Regulation of replication enzymes by metabolic enzymes in B. subtilis

所有生物都从环境中提取能量和前体，为生物合成和生物质生产提供燃料。自60年代以来，有充分的证据表明，降解和生物合成是紧密协调的，以优化细胞健康。尽管对基础、医学和生物技术科学至关重要，但这些无处不在的全球监管系统的机制仍然是一个谜。我在这项研究中的合作者Laurent Janniere进行了实验，首次揭示了分解营养物质的细胞系统的反应之间的联系，这种反应称为中央碳代谢(CCM)，它的调节器与DNA复制之间的联系。虽然我们都知道CCM从营养物质中提取的能量以某种方式促进生长，而且生长必须与DNA复制和细胞分裂有关才能产生后代细胞，但我们仍然不确定营养物质一端与DNA复制之间的这种线性联系是如何保持的。例如，是什么信号告诉细胞营养丰富，条件有利于DNA复制？本研究的目的是在分子水平上了解这些信号是什么。CCM涉及大约30个关键反应，这些反应分为糖酵解、糖异生、磷酸戊糖途径、三羧酸循环和溢出途径，这些途径构成了营养物质代谢的主要途径。这些途径受到转录因子和辅因子的严格调控，这些转录因子和辅因子动态地感知细胞的代谢状态，通过调节CCM活性来优化一系列营养物质中的能量回收。通过直接感知供需，CCM及其调节器处于战略地位，可以产生信号，使主要细胞活动适应营养丰富。由于CCM决定簇(蛋白质和代谢物)是高度保守的，并且CCM活性普遍受调节器的控制，因此可以推测，在所有活体中观察到的复制的代谢控制可能涉及与在枯草杆菌中检测到的信号系统有关的信号系统。使用不同的方法，我们发现糖酵解的末端反应和下游反应一方面由丙酮酸脱氢酶和溢出途径进行，另一方面调节CCM活性，在富媒体中对于维持营养和复制之间的通信线路是至关重要的。我们还发现，这些CCM决定因素通过多个相互缠绕的链接来调节复制的起始和延伸阶段，这些链接的主要复制目标是通用起始蛋白Dna A和三种复制酶：合成新DNA的聚合酶Dna E，形成启动DNA合成的引物的引物Dna G，以及分离亲本DNA链以揭示要复制到新DNA中的序列的解旋酶Dna C。我的实验室发现，DNAE、DNAG和DNAC在物理上相互作用，并调节彼此的活动，支持这样一种观点，即它们形成一个独特的亚复合体，专门作用于其中一条复制链，即滞后链。我们进一步表明DNAE在滞后链合成中发挥着重要作用。这些数据已经公布。根据我们的集体发现，我们假设糖酵解和下游反应的较低部分以及CCM调节器形成了一个代谢中枢，该中枢感知细胞的新陈代谢状态，并向DNA复制机器的启动和延伸阶段发送信号，以调节相对于从营养物质中提取的能量的复制速度。尽管我们已经确定了这一通讯途径中的主要蛋白质角色，但这些蛋白质如何相互通信的实际机制仍然是一个谜。我们提出了一系列实验来从分子水平上了解枯草杆菌的这些机制。

All organisms extract energy and precursors from the environment to fuel biosynthesis and biomass production. Since the 60s, it is well documented that degradation and biosynthesis are tightly coordinated for optimizing cell fitness. Although of paramount importance for the fundamental, medical and biotechnology sciences, the mechanism of these ubiquitous, global regulatory systems remains a mystery. My collaborator in this research Laurent Janniere has carried out experiments which revealed for the first time links between reactions of the cellular system that breaks down nutrients, called the central carbon metabolism (CCM), its regulators and DNA replication. Although we all understand that somehow the energy extracted from nutrients by CCM fuels growth and that growth must be related to DNA replication and cell division to produce progeny cells, we are still not sure how this linear link between nutrients at one end and DNA replication at the other end is maintained. For example, what are the signals that tell cells that nutrients are abundant and conditions are favourable for DNA replication? The aim of this research is to understand what these signals are at the molecular level.CCM involves about 30 key reactions grouped in pathways of which glycolysis, gluconeogenesis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle and the overflow pathway form the main routes for metabolizing nutrients. These pathways are tightly regulated by transcription factors and cofactors that dynamically sense the metabolic status of the cell for optimizing energy recovery in a range of nutrients by regulating CCM activity. By directly sensing the supply and demand, CCM and its regulators are at a strategic position for producing signals for adapting main cellular activities to nutrient richness. As CCM determinants (proteins and metabolites) are highly conserved and as CCM activity is ubiquitously under the control of regulators, one can speculate that the metabolic control of replication observed in all living organisms may involve signaling systems related to that detected in B. subtilis. Using different approaches, we found that terminal reactions of glycolysis and downstream reactions carried out by the pyruvate dehydrogenase and the overflow pathway on one hand, and regulators of CCM activity on the other hand, are of prime importance in rich media to maintain the communication lines between nutrients and replication. We also showed that these CCM determinants modulate the initiation and elongation phase of replication via multiple, and intertwined links and that the main replication targets of these links are the universal initiation protein DnaA and three replication enzymes: the polymerase DnaE that synthesizes new DNA, primase DnaG that forms the primers to initiate DNA synthesis and helicase DnaC that separates the parental DNA strands to reveal the sequences to be copied into new DNA. My lab discovered that DnaE, DnaG and DnaC physically interact and modulate each others' activities supporting the notion that they form a distinct subcomplex acting specifically on one of the replicating strands known as the lagging strand. We further showed that DnaE plays a major role in the lagging strand synthesis. These data have been published. As a result of our collective findings we hypothesize that the lower part of glycolysis and downstream reactions as well as CCM regulators form a metabolic hub that sense the cell's metabolic status and send signals to the initiation and elongation phase of DNA replication machineries for modulating the rate of replication with respect to the energy extracted form nutrients.Although we have identified main protein players in this communication pathway, the actual mechanisms of how these proteins communicate with each other are still a mystery. We propose a series of experiments to understand these mechanisms at the molecular level in B. subtilis.