Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with "phasin 2 motifs." To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a Delta phaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.