Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16
Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16
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DOI：
10.1128/aem.02279-15
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发表时间：
2015-12-01
影响因子：
4.4
通讯作者：
Jendrossek, Dieter
中科院分区：
文献类型：
--
作者：
Tumlirsch, Tony;Sznajder, Anna;Jendrossek, Dieter
通过双杂交分析鉴定了一个与聚3-羟基丁酸酯(PHB)解聚酶PhaZa1和PHB颗粒相关Phasin蛋白PhaP2相互作用的蛋白(Phax)。在营养肉汤-葡萄糖酸盐培养中，phax的缺失导致聚磷酸盐(Polyp)颗粒形成水平的增加和PHB利用的损害。开发了一种从细胞提取液中浓缩息肉颗粒的方法。通过蛋白质组学分析，在息肉颗粒组分中鉴定出27种在其他细胞组分中缺失的蛋白质。一种蛋白(A2437)含有I型多聚磷酸酶(PPK1)基序，两种蛋白(A1212、A1271)具有PPK2基序。通过增强型黄色荧光蛋白(EYFP)与三种多聚磷酸酶(PPKs)的C端和N端融合表达，证实了在体内与息肉颗粒的共定位。对具有PPK基序的其他蛋白质的基因组DNA序列进行筛选，发现一个具有ppk1基序的蛋白质和三个具有PPK2基序的蛋白质。四个新的PPK候选蛋白与EYFP的C-端和N-端融合的构建和随后的表达表明，只有1979(PPK2基序)与息肉颗粒共定位。除息肉外，其余3种蛋白在细胞极点附近形成荧光斑(A0997、B1019)或可溶性蛋白(A0226)。在大肠杆菌Delta PPK背景中表达了Ralstonia eutroa PPK(Reu)基因，构建了一组单染色体和多染色体缺失，发现A2437(PPK1a)和A1212(PPK2c)都参与了息肉颗粒的形成。缺失这两个基因的突变体不能产生息肉颗粒。在不同的染色体背景下，研究了PHB和息肉颗粒的形成和利用。
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 Delta 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.