Abstract：
To cope with a high-salinity environment, haloarchaea generally employ the twin-arginine translocation (Tat) pathway to transport secretory proteins across the cytoplasm membrane in a folded state, including Tat-dependent extracellular subtilases (halolysins) capable of autocatalytic activation. Some halolysins, such as SptA of Natrinema gari J7-2, are produced at late-log phase to prevent premature enzyme activation and proteolytic damage of cellular proteins in haloarchaea; however, the regulation mechanism for growth phase-dependent expression of halolysins remains largely unknown. In this study, a DNA-protein pull-down assay was performed to identify the proteins binding to the 5\'-flanking sequence of sptA encoding halolysin SptA in strain J7-2, revealing a TrmBL2-like transcription factor (NgTrmBL2). The ΔtrmBL2 mutant of strain J7-2 showed a sharp decrease in the production of SptA, suggesting that NgTrmBL2 positively regulates sptA expression. The purified recombinant NgTrmBL2 mainly existed as a dimer although monomeric and higher-order oligomeric forms were detected by native-PAGE analysis. The results of electrophoretic mobility shift assays (EMSAs) showed that NgTrmBL2 binds to the 5\'-flanking sequence of sptA in a non-specific and concentration-dependent manner and exhibits an increased DNA-binding affinity with the increase in KCl concentration. Moreover, we found that a distal cis-regulatory element embedded in the neighboring upstream gene negatively regulates trmBL2 expression and thus participates in the growth phase-dependent biosynthesis of halolysin SptA.
OBJECTIVE: Extracellular proteases play important roles in nutrient metabolism, processing of functional proteins, and antagonism of haloarchaea, but no transcription factor involved in regulating the expression of haloaechaeal extracellular protease has been reported yet. Here we report that a TrmBL2-like transcription factor (NgTrmBL2) mediates the growth phase-dependent expression of an extracellular protease, halolysin SptA, of haloarchaeon Natrinema gari J7-2. In contrast to its hyperthermophilic archaeal homologs, which are generally considered to be global transcription repressors, NgTrmBL2 functions as a positive regulator for sptA expression. This study provides new clues about the transcriptional regulation mechanism of extracellular protease in haloarchaea and the functional diversity of archaeal TrmBL2.
OBJECTIVE: Extracellular proteases play important roles in nutrient metabolism, processing of functional proteins, and antagonism of haloarchaea, but no transcription factor involved in regulating the expression of haloaechaeal extracellular protease has been reported yet. Here we report that a TrmBL2-like transcription factor (NgTrmBL2) mediates the growth phase-dependent expression of an extracellular protease, halolysin SptA, of haloarchaeon Natrinema gari J7-2. In contrast to its hyperthermophilic archaeal homologs, which are generally considered to be global transcription repressors, NgTrmBL2 functions as a positive regulator for sptA expression. This study provides new clues about the transcriptional regulation mechanism of extracellular protease in haloarchaea and the functional diversity of archaeal TrmBL2.
摘要：
为了应对高盐度环境，卤代古菌通常采用双精氨酸易位（Tat）途径以折叠状态跨细胞质膜运输分泌蛋白，包括能够自动催化活化的Tat依赖性细胞外枯草杆菌酶(卤代溶素)。一些卤溶素，例如NatrinemagariJ7-2的SptA，是在对数后期产生的，以防止卤代古菌中细胞蛋白的过早酶激活和蛋白水解损伤；但是，生长阶段依赖的卤溶素表达的调节机制仍然未知。在这项研究中,进行DNA-蛋白质下拉测定以鉴定与菌株J7-2中编码卤溶素SptA的sptA的5'侧翼序列结合的蛋白质，揭示了TrmBL2样转录因子（NgTrmBL2）。菌株J7-2的ΔtrmBL2突变体显示SptA的产量急剧下降，表明NgTrmBL2正调控sptA表达。纯化的重组NgTrmBL2主要以二聚体形式存在，尽管通过天然PAGE分析检测到单体和高级寡聚形式。电泳迁移率变化测定（EMSAs）的结果表明，NgTrmBL2以非特异性和浓度依赖性方式与sptA的5'侧翼序列结合，并随着KCl浓度的增加而表现出增加的DNA结合亲和力。此外，我们发现嵌入相邻上游基因中的远端顺式调节元件负调节trmBL2的表达，从而参与了依赖生长期的卤代溶素SptA的生物合成。
目的：胞外蛋白酶在营养代谢中发挥重要作用，功能性蛋白质的加工，和卤代菌的拮抗作用，但是尚未报道过参与调节细胞外蛋白酶表达的转录因子。在这里，我们报道了TrmBL2样转录因子（NgTrmBL2）介导了细胞外蛋白酶的生长阶段依赖性表达，halolysinSptA,haloarchaeonNatrinemagariJ7-2。与其超嗜热古细菌同源物相反，通常被认为是全局转录抑制因子，NgTrmBL2充当sptA表达的正调节因子。本研究为盐生古细菌胞外蛋白酶的转录调控机制和古细菌TrmBL2的功能多样性提供了新的线索。
目的：胞外蛋白酶在营养代谢中发挥重要作用，功能性蛋白质的加工，和卤代菌的拮抗作用，但是尚未报道过参与调节细胞外蛋白酶表达的转录因子。在这里，我们报道了TrmBL2样转录因子（NgTrmBL2）介导了细胞外蛋白酶的生长阶段依赖性表达，halolysinSptA,haloarchaeonNatrinemagariJ7-2。与其超嗜热古细菌同源物相反，通常被认为是全局转录抑制因子，NgTrmBL2充当sptA表达的正调节因子。本研究为盐生古细菌胞外蛋白酶的转录调控机制和古细菌TrmBL2的功能多样性提供了新的线索。
