Glucosylglycerate Biosynthesis in the Deepest Lineage of the Bacteria : Characterization of the Thermophilic Proteins GpgS and GpgP from Persephonella marina

Abstract

The pathway for the synthesis of glucosylglycerate (GG) in the thermophilic bacterium Persephonella marina is proposed based on the activities of recombinant glucosyl-3-phosphoglycerate (GPG) synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP). The sequences of gpgS and gpgP from the cold-adapted bacterium Methanococcoides burtonii were used to identify the homologues in the genome of P. marina, which were separately cloned and overexpressed as His-tagged proteins in Escherichia coli. The recombinant GpgS protein of P. marina, unlike the homologue from M. burtonii, which was specific for GDP-glucose, catalyzed the synthesis of GPG from UDP-glucose, GDP-glucose, ADP-glucose, and TDP-glucose (in order of decreasing efficiency) and from D-3-phosphoglycerate, with maximal activity at 90°C. The recombinant GpgP protein, like the M. burtonii homologue, dephosphorylated GPG and mannosyl-3-phosphoglycerate (MPG) to GG and mannosylglycerate, respectively, yet at high temperatures the hydrolysis of GPG was more efficient than that of MPG. Gel filtration indicates that GpgS is a dimeric protein, while GpgP is monomeric. This is the first characterization of genes and enzymes for the synthesis of GG in a thermophile.The compatible solute ␣-glucosylglycerate (GG) has been identified in the cyanobacterium Agmenellum quadruplicatum strain PCC7002, in the archaeon Methanohalophilus portucalensis strain FDF-1, in a salt-sensitive mutant of Halomonas elongata, and in the ␥-proteobacterium Erwinia chrysanthemi strain 3937, where it behaves as a compatible solute during osmotic stress under nitrogen-limiting conditions (8,21,26,33). This compatible solute is chemically related to mannosylglycerate (MG), which is widespread in (hyper)thermophilic bacteria and archaea and has been shown to serve as a compatible solute under salt stress in several of these organisms (1,35,36). However, MG has also been encountered in marine red algae, and the genes for the synthesis of MG have been found in the mesophilic bacterium "Dehalococcoides ethenogenes" (proposed name) (16,25). On the other hand, the accumulation of GG had been detected only in mesophilic bacteria and archaea (8,21,26,33). However, GG was recently shown to accumulate in Persephonella marina (H. Santos, personal communication), a thermophilic, strictly chemolithoautotrophic, microaerophilic, hydrogen-oxidizing bacterium isolated from a deep-sea hydrothermal vent which is a member of the order Aquificales (20). This bacterium has a temperature range for growth of between 55 and 80°C (optimum at 73°C) and grows optimally in media containing 2.5% (wt/vol) NaCl (20). The identification of GG in this organism, where it may have a role in osmoadaptation, prompted us to examine the pathway for its synthesis.The biosynthetic pathway for the synthesis of GG in Methanococcoides burtonii proceeds via a two-step pathway involving glucosyl-3-phosphoglycerate synthase (GpgS), which catalyzes the conversion of GDP-glucose and D-3-phosphoglycerate (3-PGA) to glucosyl-3-phosphogl...

To be or not to be a compatible solute: Bioversatility of mannosylglycerate and glucosylglycerate

“…The purification of the native enzyme allowed the identification of a highly divergent MpgS from which sequence homology to the known MpgSs had been extensively erased (Empadinhas et al, unpublished results). Adjacent to this mpgS gene we detected genes encoding two unspecific phosphatases, leading to the hypothesis that one or both could complete the two-step synthesis of MG. On the other hand, this atypical MpgS had high homology with one enzyme found in mycobacteria and related Actinobacteria, later found to be a glucosyl-3-phosphoglycerate synthase (GpgS) with low homology to the homo-functional GpgSs from M. burtonii and P. marina [8,9,18]. In fact, the R. xylanophilus MpgS could synthesize GPG, the phosphorylated precursor of GG, with higher efficiency than that for MPG, although free GG has never been detected in R. xylanophilus.…”

“…GG was also recently shown to behave as a compatible solute in the g-proteobacterium Erwinia chrysanthemi under combined salt stress and nitrogen-limiting conditions, replacing glutamate and glutamine, the major compatible solutes when abundant sources of nitrogen are present in the medium [22] GG also accumulates during salt stress in other microorganisms where the corresponding genes have been detected, under conditions when limiting nitrogen has to be mobilized for the synthesis of other nitrogencontaining cell components [9].…”

“…3) [9]. A second operon-like structure located immediately upstream contains genes related to phosphate uptake leading to the speculation that the synthesis of GG might be controlled by phosphate availability.…”

“…In fact, GG has not been detected in P. miotherma in any of the conditions tested and the precursor for MGG biosynthesis has recently been identified to be glucosyl-3-phosphoglycerate (GPG) (Fernandes, unpublished results). This compound is the phosphorylated precursor for the GG found in several organisms [8,9].…”

“…3. Genetic organization and alternative pathways for synthesis of glucosylglycerate in Persephonella marina [9,19]. GpgP, glucosyl-3-phosphoglycerate phosphatase; Ggs, glucosylglycerate synthase; GpgS, glucosyl-3-phosphoglycerate synthase; gK, putative gycerate kinase; phoR, putative histidine kinase regulator; 3-PGA, 3-phosphoglycerate; NDP, nucleoside diphosphate.…”