Regulation of replication enzymes by metabolic enzymes in B. subtilis

枯草芽孢杆菌中代谢酶对复制酶的调节

• 批准号: BB/R013357/1
• 负责人: Panos Soultanas
• 金额: $ 51.46万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR013357%2F1
• 关键词: Regulation; replication; enzymes; metabolic; B.

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.

所有生物都从环境中提取能量和前体，以促进生物合成和生物质生产。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，以优化细胞适应性。虽然对基础科学、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反应之间的联系，称为中央碳代谢（CCM），其调节剂和DNA复制。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，并且生长必须与DNA复制和细胞分裂有关以产生后代细胞，但我们仍然不确定一端的营养物质和另一端的DNA复制之间的线性联系是如何维持的。例如，是什么信号告诉细胞营养丰富，条件有利于DNA复制？CCM涉及约30个关键反应，其中糖酵解、糖异生、磷酸戊糖途径、三羧酸循环和溢流途径是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节，这些转录因子和辅因子动态地感测细胞的代谢状态，以通过调节CCM活性来优化一系列营养物质中的能量回收。通过直接感知供应和需求，CCM及其调节剂处于产生信号的战略地位，使主要细胞活动适应营养丰富度。由于CCM决定簇（蛋白质和代谢物）高度保守，并且CCM活性普遍受调节因子控制，因此可以推测，在所有活生物体中观察到的复制代谢控制可能涉及与B中检测到的信号传导系统相关的信号传导系统。枯草芽孢杆菌使用不同的方法，我们发现，糖酵解的终端反应和下游反应进行的丙酮酸脱氢酶和溢流途径，另一方面，CCM活性的调节剂，是最重要的丰富的媒体，以保持营养和复制之间的通信线路。我们还表明，这些CCM决定因素通过多个相互交织的链接调节复制的起始和延伸阶段，这些链接的主要复制靶点是通用起始蛋白DnaA和三种复制酶：合成新DNA的聚合酶DnaE，引物酶DnaG形成引物以启动DNA合成，解旋酶DnaC分离亲本DNA链以揭示待合成的序列。复制到新的DNA中。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，这支持了这样一种观点，即它们形成了一种独特的亚复合物，专门作用于一条复制链，称为滞后链。我们进一步表明，DnaE在滞后链合成中起着重要作用。这些数据已经公布。由于我们的集体研究结果，我们假设糖酵解和下游反应的下部以及CCM调节剂形成一个代谢中心，该中心感知细胞的代谢状态，并向DNA复制机制的起始和延伸阶段发送信号，以调节从营养物中提取的能量的复制速率。这些蛋白质如何相互交流的实际机制仍然是一个谜。我们提出了一系列的实验，以了解这些机制在分子水平上的B。枯草杆菌。

项目成果

DNA replication initiation in Bacillus subtilis ; Structural and functional characterisation of the essential DnaA-DnaD interaction

枯草芽孢杆菌中 DNA 复制起始；

• DOI: 10.1101/444885
• 发表时间: 2018
• 作者: Martin E

The metabolic control of DNA replication: mechanism and function.

• DOI: 10.1098/rsob.230220
• 发表时间: 2023-08
• 期刊: OPEN BIOLOGY
• 影响因子: 5.8
• 作者: Soultanas, Panos;Janniere, Laurent
• 通讯作者: Janniere, Laurent

Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication.

• DOI: 10.1186/s12915-022-01278-3
• 发表时间: 2022-04-13
• 期刊: BMC BIOLOGY
• 影响因子: 5.4
• 作者: Horemans, Steff;Pitoulias, Matthaios;Holland, Alexandria;Pateau, Emilie;Lechaplais, Christophe;Ekaterina, Dariy;Perret, Alain;Soultanas, Panos;Janniere, Laurent
• 通讯作者: Janniere, Laurent

SirA inhibits the essential DnaA:DnaD interaction to block helicase recruitment during Bacillus subtilis sporulation

SirA 抑制必需的 DnaA:DnaD 相互作用，以阻止枯草芽孢杆菌孢子形成过程中解旋酶的招募

• DOI: 10.1101/2022.04.18.488658
• 发表时间: 2022
• 作者: Winterhalter C

Ferric quinate (QPLEX) interacts with the major outer membrane protein (MOMP) of Campylobacter jejuni and enters through the porin channel into the periplasmic space.

• DOI: 10.1016/j.csbj.2022.09.032
• 发表时间: 2022
• 期刊: COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL
• 影响因子: 6
• 作者: Okoye, Jennifer C.;Bellamy-Carter, Jeddidiah;Oldham, Neil J.;Oldfield, Neil J.;Mahdavi, Jafar;Soultanas, Panos
• 通讯作者: Soultanas, Panos