Li Jie, Zheng Xin, Guo Xiaopeng, Qi Lei, Dong Xiuzhu
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences, Beijing, China.
PLoS One. 2014 Apr 18;9(4):e95502. doi: 10.1371/journal.pone.0095502. eCollection 2014.
Two-component signal transduction systems (TCSs) are a major mechanism used by bacteria in response to environmental changes. Although many sequenced archaeal genomes encode TCSs, they remain poorly understood. Previously, we reported that a methanogenic archaeon, Methanosaeta harundinacea, encodes FilI, which synthesizes carboxyl-acyl homoserine lactones, to regulate transitions of cellular morphology and carbon metabolic fluxes. Here, we report that filI, the cotranscribed filR2, and the adjacent filR1 constitute an archaeal TCS. FilI possesses a cytoplasmic kinase domain (histidine kinase A and histidine kinase-like ATPase) and its cognate response regulator. FilR1 carries a receiver (REC) domain coupled with an ArsR-related domain with potential DNA-binding ability, while FilR2 carries only a REC domain. In a phosphorelay assay, FilI was autophosphorylated and specifically transferred the phosphoryl group to FilR1 and FilR2, confirming that the three formed a cognate TCS. Through chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) using an anti-FilR1 antibody, FilR1 was shown to form in vivo associations with its own promoter and the promoter of the filI-filR2 operon, demonstrating a regulatory pattern common among TCSs. ChIP-qPCR also detected FilR1 associations with key genes involved in acetoclastic methanogenesis, acs4 and acs1. Electrophoretic mobility shift assays confirmed the in vitro tight binding of FilR1 to its own promoter and those of filI-filR2, acs4, and mtrABC. This also proves the DNA-binding ability of the ArsR-related domain, which is found primarily in Archaea. The archaeal promoters of acs4, filI, acs1, and mtrABC also initiated FilR1-modulated expression in an Escherichia coli lux reporter system, suggesting that FilR1 can up-regulate both archaeal and bacterial transcription. In conclusion, this work identifies an archaeal FilI/FilRs TCS that regulates the methanogenesis of M. harundinacea.
双组分信号转导系统（TCSs）是细菌用于响应环境变化的主要机制。尽管许多已测序的古菌基因组编码TCSs，但人们对它们的了解仍然很少。此前，我们报道产甲烷古菌哈尔氏甲烷八叠球菌编码合成羧基酰基高丝氨酸内酯的FilI，以调节细胞形态转变和碳代谢通量。在此，我们报道filI、共转录的filR2和相邻的filR1构成一个古菌TCS。FilI具有一个细胞质激酶结构域（组氨酸激酶A和组氨酸激酶样ATP酶）及其同源应答调节因子。FilR1带有一个接收器（REC）结构域，与具有潜在DNA结合能力的ArsR相关结构域相连，而FilR2仅带有一个REC结构域。在磷酸传递分析中，FilI发生自磷酸化，并将磷酸基团特异性转移至FilR1和FilR2，证实这三者形成了一个同源TCS。通过使用抗FilR1抗体进行染色质免疫沉淀-定量聚合酶链反应（ChIP-qPCR），结果显示FilR1在体内与其自身启动子以及filI-filR2操纵子的启动子形成关联，这证明了TCSs中常见的一种调控模式。ChIP-qPCR还检测到FilR1与参与乙酸裂解产甲烷作用的关键基因acs4和acs1存在关联。电泳迁移率变动分析证实了FilR1在体外与其自身启动子以及filI-filR2、acs4和mtrABC的启动子紧密结合。这也证明了主要存在于古菌中的ArsR相关结构域的DNA结合能力。acs4、filI、acs1和mtrABC的古菌启动子在大肠杆菌荧光素酶报告系统中也启动了FilR1调节的表达，这表明FilR1可以上调古菌和细菌的转录。总之，这项工作鉴定出一个调节哈尔氏甲烷八叠球菌产甲烷作用的古菌FilI/FilRs TCS。
