Regulation of replication enzymes by metabolic enzymes in B. subtilis

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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.