  Cells have evolved a metabolic control of DNA replication to respond to a
wide range of nutritional conditions. Accumulating data suggest that this
poorly understood control depends, at least in part, on Central Carbon
Metabolism (CCM). In Bacillus subtilis , the glycolytic pyruvate kinase (PykA)
is intricately linked to replication. This 585 amino-acid-long enzyme comprises
a catalytic (Cat) domain that binds to phosphoenolpyruvate (PEP) and ADP to
produce pyruvate and ATP, and a C-terminal domain of unknown function.
Interestingly, the C-terminal domain termed PEPut interacts with Cat and is
homologous a domain that, in other metabolic enzymes, are phosphorylated at a
conserved TSH motif at the expense of PEP and ATP to drive sugar import and
catalytic or regulatory activities. To gain insights into the role of PykA in
replication, DNA synthesis was analyzed in various Cat and PEPut mutants grown
in a medium where the metabolic activity of PykA is dispensable for growth.
Measurements of replication parameters ( ori/ter ratio, C period and fork
speed) and of the pyruvate kinase activity showed that PykA mutants exhibit
replication defects resulting from side chain modifications in the PykA protein
rather than from a reduction of its metabolic activity. Interestingly, Cat and
PEPut have distinct commitments in replication: while Cat impacts positively
and negatively replication fork speed, PEPut stimulates initiation through a
process depending on Cat-PEPut interaction and growth conditions. Residues
binding to PEP and ADP in Cat, stabilizing the Cat-PEPut interaction and
belonging to the TSH motif of PEPut were found important for the commitment of
PykA in replication. In vitro , PykA affects the activities of replication
enzymes (the polymerase DnaE, helicase DnaC and primase DnaG) essential for
initiation and elongation and genetically linked to pykA . Our results thus
connect replication initiation and elongation to CCM metabolites (PEP, ATP and
ADP), critical Cat and PEPut residues and to multiple links between PykA and
the replication enzymes DnaE, DnaC and DnaG. We propose that PykA is endowed
with a moonlighting activity that senses the concentration of signaling
metabolites and interacts with replication enzymes to convey information on the
cellular metabolic state to the replication machinery and adjust replication
initiation and elongation to metabolism. This defines a new type of replication
regulator proposed to be part of the metabolic control that gates replication
in the cell cycle.
