Enhanced catabolism of glycine betaine and derivatives provides improved osmotic stress protection in Methylorubrum extorquens PA1

Abstract

Integration of metabolites into the overall metabolic network of a cell requires careful coordination dependent upon the ultimate usage of the metabolite. Different stoichiometric needs, and thus pathway fluxes, must exist for compounds destined for diverse uses, such as carbon sources, nitrogen sources, or stress-protective agents. Herein, we expand upon our previous work that highlighted the nature of glycine betaine (GB) metabolism in Methylobacteria to examine the utilization of GB-derivative compounds dimethylglycine (DMG) and sarcosine into Methylorubrum extorquens in different metabolic capacities, including as sole nitrogen and/or carbon sources. We isolated gain-of-function mutations that allowed M. extorquens PA1 to utilize dimethylglycine as a carbon source and dimethylglycine and sarcosine as nitrogen source. Characterization of mutants demonstrated selection for variants of the AraC-like regulator Mext_3735 that confer constitutive expression of the GB metabolic gene cluster, allowing direct utilization of the downstream GB derivatives. Finally, among the distinct isolates examined, we found that catabolism of the osmoprotectant used for selection (GB or dimethylglycine) enhanced osmotic stress resistance provided in the presence of that particular osmolyte. Thus, access to the carbon and nitrogen and osmoprotective effects of GB and DMG are made readily accessible through adaptive mutations. In M. extorquens PA1, the limitations to exploiting this group of compounds appear to exist predominantly at the levels of gene regulation and functional activity, rather than being constrained by transport or toxicity.IMPORTANCEOsmotic stress is a common challenge for bacteria colonizing the phyllosphere, where glycine betaine (GB) can be found as a prevalent osmoprotectant. Though Methylorubrum extorquens PA1 cannot use GB or its demethylation products, dimethylglycine (DMG) and sarcosine, as a sole carbon source, utilization is highly selectable via single nucleotide changes for both GB and DMG growth. The innate inability to use these compounds is due to limited flux through steps in the pathway and regulatory constraints. Herein, the characterization of the transcriptional regulator, Mext_3735 (GbdR), expands our understanding of the various roles in which GB derivatives can be used in M. extorquens PA1. Interestingly, increased catabolism of GB and derivatives does not interfere with, but rather improves, the ability of cells to thrive under increased salt stress conditions, suggesting that metabolic flux improves stress tolerance rather than providing a distinct tension between uses.

Keywords: dimethylglycine; experimental evolution; glycine betaine; metabolic homeostasis; osmolytes; osmotic stress.

Fig 1

Glycine betaine catabolism. Diagram of GB utilization pathways in M. extorquens PA1. The specific enzymes catalyzing each demethylation reaction are listed above the arrows.

Fig 2

M. extorquens PA1 is unable to utilize dimethylglycine or sarcosine as a sole source of carbon. Growth of M. extorquens PA1 strains [wild type (black) and dgcB^P30L (red)], as well as the closely related M. extorquens AM1 (gray) strain in liquid MP media supplemented with (A) 8 mM DMG or (B) 8 mM sarcosine as a carbon source. Error bars represent 95% confidence intervals based on three biological replicates.

Fig 3

M. extorquens PA1 is unable to utilize dimethylglycine or sarcosine as a sole source of nitrogen. Growth of M. extorquens PA1 strains (wild type and dgcB^P30L), as well as the closely related M. extorquens AM1 strain in liquid MP media supplemented with 15 mM methanol as a carbon source and the following sole nitrogen sources: (A) glycine betaine, (B) dimethylglycine, and (C) sarcosine. Experimental controls including ammonium, glycine, and no additional nitrogen are shown in Fig. S1. Error bars represent the 95% confidence intervals based on three biological replicates.

Fig 4

Minimum inhibitory concentrations of glycine betaine, dimethylglycine, and sarcosine. The wild-type M. extorquens PA1 strain was exposed to a range of concentrations of glycine betaine (G), dimethylglycine (D), and sarcosine (S). Growth was measured after 48 hours of incubation in liquid MP media supplemented with 15 mM methanol and the indicated GB-related compound and concentration. Error bars represent 95% confidence intervals based on three biological replicates.

Fig 5

Selection for mutations in M. extorquens dgcB^P30L allows dimethylglycine utilization as a sole source of carbon and energy. Growth of M. extorquens GB+ background strain dgcB^P30L (red circles), DMG+ evolved isolates dgcB^P30L Mext_3735^D167N (JB528, yellow closed circles), dgcB^P30L Mext_3735^G165S (JB529, olive squares), and dgcB^P30L Mext_3735^E170V (JB533, green open circles) was quantified in liquid MP medium supplemented with 8 mM DMG. Error bars represent 95% confidence intervals based on three biological replicates.

Fig 6

Mutations in gbdR lead to constitutive activation of GB cluster genes, regardless of ligand presence. The relative expression of representative genes from each operon of the M. extorquens GB gene cluster was examined, including proV, soxA, gbcB, sdaB, and dgcB. Growth was carried out in MP supplemented with 15 mM MeOH with or without 1 mM GB or DMG. (A) Expression of genes in the GB cluster in PA1 dgcB^P30L mutant, comparing conditions with versus without DMG, shows that DMG cannot induce the expression of GB cluster genes, in contrast to GB itself (42). (B) Expression of genes in the GB cluster in the dgcB^P30L Mext_3735^D167N mutant in comparison to PA1 dgcB^P30L grown in MP supplemented with only 15 mM MeOH. (C) Expression of genes in the GB cluster in the dgcB^P30L ΔMext_3735 mutant in comparison to PA1 wild type grown in MP supplemented with only 15 mM MeOH. Individual values shown are averages of three technical replicates. Error bars represent the standard deviation of three biological replicates.

Fig 7

Constitutive expression of GB cluster allows utilization of DMG and sarcosine as sole nitrogen sources. Final OD₆₀₀ readings of M. extorquens strains grown in MP media lacking nitrogen supplemented with 15 mM methanol and 5 mM of the indicated nitrogen sources. Bars represent the mean value of three independent biological replicates (shown as circles). Error bars represent 95% confidence intervals based on three biological replicates.

Fig 8

Predicted protein structure of M. extorquens PA1 GbdR. (A) AlphaFold 2 predicted structure of wild-type GbdR (gray) with variant sites mutated in the selected DMG+ evolved strains highlighted in distinct colors. (B) Wild-type GbdR structure shown with the electrostatic potential surface. Each color indicates the following: red, negatively charged surface; blue, positively charged surface; and white, hydrophobic. Black circle highlights the location of three variant sites on the electrostatic potential surface. The model file can be found at https://github.com/BazurtoLab/Glycine-betaine-osmotic-stress/blob/main/IFD_glycine_betaine_top.pdb.

Fig 9

Root mean square deviation of wild-type GbdR and three variants during molecular dynamics simulations. The RMSD plot shows the root mean square deviation of three protein variants (G165S, D167N, and E170V) compared to the wild type (“WT”) over time. The x-axis shows the time in nanoseconds (ns), and the y-axis shows the root mean square deviation in Angstroms. The wild-type GbdR exhibits a higher RMSD after 70 ns compared to the three variants, indicating that the wild-type form undergoes a large conformational change, which is not seen for the three variants.

Fig 10

Docking of GB to GbdR. (A) This panel shows the top docked pose of GB to GbdR in electrostatic surface representation. The molecule is colored according to its electrostatic potential, with red representing negative charges and blue representing positive charges. The circle around the docked GB represents the binding pocket of GbdR. (B) This panel shows the amino acid residues in the predicted binding pocket of GbdR, with dashed lines indicating hydrogen bonds between the residues and GB (gray). The residues (green) are labeled with their one-letter amino acid codes and position numbers.

Fig 11

Minimum inhibitory concentrations of NaCl. Different genotypes were exposed to a range of concentrations of up to 500 mM NaCl and (A) without or (B) with supplementation of 1 mM GB. Growth was measured after 48 hours of incubation in liquid MP media supplemented with 15 mM methanol and the indicated NaCl concentration. Error bars represent 95% confidence intervals derived from three biological replicates.

Fig 12

Regulated GB utilization improves competitive fitness under osmotic stress conditions. Relative Malthusian fitness scores of M. extorquens PA1 wild type (blue), dgcB^P30L (red), dgcB^P30L gbdR^D167N (green), dgcB^P30L ΔgbdR (yellow), dgcB^P30L ΔgbcBA (cyan), and dgcB^P30L ΔproVUX (magenta) following a 24-hour competition period against a fluorescently tagged wild-type PA1 strain under growth conditions in liquid MP media supplemented with 15 mM methanol and addition of (A) neither NaCl nor GB, (B) 1 mM GB, (C) 200 mM NaCl, and (D) 200 mM NaCl and 1 mM GB. Error bars represent the 95% confidence interval of three independent biological replicates. ns, no significant difference. ****P < 0.0001.

Fig 13

DMG utilization improves competitive fitness under osmotic stress conditions for strains with increased GB pathway flux. Relative Malthusian fitness scores of M. extorquens PA1 wild type (blue), dgcB^P30L (red), dgcB^P30L gbdR^D167N (yellow), dgcB^P30L ΔgbdR (green), dgcB^P30L ΔdgcBA (blue), and dgcB^P30L ΔproVUX (magenta) following a 24-hour competition period against a fluorescently tagged wild-type PA1 strain under growth conditions in liquid MP media supplemented with 15 mM methanol and addition of (A) neither NaCl nor DMG, (B) 1 mM DMG, (C) 200 mM NaCl, and (D) 200 mM NaCl and 1 mM DMG. Error bars represent the 95% confidence interval of three independent biological replicates. ns, no significant difference. ***P < 0.001 and ****P < 0.0001.