Functional and Metabolic Effects of Adaptive Glycerol Kinase (GLPK) Mutants in Escherichia coli

Abstract

Herein we measure the effect of four adaptive non-synonymous mutations to the glycerol kinase (glpK) gene on catalytic function and regulation, to identify changes that correlate to increased fitness in glycerol media. The mutations significantly reduce affinity for the allosteric inhibitor fructose-1,6-bisphosphate (FBP) and formation of the tetramer, which are structurally related, in a manner that correlates inversely with imparted fitness during growth on glycerol, which strongly suggests that these enzymatic parameters drive growth improvement. Counterintuitively, the glpK mutations also increase glycerol-induced auto-catabolite repression that reduces glpK transcription in a manner that correlates to fitness. This suggests that increased specific GlpK activity is attenuated by negative feedback on glpK expression via catabolite repression, possibly to prevent methylglyoxal toxicity. We additionally report that glpK mutations were fixed in 47 of 50 independent glycerol-adapted lineages. By far the most frequently mutated locus (nucleotide 218) was mutated in 20 lineages, strongly suggesting this position has an elevated mutation rate. This study demonstrates that fitness correlations can be used to interrogate adaptive processes at the protein level and to identify the regulatory constraints underlying selection and improved growth.

Global metabolic network reorganization by adaptive mutations allows fast growth of Escherichia coli on glycerol

“…3c). A previous study showed reduced intracellular cAMP level in the GLPK strain, and this result was consistent in three other knock-in strains with different adaptive glpK mutations 10 . This observation is further supported at the proteome level by a twofold decrease of cAMP receptor protein (Crp) in GLPK and DKI strains ( Supplementary Fig.…”

“…Since cAMP-Crp activates tricarboxylic acid (TCA) cycle genes, acetate formation ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms4233 is limited on glycerol, a normally non-fermentable carbon source. On the other hand, the glycerol uptake rate was enhanced in the glpK mutant most probably because of reduced inhibitory effect of fructose 1,6-bisphosphate on GlpK, as shown previously for this mutant 10 . This results in increased transfer of phosphate from phosphorylated EIIA Glc to produce glycerol-3-phosphate 28 .…”

“…We note that these changes were not observed in the RPOC strain that displays an intermediate growth rate between the GLPK and DKI strains. The increased carbon flow is probably due to the improved kinetics of mutant GlpK, as well as reduced inhibition by fructose 1,6-bisphosphate, although the level of GlpK actually decreases in the GLPK strain (Supplementary Table 1) 10 . Reduced GlpK expression may appear as a counterintuitive response but it has previously been suggested to be linked to catabolite repression of glpFKX operon owing to reduced cAMP levels and suggested to be important to limit methylglyoxal toxicity 10 .…”

“…Most of the acquired mutations affecting growth phenotype can be grouped into those affecting condition-specific function (for example, glycerol kinase or glpK) or global transcription patterns (for example, RNA polymerase b 0 subunit encoded by rpoC). Although the biochemical characteristics of some of the naturally occurring mutations in glycerol kinase and RNA polymerase have been studied 10,11 , the underlying molecular and network-level consequences and mechanisms promoting cell growth in vivo have not been uncovered. While adaptive evolution and identification of mutations by resequencing has been made easy and cheap, the elucidation of network-level mechanisms remains a major bottleneck in the study of adaptive evolution 4,12 .…”

“…However, the genotype-phenotype relationship is multilayered and hierarchical and the measured genotypes and phenotypes are the opposite ends of this hierarchy. The in vitro assessment of the effects of the mutated gene products relative to the wild type (WT) can help determine how molecular properties change 10,11 but studies at the level of individual gene-product properties give limited insights into the mechanisms that change the phenotype. Network-level changes resulting from the introduction of causal mutations can now be characterized through the generation and integrative analysis of polyomic data sets.…”