Regulation of replication enzymes by metabolic enzymes in B. subtilis

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.

It is well established that DNA replication and growth in uni- and multi-cellular organisms is responsive to nutrient availability. In most organisms, DNA replication occurs in a specific window of time during the cell cycle, and this temporal compartmentalization of replication is under the control of growth rate and linked to nutrient availability. The way DNA replication is linked to nutrient availability and the central carbon metabolism is of fundamental and medical importance, but the precise molecular mechanism(s) underpinning this link remains largely unknown.
Work in Laurent Janniere's lab (collaborator in this project) revealed a strong genetic link between glycolysis and DNA replication in the model gram positive bacterium Bacillus subtilis. The second part of glycolysis, known as the C-3 (carbon-3), converts D-glyceraldehyde-3-phospate to pyruvate through a 5-step series of metabolic reactions catalyzed sequentially by 5 enzymes GapA, Pgk, Pgm, Eno and PykA. During a genetic search for suppressors of thermosensitive DNA replication mutants spontaneous suppressive mutations were identified in genes coding for these 5 enzymes. Further detailed characterization of these mutants revealed that their suppressive effects were limited to three replication elongation factors (the DnaE polymerase, DnaG primase and DnaC helicase) specializing in lagging strand replication. These data provided the first evidence that links DNA chain elongation to glycolysis and may have revealed a universally conserved regulatory hub modulating DNA replication in response to energy provided by environmental nutrients. Based upon these results, our working hypothesis is that this regulatory hub generates signals, in response to nutrient availability, that drive key structural/functional changes in key replication enzymes, DnaE, DnaG and DnaC, as well as the replication initiator DnaA. We propose to elucidate the molecular and biochemical mechanisms that underpin this regulatory hub.

Understanding how bacteria respond to environmental stimuli and nutritional challenges is of paramount importance and with potential impact across a wide range of applied and theoretical scientific fields.

Health and Bioengineering
Theoreticians and modellers will be able to use our data in order to mathematically model a variety of different nutritional versus growth scenaria in system approaches to biosciences (through metabolomics) and synthetic biology, both of which are explicit strategic priorities in the BBSRC research agenda. Such approaches will have a direct beneficial impact in the Health and Biotechnology sectors. Bacilli are major producers for the detergent, vitamin and biocompatible insecticide industries. Information on how to affect growth through nutritional modulation would be particularly useful to bioprocessing in industrial biotechnology. Bioengineers will be able to modify growth media to maximize or control output. Insights into nutritional challenges and cell growth will help us design and implement new strategic approaches to a wide range of bacteria-driven bioprocesses.

In the health sector, new innovative approaches could be considered to manage and combat infections through nutritional control (high or low glucose diet) where appropriate.

Food
Our work will have direct impact in the food sector. Bacillus subtilis, the model organism that we will be working in this project, and its close relatives are the biggest food spoilers in the food industry. Data from our work will potentially help to design new innovative approaches to improve food safety which is another strategic priority of the BBSRC research agenda (Healthy and Safe Food).

Cancer
The Nobel laureate Otto Heinrich Warburg in 1924 suggested that rapidly growing cancer cells are metabolically different than normal cells. They generate their energy through the non-oxidative breakdown of glucose during glycolysis rather than through the ribosomally driven oxidative phosphorylation through the Kreb's cycle. This bears a striking resemblance to the genetic link between the lower C-3 part of glycolysis and cell growth that we propose to investigate here. Data from our studies on a simple model organism such as Bacillus subtilis will have a direct impact on understanding further the "Warburg Effect" at the molecular level through translational routes. Many aspects of DNA replication at the mechanistic and regulatory levels show a striking conservation between different species, and regulatory mechanisms that will potentially be elucidated here may be applicable to cancer cells. New lines of investigations into cancer metabolism and cell proliferation are likely to emerge, particularly as the Bacillus subtilis replisome is more similar to the eukaryotic replisome than the Escherichia coli replisome. For example, both the B. subtilis and the eukaryotic replisomes utilize lagging strand specific DNA polymerases, DnaE and pol alpha, respectively. DnaE is hypothesized to be a potential regulatory end point linking glycolysis and DNA replication and pol alpha could be under similar regulation in rapidly growing cancer cells.

International Collaboration
A strategic priority of BBSRC is to harness mutual benefit that derives from international collaboration. We will gain access to a wealth of resources in the form of plasmids and strains that have been developed by our project partner in France. He will be engaging in a parallel modelling systems approach to investigate the whole network including global supercoiling effects and not just the glycolysis/replication link. He is working with collaborating physicists and mathematicians to develop new tools in order to model regulatory loops individually and collectively. Iterative cycles of prediction and experimentation will be conducted to generate an integrated understanding of the system. We will maintain a two-way communication during the project for our mutual benefit.