The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and D-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and "Dehalococcoides ethenogenes." GpgS had maximal activity at 50°C. The maximal activity of GpgP was at 50°C with GPG as the substrate and at 60°C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate.Most microorganisms capable of osmotic adjustment accumulate low-molecular-weight organic compounds, commonly designated compatible solutes, which can be taken up from the environment or synthesized de novo (6). Although the uptake of organic solutes such as glycine betaine and trehalose, among others, by microorganisms under salt stress is preferred because it is energetically favorable (10), many organisms synthesize specific compatible solutes because these are not present in the environment or those that are present do not fulfill the specific requirements of the organisms.Ectoine, glycine betaine, trehalose, and glutamate are among the most common compatible solutes of bacteria and archaea, where they normally accumulate in response to stress imposed by salt (10). Mannosylglycerate (MG), di-myo-inositol-phosphate, and diglycerol-phosphate have a more restricted distribution, being found primarily in (hyper)thermophilic prokaryotes. Mannosylglycerate is widespread in (hyper)thermophilic bacteria and archaea and is also found in some red algae (21, 35). Cyclic 2,3-bisphosphoglycerate has only been encountered in methanogens (20,25,36).The rare solute glucosylglycerate (GG) was originally identified in the cyanobacterium Agmenellum quadruplicatum strain PCC7002 grown under nitrogen-limiting conditions (22). This solute was also detected in the archaeon Methanohalophilus portucalensis strain FDF...

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...

Groundwater samples (111) from six different boreholes located in two geographical areas were examined for the presence of legionellae over a 7-year period. The number of Legionella isolates detected was generally low. The colonization of the aquifers was not uniform, and the persistence of Legionella was independent of the hydraulic pumps and the plumbing system present in the borehole. A total of 374 isolates identified by fatty acid methyl ester analysis belonged to Legionella pneumophila, L. oakridgensis, L. sainthelensi, and L. londiniensis. In area 1, L. oakridgensis constituted the major population detected, exhibiting only one random amplified polymorphic DNA (RAPD)-PCR profile. L. sainthelensi strains were less frequently isolated and also displayed a single RAPD profile, while L. pneumophila was only sporadically detected. In contrast, L. pneumophila comprised the vast majority of the isolates in area 2 and exhibited six distinct RAPD patterns, indicating the presence of different genetic groups; three L. londiniensis RAPD types were also detected. Two of the L. pneumophila and one of the L. londiniensis RAPD types were persistent in this environment for at least 12 years. The genetic structure of L. pneumophila groundwater populations, inferred from rpoB and dotA gene sequences, was peculiar, since the majority of the isolates were allied in a discrete group different from the lineages containing most of the type and reference strains of the three subspecies of L. pneumophila. Furthermore, gene exchange events related to the dotA allele could be envisioned.

The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and D-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis.Thermophilic and hyperthermophilic organisms, like the vast majority of other microorganisms, accumulate compatible solutes in response to water stress imposed by salt. In fact, many of the (hyper)thermophiles known were isolated from geothermal areas venting seawater (36). However, the compatible solutes of thermophilic and hyperthermophilic prokaryotes are generally different from those of their mesophilic counterparts and some, namely, di-myo-inositol-phosphate (DIP), mannosyl-di-myo-inositol-phosphate (MDIP), diglycerol phosphate, and mannosylglyceramide, are confined to organisms that grow at extremely high temperatures (19,22,34,38). Mannosylglycerate (2-␣-D-mannosylglycerate; MG), for example, is a common compatible solute of thermophiles and hyperhermophiles (23, 27, 38) but has also been found in mesophilic organisms, such as red algae, where it was first identified (6). It should also be noted that there is a growing awareness that compatible solutes are involved in other types of stress; trehalose, for example, plays a role in osmotic stress, heat stress, desiccation, and freezing (9). Some compatible solutes of ...

Mannosylglycerate (MG) is a common compatible solute found in thermophilic and hyperthermophilic prokaryotes. In this study we characterized a mesophilic and bifunctional mannosylglycerate synthase (MGSD) encoded in the genome of the bacterium Dehalococcoides ethenogenes. mgsD encodes two domains with extensive homology to mannosyl-3-phosphoglycerate synthase (MPGS, EC 2.4.1.217) and to mannosyl-3-phosphoglycerate phosphatase (MPGP, EC 3.1.3.70), which catalyze the consecutive synthesis and dephosphorylation of mannosyl-3-phosphoglycerate to yield MG in Pyrococcus horikoshii, Thermus thermophilus, and Rhodothermus marinus. The bifunctional MGSD was overproduced in Escherichia coli, and we confirmed the combined MPGS and MPGP activities of the recombinant enzyme. The optimum activity of the enzyme was at 50°C. To examine the properties of each catalytic domain of MGSD, we expressed them separately in E. coli. The monofunctional MPGS was unstable, while the MPGP was stable and was characterized. Dehalococcoides ethenogenes cannot be grown sufficiently to identify intracellular compatible solutes, and E. coli harboring MGSD did not accumulate MG. However, Saccharomyces cerevisiae expressing mgsD accumulated MG, confirming that this gene product can synthesize this compatible solute and arguing for a role in osmotic adjustment in the natural host. We did not detect MGSD activity in cell extracts of S. cerevisiae. Here we describe the first gene and enzyme for the synthesis of MG from a mesophilic microorganism and discuss the possible evolution of this bifunctional MGSD by lateral gene transfer from thermophilic and hyperthermophilic organisms.Several compatible solutes such as ectoine, glycine betaine, and trehalose are widespread in mesophilic bacteria, where they accumulate in response to stress imposed by salt or heat. Thermophilic and hyperthermophilic bacteria and archaea generally accumulate unusual compatible solutes, such as dimyo-inositol-phosphate, di-mannosyl-di-myo-inositol-phosphate, diglycerol-phosphate, and mannosylglycerate, that have not yet been identified in mesophilic prokaryotes (40). Mannosylglycerate (MG) has been encountered in several thermophilic bacteria, such as Thermus thermophilus, Rhodothermus marinus, and Rubrobacter xylanophilus, and in hyperthermophilic archaea such as Aeropyrum pernix, Pyrococcus spp., and Thermococcus spp. and some strains of the species Archaeoglobus (40). Despite its scattered distribution, MG has been primarily associated with prokaryotes that grow at high temperatures (13,40). While the role of MG during osmotic stress is uncontroversial in some of these organisms (42), in vitro evidence indicates a role for this compatible solute on the protection of proteins against thermal denaturation (5, 14, 36). However, MG was also encountered in marine red algae and is not therefore restricted to thermophilic and hyperthermophilic prokaryotes (25).Identification of the genes and biosynthetic pathways involved in the synthesis of MG is essential to elucidate the physio...