A repressor protein, PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct tnteraction with PHA
A repressor protein, PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct tnteraction with PHA
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DOI：
10.1128/jb.184.14.3992-4002.2002
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发表时间：
2002-07-01
影响因子：
3.2
通讯作者：
Doi, Y
中科院分区：
文献类型：
--
作者：
Maehara, A;Taguchi, S;Doi, Y
Phasins（PhaP）主要是聚羟基链烷酸酯（PHA）颗粒相关蛋白，其积极影响PHA合成。最近，我们报道了phaR基因，这是位于phaP的下游在Paracoccus pacificans，编码负调控参与PhaP的表达。在这项研究中，DNase I足迹显示PhaR特异性结合位于phaP和phaR的上游的两个区域，表明PhaR在调节phaP表达以及自身调节中起作用。在PhaR识别的上游元件中发现了许多富含TGC的序列。重组大肠杆菌粗裂解物中的PhaR能够特异性地与聚[（R）-3-羟基丁酸酯] [P（3 HB）]颗粒再结合。此外，人工P（3 HB）颗粒和3 HB寡聚体引起PhaR从PhaR-DNA复合物中解离，而天然PHA颗粒被PhaP或其他非特异性蛋白质覆盖，不引起解离。这些结果表明，PhaR能够通过直接结合PHA来感知PHA合成的开始和颗粒的增大。然而，游离PhaR可能无法感知已经被PhaP和/或其他蛋白质充分覆盖的成熟PHA颗粒。体外表达实验表明，phaP的表达被抑制的PhaR的加入和被解抑制的P（3 HB）的加入。基于这些发现，我们在这里提出了一个可能的模型占PhaR介导的PHA合成机制。PhaR同源物在短链PHA生产细菌中的广泛分布表明PhaR介导的PHA合成调控具有共同而重要的作用。
Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA, synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.