Lawrence Adam G, Schoenheit Joerg, He Aimin, Tian Jiamin, Liu Pinghua, Stubbe Joanne, Sinskey Anthony J
Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, USA.
Appl Microbiol Biotechnol. 2005 Sep;68(5):663-72. doi: 10.1007/s00253-005-1969-3. Epub 2005 Oct 26.
Poly-(R)-3-hydroxybutyrate (PHB) homeostasis in Ralstonia eutropha takes place at the interface of the cytosol and the hydrophobic PHB granule. PHB synthesis and degradation are therefore intimately linked to the process of granule assembly and breakdown. Unraveling this time-dependent three-dimensional process requires an understanding of the kinetics of synthesis of relevant proteins. Reverse transcriptase quantitative PCR and quantitative Western blotting were carried out on batch cultures of R. eutropha H16 in order to gain insight into how expression of the PHB-related genes phaA, phaB, phaC, phaP, phaR, phaZ1a, phaZ1b, and phaZ1c changed during a cell growth phase, a PHB production phase, and a PHB utilization phase. phaA, phaB, phaC, phaR, and phaZ1a were transcribed throughout cell growth, PHB production, and PHB degradation. PHB-mediated induction of PhaP expression was shown to occur at the transcriptional level, with transcript levels increasing during PHB production and decreasing during PHB utilization. Levels of PhaP correlated strongly with levels of PHB. Levels of phaZ1b transcript and protein increased sharply during production and decreased during degradation, but transcript accumulation did not depend on PHB production as in the case of phaP. No evidence of phaZ1c expression was found under the experimental conditions used in this study.
在真养产碱杆菌中，聚（R）-3-羟基丁酸酯（PHB）的稳态发生在细胞质溶胶与疏水性PHB颗粒的界面处。因此，PHB的合成和降解与颗粒组装和分解过程密切相关。要弄清楚这个随时间变化的三维过程，需要了解相关蛋白质的合成动力学。对真养产碱杆菌H16的分批培养物进行了逆转录酶定量PCR和定量蛋白质免疫印迹分析，以便深入了解PHB相关基因phaA、phaB、phaC、phaP、phaR、phaZ1a、phaZ1b和phaZ1c在细胞生长阶段、PHB产生阶段和PHB利用阶段的表达变化情况。phaA、phaB、phaC、phaR和phaZ1a在整个细胞生长、PHB产生和PHB降解过程中均有转录。结果表明，PHB介导的PhaP表达诱导发生在转录水平，转录水平在PHB产生期间增加，在PHB利用期间降低。PhaP的水平与PHB的水平密切相关。phaZ1b转录本和蛋白质的水平在产生期间急剧增加，在降解期间降低，但转录本积累不像phaP那样依赖于PHB的产生。在本研究使用的实验条件下，未发现phaZ1c表达的证据。
