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首页> 外文期刊>Applied Microbiology >Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16
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Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16

机译：富营养小球藻H16菌株中多磷酸盐激酶形成多磷酸盐及其与聚3-羟基丁酸积累的关系

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A protein (PhaX) that interacted with poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 and with PHB granule-associated phasin protein PhaP2 was identified by two-hybrid analysis. Deletion of phaX resulted in an increase in the level of polyphosphate (polyP) granule formation and in impairment of PHB utilization in nutrient broth-gluconate cultures. A procedure for enrichment of polyP granules from cell extracts was developed. Twenty-seven proteins that were absent in other cell fractions were identified in the polyP granule fraction by proteome analysis. One protein (A2437) harbored motifs characteristic of type 1 polyphosphate kinases (PPK1s), and two proteins (A1212, A1271) had PPK2 motifs. In vivo colocalization with polyP granules was confirmed by expression of C- and N-terminal fusions of enhanced yellow fluorescent protein (eYFP) with the three polyphosphate kinases (PPKs). Screening of the genome DNA sequence for additional proteins with PPK motifs revealed one protein with PPK1 motifs and three proteins with PPK2 motifs. Construction and subsequent expression of C- and N-terminal fusions of the four new PPK candidates with eYFP showed that only A1979 (PPK2 motif) colocalized with polyP granules. The other three proteins formed fluorescent foci near the cell pole (apart from polyP) (A0997, B1019) or were soluble (A0226). Expression of the Ralstonia eutropha ppk ( ppk_(Reu) ) genes in an Escherichia coli Δ ppk background and construction of a set of single and multiple chromosomal deletions revealed that both A2437 (PPK1a) and A1212 (PPK2c) contributed to polyP granule formation. Mutants with deletion of both genes were unable to produce polyP granules. The formation and utilization of PHB and polyP granules were investigated in different chromosomal backgrounds.
机译：通过两杂交分析鉴定了与聚（3-羟基丁酸）（PHB）解聚酶PhaZa1和与PHB颗粒相关的相蛋白PhaP2相互作用的蛋白（PhaX）。删除phaX会导致营养肉汤-葡萄糖酸盐培养物中多磷酸盐（polyP）颗粒形成水平的增加，并损害PHB的利用。开发了一种从细胞提取物中富集polyP颗粒的方法。通过蛋白质组分析，在polyP颗粒级分中鉴定出其他细胞级分中不存在的27种蛋白质。一种蛋白质（A2437）带有1型多磷酸激酶（PPK1s）的特征基序，而两种蛋白质（A1212，A1271）具有PPK2基序。通过表达增强型黄色荧光蛋白（eYFP）与三种多磷酸激酶（PPK）的C和N端融合蛋白，证实了与polyP颗粒的体内共定位。对基因组DNA序列进行筛选，寻找具有PPK基序的其他蛋白质，发现一种具有PPK1基序的蛋白质和三种具有PPK2基序的蛋白质。四种新的PPK候选物与eYFP的C和N末端融合体的构建和随后的表达表明，只有A1979（PPK2母题）与polyP颗粒共定位。其他三种蛋白质在细胞极附近形成荧光病灶（除polyP以外）（A0997，B1019）或可溶（A0226）。 Ralstonia eutropha ppk（ppk_（Reu））基因在大肠杆菌Δppk背景中的表达以及一组单个和多个染色体缺失的构建表明A2437（PPK1a）和A1212（PPK2c）都有助于polyP颗粒的形成。具有两个基因缺失的突变体不能产生polyP颗粒。在不同的染色体背景下研究了PHB和polyP颗粒的形成和利用。

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