Functional and physiological consequences of variations in DgoR, a transcriptional repressor of D-galactonate metabolism in Escherichia coli

Abstract

GntR/FadR family of transcriptional regulators (TRs), constituting one of the largest TR families, features an N-terminal winged helix-turn-helix DNA-binding domain and a C terminal α-helical effector-binding and oligomerization domain. Several GntR/FadR TRs govern sugar acid metabolism and although effectors are known for a few TRs, the unavailability of relevant structures has left their allosteric mechanism unexplored. Here, using Escherichia coli DgoR, a transcriptional repressor of D-galactonate metabolism and its four superrepressor alleles, we probed allostery in the GntR/FadR family sugar acid TR. Genetic and biochemical studies established compromised response to D-galactonate as the reason for the superrepressor behavior of the mutants: one mutant does not bind D-galactonate, and while the other three mutants bind D-galactonate, effector binding does not induce a conformational change required for DNA release, suggesting altered allosteric behavior. For detailed mechanistic insights into allosteric communication, we performed extensive molecular dynamics simulations of the modeled DgoR structure in various allosteric states for both the wild-type and mutant proteins. We found that each mutant exhibits unique dynamics disrupting the intrinsic allosteric communication pathways, thereby impacting DgoR function. We also validated the allosteric communication model by testing in silico predictions with experimental data. Our work offers a basis for examining the allosteric behavior of other GntR/FadR family TRs to further improve our understanding of transcriptional regulation. D-galactonate is a widely prevalent sugar acid, and its metabolism is extensively implicated in the interaction of enteric bacteria with their hosts. Importantly, in multiple evolutionary studies, E. coli isolates adapted to the mouse gut accumulated inactivating mutations in dgoR, suggesting that genetic variations in dgoR might determine the colonization potential of natural E. coli isolates in the complex microbial community of the host. Here, we analyzed a panel of 340 sequenced natural E. coli isolates for variations in dgoR and determined their impact on repressor function. Genetic and biochemical analyses identified variants with a partial loss of DNA-binding ability as well as variants that exhibit a decreased response to D-galactonate. Interestingly, a variation resulting in reduced sensitivity to D galactonate is located in the linker region while a variation leading to a defect in DNA binding is located in the effector-binding domain, suggesting altered allostery as the reason for their compromised function. We thus investigated the behavior of these variants in simulation studies. Importantly, corroborating their effect on the repressor function, these variations impacted the growth of natural isolates in D-galactonate. Our studies provide a rationale for examining the effect of these genetic variations on the colonization of isolates inside their hosts. Because the dgo operon is present in enterobacterial strains, including both commensals and pathogens, these studies can be extended beyond E. coli. Research in these directions will help envision novel strategies for promoting the growth of commensal strains to outcompete their pathogenic counterparts.