Cai Shuangfeng, Cai Lei, Zhao Dahe, Liu Guiming, Han Jing, Zhou Jian, Xiang Hua
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China University of Chinese Academy of Sciences, Beijing, People's Republic of China.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, People's Republic of China.
Appl Environ Microbiol. 2015 Jan;81(1):373-85. doi: 10.1128/AEM.02878-14. Epub 2014 Oct 24.
Polyhydroxyalkanoates (PHAs) are synthesized and assembled as PHA granules that undergo well-regulated formation in many microorganisms. However, this regulation remains unclear in haloarchaea. In this study, we identified a PHA granule-associated regulator (PhaR) that negatively regulates the expression of both its own gene and the granule structural gene phaP in the same operon (phaRP) in Haloferax mediterranei. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated a significant interaction between PhaR and the phaRP promoter in vivo. Scanning mutagenesis of the phaRP promoter revealed a specific cis-element as the possible binding position of the PhaR. The haloarchaeal homologs of the PhaR contain a novel conserved domain that belongs to a swapped-hairpin barrel fold family found in AbrB-like proteins. Amino acid substitution indicated that this AbrB-like domain is critical for the repression activity of PhaR. In addition, the phaRP promoter had a weaker activity in the PHA-negative strains, implying a function of the PHA granules in titration of the PhaR. Moreover, the H. mediterranei strain lacking phaR was deficient in PHA accumulation and produced granules with irregular shapes. Interestingly, the PhaR itself can promote PHA synthesis and granule formation in a PhaP-independent manner. Collectively, our results demonstrated that the haloarchaeal PhaR is a novel bifunctional protein that plays the central role in the regulation of PHA accumulation and granule formation in H. mediterranei.
聚羟基脂肪酸酯（PHA）作为PHA颗粒在许多微生物中合成并组装，其形成过程受到良好调控。然而，在嗜盐古菌中这种调控机制仍不清楚。在本研究中，我们鉴定出一种PHA颗粒相关调节因子（PhaR），它对地中海嗜盐嗜盐菌同一操纵子（phaRP）中自身基因和颗粒结构基因phaP的表达均起负调控作用。染色质免疫沉淀-定量PCR（ChIP-qPCR）分析表明，PhaR与phaRP启动子在体内存在显著相互作用。对phaRP启动子进行扫描诱变，发现一个特定的顺式元件可能是PhaR的结合位点。PhaR的嗜盐古菌同源物包含一个新的保守结构域，属于在AbrB样蛋白中发现的交换发夹桶折叠家族。氨基酸替换表明，这个AbrB样结构域对PhaR的抑制活性至关重要。此外，phaRP启动子在PHA阴性菌株中的活性较弱，这意味着PHA颗粒在滴定PhaR中发挥作用。此外，缺失phaR的地中海嗜盐菌菌株PHA积累不足，产生的颗粒形状不规则。有趣的是，PhaR本身可以以不依赖PhaP的方式促进PHA合成和颗粒形成。总的来说，我们的结果表明，嗜盐古菌PhaR是一种新型双功能蛋白，在地中海嗜盐菌PHA积累和颗粒形成的调控中起核心作用。
