  The relationship between sequence variation and phenotype is poorly
understood. Here we use metabolomic analysis to elucidate the molecular
mechanism underlying the filamentous phenotype of E. coli strains that carry
destabilizing mutations in the Dihydrofolate Reductase (DHFR). We find that
partial loss of DHFR activity causes SOS response indicative of DNA damage and
cell filamentation. This phenotype is triggered by an imbalance in deoxy
nucleotide levels, most prominently a disproportionate drop in the
intracellular dTTP. We show that a highly cooperative (Hill coefficient 2.5) in
vivo activity of Thymidylate Kinase (Tmk), a downstream enzyme that
phosphorylates dTMP to dTDP, is the cause of suboptimal dTTP levels. dTMP
supplementation in the media rescues filamentation and restores in vivo Tmk
kinetics to almost perfect Michaelis-Menten, like its kinetics in vitro.
Overall, this study highlights the important role of cellular environment in
sculpting enzymatic kinetics with system level implications for bacterial
phenotype.
