Kanai Tamotsu, Akerboom Jasper, Takedomi Shogo, van de Werken Harmen J G, Blombach Fabian, van der Oost John, Murakami Taira, Atomi Haruyuki, Imanaka Tadayuki
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands.
J Biol Chem. 2007 Nov 16;282(46):33659-33670. doi: 10.1074/jbc.M703424200. Epub 2007 Sep 17.
We identified a novel regulator, Thermococcales glycolytic regulator (Tgr), functioning as both an activator and a repressor of transcription in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Tgr (TK1769) displays similarity (28% identical) to Pyrococcus furiosus TrmB (PF1743), a transcriptional repressor regulating the trehalose/maltose ATP-binding cassette transporter genes, but is more closely related (67%) to a TrmB paralog in P. furiosus (PF0124). Growth of a tgr disruption strain (Deltatgr) displayed a significant decrease in growth rate under gluconeogenic conditions compared with the wild-type strain, whereas comparable growth rates were observed under glycolytic conditions. A whole genome microarray analysis revealed that transcript levels of almost all genes related to glycolysis and maltodextrin metabolism were at relatively high levels in the Deltatgr mutant even under gluconeogenic conditions. The Deltatgr mutant also displayed defects in the transcriptional activation of gluconeogenic genes under these conditions, indicating that Tgr functions as both an activator and a repressor. Genes regulated by Tgr contain a previously identified sequence motif, the Thermococcales glycolytic motif (TGM). The TGM was positioned upstream of the Transcription factor B-responsive element (BRE)/TATA sequence in gluconeogenic promoters and downstream of it in glycolytic promoters. Electrophoretic mobility shift assay indicated that recombinant Tgr protein specifically binds to promoter regions containing a TGM. Tgr was released from the DNA when maltotriose was added, suggesting that this sugar is most likely the physiological effector. Our results strongly suggest that Tgr is a global transcriptional regulator that simultaneously controls, in response to sugar availability, both glycolytic and gluconeogenic metabolism in T. kodakaraensis via its direct binding to the TGM.
我们鉴定出一种新型调节因子——嗜热栖热菌糖酵解调节因子（Tgr），它在嗜热古菌柯达嗜热栖热菌KOD1中兼具转录激活因子和转录抑制因子的功能。Tgr（TK1769）与激烈火球菌TrmB（PF1743）具有相似性（28%的序列相同），TrmB是一种转录抑制因子，调控海藻糖/麦芽糖ATP结合盒转运蛋白基因，但Tgr与激烈火球菌中的TrmB旁系同源物（PF0124）关系更为密切（67%）。与野生型菌株相比，tgr缺失菌株（Δtgr）在糖异生条件下的生长速率显著降低，而在糖酵解条件下观察到的生长速率相当。全基因组微阵列分析表明，即使在糖异生条件下，Δtgr突变体中几乎所有与糖酵解和麦芽糊精代谢相关的基因转录水平都相对较高。在这些条件下，Δtgr突变体在糖异生基因的转录激活方面也存在缺陷，表明Tgr兼具激活因子和抑制因子的功能。受Tgr调控的基因包含一个先前鉴定的序列基序——嗜热栖热菌糖酵解基序（TGM）。TGM位于糖异生启动子中转录因子B反应元件（BRE）/TATA序列的上游，而在糖酵解启动子中位于其下游。电泳迁移率变动分析表明，重组Tgr蛋白特异性结合含有TGM的启动子区域。当添加麦芽三糖时，Tgr从DNA上释放，这表明这种糖很可能是生理效应物。我们的结果有力地表明，Tgr是一种全局转录调节因子，通过直接结合TGM，响应糖的可利用性，同时控制柯达嗜热栖热菌中的糖酵解和糖异生代谢。
