Metabolic response to point mutations reveals principles of modulation of in vivo enzyme activity and phenotype

Abstract

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 dihydrofolate reductase (DHFR). We find that partial loss of DHFR activity causes reversible filamentation despite SOS response indicative of DNA damage, in contrast to thymineless death (TLD) achieved by complete inhibition of DHFR activity by high concentrations of antibiotic trimethoprim. This phenotype is triggered by a disproportionate drop in intracellular dTTP, which could not be explained by drop in dTMP based on the Michaelis-Menten-like in vitro activity curve of thymidylate kinase (Tmk), a downstream enzyme that phosphorylates dTMP to dTDP. Instead, we show that a highly cooperative (Hill coefficient 2.5) in vivo activity of Tmk is the cause of suboptimal dTTP levels. dTMP supplementation rescues filamentation and restores in vivo Tmk kinetics to Michaelis-Menten. Overall, this study highlights the important role of cellular environment in sculpting enzymatic kinetics with system-level implications for bacterial phenotype.

Development of antibacterial compounds that constrain evolutionary pathways to resistance

“…Our in vivo DHFR over-expression experiment showed partial recovery from the CD15-3-induced growth inhibition, thereby validating DHFR as a target for the intracellular inhibition by CD15-3. That DHFR is being targeted by CD15-3 is further supported by our imaging results showing antibiotic-stress-induced filamentation in the WT cells, a clear sign indicating that the folate pathway is indeed impacted by the drug ( Bhattacharyya et al, 2021 ). However, the partial recovery upon DHFR overexpression strongly suggests the presence of at least one additional protein target of CD15-3.…”

A comprehensive map of transient protein-protein interactions and their structural features in theE. coli1-carbon metabolism pathway

“…The only exception in the pyrimidine pathway is Thymidylate Kinase (Tmk) which does interact with several proteins within its own pathway along with proteins from purine and folate pathways. Interestingly it was shown that Tmk overexpression is toxic, probably due to mis-interactions upon overexpression with other proteins of the folate pathway (Bhattacharyya et al, 2021; Bhattacharyya et al, 2016). Next, we checked for significant inter-pathway interactions among the three pathways.…”

“…In our previous work (Bhattacharyya et al, 2016), using immunoprecipitation experiments, we have shown that Dihydrofolate Reductase (DHFR), a central enzyme of the E. coli 1-carbon metabolism pathway, associates with multiple proteins in its functional vicinity via weak interactions, which in turn was also responsible for overexpression toxicity of DHFR. Though precise evidence of substrate channeling was not established, a more recent work (Bhattacharyya et al, 2021) presented compelling circumstantial evidence that much of these interactions involve channeling of substrates, causing diffusion limitation of the substrates inside the cell. To obtain a deeper understanding of the breadth and nature of interactions in prokaryotic enzyme assemblies, in this work we established a complete PPI map of the E. coli 1-carbon metabolism pathway using biomolecular fluorescence complementation assay.…”

The fitness effects of codon composition of the horizontally transferred antibiotic resistance genes intensify at sub-lethal antibiotic levels

“…When the product of DHFR abundance and catalytic activity ( k cat /K M ) drops below the basal level, which in E. coli constitutes 40-100 DHFR molecules per cell (Taniguchi, et al 2010), the decrease in bacterial growth rate follows Michaelis-Menten-like kinetics (Bershtein, et al 2013; Bershtein, Choi, et al 2015; Rodrigues, et al 2016; Bhattacharyya, et al 2018). The drop in functional DHFR levels was shown to cause a profound imbalance in the metabolic pools (Bershtein, Choi, et al 2015; Bhattacharyya, et al 2021), which, in turn, leads to an upregulation of folA transcription via a negative metabolic regulation loop operating through binding of TyrR transcription activator to two TyrR boxes located in folA regulatory region (Yang, et al 2007). Furthermore, the increase in DHFR abundance, well above the basal level, was also found to cause a decline in the growth rate of E. coli , owing to the formation of transient protein-protein interactions that trigger toxic metabolic imbalance (Bhattacharyya, et al 2016).…”