JOURNAL OF BACTERIOLOGY, Mar. 2007, p. 1648–1654 Vol. 189, No. 50021-9193/07/$08.00�0 doi:10.1128/JB.00841-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Glucosylglycerate Biosynthesis in the Deepest Lineage of the Bacteria:Characterization of the Thermophilic Proteins GpgS and

GpgP from Persephonella marina�

Joana Costa,1 Nuno Empadinhas,1 and Milton S. da Costa2*Centro de Neurociencias e Biologia Celular, Departamento de Zoologia, Universidade de Coimbra, 3004-517 Coimbra,

Portugal,1 and Departamento de Bioquımica, Universidade de Coimbra, 3001-401 Coimbra, Portugal2

Received 13 June 2006/Accepted 7 December 2006

The pathway for the synthesis of glucosylglycerate (GG) in the thermophilic bacterium Persephonella marinais proposed based on the activities of recombinant glucosyl-3-phosphoglycerate (GPG) synthase (GpgS) andglucosyl-3-phosphoglycerate phosphatase (GpgP). The sequences of gpgS and gpgP from the cold-adaptedbacterium Methanococcoides burtonii were used to identify the homologues in the genome of P. marina, whichwere separately cloned and overexpressed as His-tagged proteins in Escherichia coli. The recombinant GpgSprotein of P. marina, unlike the homologue from M. burtonii, which was specific for GDP-glucose, catalyzed thesynthesis of GPG from UDP-glucose, GDP-glucose, ADP-glucose, and TDP-glucose (in order of decreasingefficiency) and from D-3-phosphoglycerate, with maximal activity at 90°C. The recombinant GpgP protein, likethe M. burtonii homologue, dephosphorylated GPG and mannosyl-3-phosphoglycerate (MPG) to GG andmannosylglycerate, respectively, yet at high temperatures the hydrolysis of GPG was more efficient than thatof MPG. Gel filtration indicates that GpgS is a dimeric protein, while GpgP is monomeric. This is the firstcharacterization of genes and enzymes for the synthesis of GG in a thermophile.

The compatible solute �-glucosylglycerate (GG) has beenidentified in the cyanobacterium Agmenellum quadruplicatumstrain PCC7002, in the archaeon Methanohalophilus portu-calensis strain FDF-1, in a salt-sensitive mutant of Halomonaselongata, and in the �-proteobacterium Erwinia chrysanthemistrain 3937, where it behaves as a compatible solute duringosmotic stress under nitrogen-limiting conditions (8, 21, 26,33). This compatible solute is chemically related to mannosyl-glycerate (MG), which is widespread in (hyper)thermophilicbacteria and archaea and has been shown to serve as a com-patible solute under salt stress in several of these organisms (1,35, 36). However, MG has also been encountered in marinered algae, and the genes for the synthesis of MG have beenfound in the mesophilic bacterium “Dehalococcoides etheno-genes” (proposed name) (16, 25). On the other hand, the ac-cumulation of GG had been detected only in mesophilic bac-teria and archaea (8, 21, 26, 33). However, GG was recentlyshown to accumulate in Persephonella marina (H. Santos, per-sonal communication), a thermophilic, strictly chemolithoau-totrophic, microaerophilic, hydrogen-oxidizing bacterium iso-lated from a deep-sea hydrothermal vent which is a member ofthe order Aquificales (20). This bacterium has a temperaturerange 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 mayhave a role in osmoadaptation, prompted us to examine thepathway for its synthesis.

The biosynthetic pathway for the synthesis of GG in Methano-coccoides burtonii proceeds via a two-step pathway involving glu-

cosyl-3-phosphoglycerate synthase (GpgS), which catalyzes theconversion of GDP-glucose and D-3-phosphoglycerate (3-PGA)to glucosyl-3-phosphoglycerate (GPG), which is subsequentlyconverted to GG by a GPG phosphatase (GpgP) (9). The mostcommon pathway for the synthesis of MG also proceeds via aphosphorylated intermediate which is dephosphorylated bymannosylglycerate-3-phosphoglycerate (MPG) phosphatase.The MPG phosphatases of Thermus thermophilus and Pyrococ-cus horikoshii share high amino acid identity with GpgP fromM. burtonii and dephosphorylate MPG and GPG (5, 9, 14, 15).The present work describes the characterization of GpgS andGpgP, which catalyze the synthesis of GG at high temperaturesin P. marina, and is the first report of the characteristics ofthese enzymes in a thermophilic organism.

MATERIALS AND METHODS

Strains, identification, cloning, and functional overexpression of gpgS andgpgP from Persephonella marina. P. marina strain EX-H1T (DSM 14350T) wasobtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen,Braunschweig, Germany. To identify the genes responsible for GG biosynthesisin P. marina, we used the sequences of glucosyl-3-phosphoglycerate synthase(GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from M. burtoniifor BLAST searches of the P. marina genome. Preliminary sequence data wereobtained from TIGR at http://www.tigr.org.

Two open reading frames with high homology to the gpgS and gpgP genes fromM. burtonii were detected. The full gpgS and gpgP genes were amplified by usingforward and reverse primers. Chromosomal DNA of P. marina was isolatedaccording to the method of Rainey et al. (32). Forward primers were constructedwith additional NcoI recognition sequences, reverse primers were constructed byadding XhoI sites, and PCR amplifications were carried out as previously de-scribed (9). Amplification products were visualized on 1% agarose gels, purifiedby band excision (Promega), and after digestion with the suitable restrictionenzymes, ligated into the corresponding sites of the expression vector pET-30a(Novagen), carrying an N-terminal His tag coding sequence. Most DNA manip-ulations followed standard molecular techniques (34). The constructs were se-quenced by AGOWA (Berlin, Germany).

Escherichia coli BL21 was used as the host for overexpression. E. coli cells

* Corresponding author. Mailing address: Departamento de Bio-quımica, Universidade de Coimbra, 3001-401 Coimbra, Portugal.Phone: 351-239824024. Fax: 351-239826798. E-mail: milton@ci.uc.pt.

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carrying the plasmids were grown to the mid-exponential growth phase (opticaldensity at 610 nm � 1.0) in LB medium at 37°C, induced with 1 mM isopropyl-�-D-thiogalactopyranoside (IPTG), and grown for another 5 h. Kanamycin wasadded to a final concentration of 30 �g/ml. Cells were harvested by centrifuga-tion (7,000 � g, 10 min, 4°C), and pellets were suspended in 20 mM sodiumphosphate buffer, pH 7.4, with 0.5 M NaCl, 20 mM imidazole, and a proteaseinhibitor cocktail (Roche), frozen, thawed, and disrupted by sonication, followedby centrifugation (18,000 � g, 30 min, 4°C) to remove debris.

GpgS and GpgP activities were confirmed in cell extracts with the assaysdescribed below. A cell extract from E. coli with empty pET-30a was used as anegative control. The supernatants were filtered and used for purification of theenzymes. The protein contents of all samples were determined by the Bradfordassay (7).

Synthesis of GPG and MPG. GPG was synthesized using the partially purifiedrecombinant GpgS protein from P. marina in a mixture containing 10 mMUDP-glucose and 10 mM 3-PGA (sodium salt) in 25 mM Tris-HCl buffer (pH8.0) with 1 mM MnCl2. The synthesis proceeded for 30 min at 70°C. Afterremoval of denatured host proteins by centrifugation, the GPG-containing su-pernatant was used for the GpgP assays. MPG was synthesized using the partiallypurified recombinant MpgS protein from T. thermophilus as previously described,and the MPG-containing supernatant was used for the GpgP assays (9). Theconcentrations of GPG and MPG were determined as previously described (9).

Enzyme assays during purification of proteins. The activity of GpgS in E. colicell extracts and during purification was detected in reaction mixtures (50 �l)containing 15 �l cell extract, 5.0 mM (each) of UDP-glucose and 3-PGA, and 10mM MgCl2 in 25 mM bis-Tris-propane (BTP) buffer (pH 8.0) which were incu-bated at 90°C for 5 min, cooled on ice, and treated with 2 U of alkalinephosphatase (Sigma-Aldrich) for 20 min at 37°C. The products were visualized bythin-layer chromatography (TLC) (14). Cell extracts from E. coli carrying theempty vector were used as negative controls. Standards of GG, GPG, D-glucose,3-PGA, and UDP-glucose were used for comparative purposes.

The activity of GpgP was detected in reaction mixtures (50 �l) containing 15�l of the eluted fraction, 1.0 mM MPG, 25 mM morpholineethanesulfonic acid(MES) buffer (pH 6.0), and 10 mM MgCl2 at 85°C for 5 min and cooled on ice.All products were visualized by TLC, and standards of MG and MPG were usedfor comparative purposes (6).

Purification of recombinant GpgS. The His-tagged recombinant GpgS proteinwas partially purified in a prepacked Ni-Sepharose high-performance column(His-Prep 5 ml) equilibrated with 20 mM sodium phosphate, pH 7.4, 0.5 M NaCl,and 20 mM imidazole. Elution was carried out with a linear imidazole gradient(20 to 500 mM), and activity was located as described above. The purity of thefractions was determined by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE). The purest active pool was concentrated by ultracentrif-ugation (30-kDa cutoff) and equilibrated with 20 mM Tris-HCl, pH 8.0. Thesample was loaded onto a Q-Sepharose fast-flow column (Hi-Load FF 16/10)equilibrated with the same buffer and eluted by a linear gradient of NaCl (0.0 to1.0 M). Active fractions were concentrated by ultracentrifugation (30-kDa cut-off), equilibrated with 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl, and loadedinto a Superdex 200 fast-flow column equilibrated with the same buffer. Activefractions were concentrated by ultracentrifugation (30-kDa cutoff) and equili-brated with 50 mM Tris-HCl, pH 8.0, and the purity of the fractions was deter-mined by SDS-PAGE.

To determine the effect of Ni2� ions on His-tagged GpgS, an aliquot of theenzyme was treated with enterokinase according to the manufacturer’s protocol(Novagen) and loaded onto a Ni-Sepharose high-performance column equili-brated as described above. The flowthrough containing the enzyme without theHis tag was concentrated and equilibrated as described above, and the decreasein molecular weight of the protein was assessed by SDS-PAGE.

Purification of recombinant GpgP. The His-tagged recombinant GpgP proteinwas partially purified as described above. Active fractions were equilibrated with20 mM MES, pH 6.0, loaded onto a Q-Sepharose fast-flow column equilibratedwith the same buffer, and eluted by a linear gradient of NaCl (0.0 to 1.0 M). Theactive fractions were concentrated and equilibrated with 50 mM MES, pH 6.0.The purity of the fractions was determined by SDS-PAGE. Removal of the Histag and purification of the recombinant GpgP protein were carried out as de-scribed for GpgS.

Characterization of recombinant GpgS. The substrate specificity of GpgS wasdetermined using ADP-ribose, UDP-acetylgalactosamine, UDP-glucuronic acid,UDP-galactose, GDP-fucose, GDP-mannose, UDP-mannose, ADP-mannose,GDP-glucose, UDP-glucose, TDP-glucose, and ADP-glucose as possible sugar do-nors and glycerol, 3-PGA, D-2-phosphoglycerate, L-glycerol-3-phosphate, 2,3-diphos-pho-D-glycerate, and phosphoenolpyruvate as possible sugar acceptors (all fromSigma-Aldrich). Reaction mixtures (50 �l) containing 15 �l cell extract, a 2.5 mM

concentration of each substrate, and 10 mM MgCl2 in 25 mM Tris-HCl buffer (pH8.0) were incubated at 70°C for 15 min, followed by incubation with 2 U of alkalinephosphatase for 20 min at 37°C. The products were visualized by TLC. Cell extractsfrom E. coli carrying the empty vector were used as negative controls.

Temperature and pH profiles of GpgS, effects of cations on the protein, andthe thermostability of GpgS were determined by the addition of 0.05 �g of GpgSand an excess of MpgP (2.0 �g) from T. thermophilus HB27 to reaction mixturescontaining the appropriate buffer, 2.0 mM UDP-glucose, and 2.0 mM 3-PGA,with the reactions being stopped at different times by cooling on ethanol-ice, andfree phosphate was quantified with the Ames test (2, 9). The specific activity foreach sugar donor used was determined as described above, with a 2.5 mMconcentration of each substrate at 70°C in 25 mM Tris-HCl, pH 8.0. The tem-perature profile for GpgS was determined between 30 and 100°C in 25 mMTris-HCl buffer (pH 8.0) with 1.0 mM MnCl2. The effects of pH on the activitieswere determined in 25 mM acetate buffer (pH 4.0 to 5.5), 25 mM MES buffer(pH 6.0 to 6.5), BTP buffer (pH 6.5 to 9.5), 25 mM Tris-HCl buffer (pH 7.0 to9.0), and 25 mM CAPS buffer (3-[cyclohexylamino]-1-propanesulfonic acid; pH9.0 to 11.0), with 1.0 mM MnCl2, at 90°C for GpgS. All pH values were measuredat room temperature (25°C); pH values at 85 and 90°C were calculated using aconversion factor (�pKa/�T [°C]) of �0.02 for acetate buffer, 0.015 for BTPbuffer, and 0.03 for Tris-HCl buffer (19, 29). The effects of cations on theactivity of the enzyme were examined by incubating the sample with the appro-priate substrates, with a 0.01 mM, 0.1 mM, 1 mM, 10 mM, or 100 mM concen-tration of the chloride salt of Mg2�, Mn2�, Co2�, Ni2�, K�, Ca2�, Sr2�, Cu2�,Ba2�, or Zn2�, without cations, or with 0.1 or 2.0 mM of EDTA (4, 17) at 90°C.To determine the effect of Ni2� ions on the His-tagged recombinant GpgSprotein, an aliquot of the enzyme was treated with enterokinase as describedabove, and the activity was determined at 70°C in 25 mM Tris-HCl buffer at pH8.0, with a 0.1, 1.0, or 10 mM Ni2� concentration. The thermal stability wasdetermined at 70 and 90°C in 25 mM Tris-HCl (pH 8.0) and examined forresidual activities at 50°C and 70°C, respectively.

The kinetic parameters of GpgS were determined by the dephosphorylation ofGPG formed in a coupled enzyme assay where the amount of phosphate releasedwas quantified (2). The Km value for each substrate of GpgS was determined asfollows: the reactions were initiated by the addition of 0.05 �g GpgS to 25 mMTris-HCl solutions (pH 8.0) with 1.0 mM MnCl2 containing either UDP-glucose(1.0 to 5.0 mM) plus 3-PGA (5.0 mM) or UDP-glucose (5.0 mM) plus 3-PGA(0.05 to 5.0 mM) and an excess of MpgP (5.0 �g) from T. thermophilus (9). Thereactions were performed at 70°C, 85°C, and 90°C and were stopped at 1-minintervals by cooling on ice-ethanol. All experiments were performed in triplicate.

The molecular mass of the recombinant GpgS protein was estimated by gelfiltration on a Superdex 200 column, with the molecular mass standards albumin(67 kDa), aldolase (158 kDa), catalase (232 kDa), ferritin (440 kDa), and thy-roglobulin (669 kDa), and blue dextran 2000 was used to determine the voidvolume (Amersham).

Characterization of recombinant GpgP. Several sugar phosphates, namely,GPG, MPG, mannose-1-phosphate, mannose-6-phosphate, glucose-1-phos-phate, glucose-6-phosphate, glucose-1,6-bisphosphate, fructose-1-phosphate,fructose-6-phosphate, and trehalose-6-phosphate, as well as GDP and GMP (allfrom Sigma-Aldrich), were examined as possible substrates for GpgP in mixturescontaining a 2.5 mM concentration of each substrate, 25 mM MES buffer (pH6.0), and 10 mM MgCl2. The mixtures were incubated at 70°C for 10 min andcooled on ice, followed by TLC separation (14). Cell extracts from E. colicontaining the plasmid without insert were used as negative controls.

To determine the temperature profile, pH dependence, and thermal stabilityof GpgP and the effects of cations on the protein, the activity of the enzyme wascalculated based on the release of inorganic phosphate from GPG and MPG withthe following protocol: reactions were initiated by the addition of 1.0 �g of GpgPto reaction mixtures containing 2.0 mM MPG or of 0.05 �g of GpgP to reactionmixtures containing 2.0 mM GPG in the appropriate buffer, with the reactionsbeing stopped at different times by cooling on ethanol-ice. The free phosphatewas quantified by the Ames reaction (2). Temperature profiles were determinedbetween 30 and 100°C in 25 mM MES buffer (pH 6.0) with 0.01 mM MgCl2 forboth substrates. The effects of pH and cations on the activity of GpgP weredetermined at 85°C as described above. The thermal stability was determined at70 and 85°C by incubation of GpgP aliquots (20 �l of a solution at 0.7 �g/�l) in25 mM MES (pH 6.0) for the assays described above. At appropriate times,samples were withdrawn and immediately examined for residual activities at50°C and 70°C for the thermal stability at 70°C and 85°C, respectively.

Kinetic parameters of GpgP using GPG as a substrate were determined in reac-tion mixtures containing GPG (0.3 to 2.0 mM) in 25 mM BTP buffer (pH 7.0) with0.1 mM MgCl2 and 0.0125 �g of the enzyme at 85°C or 0.025 �g of the enzyme at70°C. The kinetic parameters of GpgP using MPG as a substrate were determined

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in reaction mixtures containing 0.25 �g of the enzyme and MPG (0.3 to 2.0 mM) in25 mM BTP buffer (pH 7.0) with 10 mM MgCl2 at 70°C, 85°C, and 90°C. The proteincontents of the samples were determined by the Bradford assay (7). The molecularmass of the recombinant GpgP protein was estimated by gel filtration as describedfor GpgS. All experiments were performed in triplicate.

RESULTS

Identification of genes encoding GpgS and GpgP in P.marina. Based on the amino acid sequences of GpgS and GpgPfrom M. burtonii, BLAST searches were performed with the P.marina genome database, resulting in the identification of twoopen reading frames with high homology to gpgS and gpgPgenes, which were then functionally expressed in E. coli. TheGpgS gene contains 1,212 bp, coding for a polypeptide with403 amino acids and a calculated molecular mass of 46.7 kDa.Gel filtration experiments indicated that the recombinant His-tagged GpgS protein behaves as a dimeric protein with a mo-lecular mass of around 105.4 4.3 kDa.

The alignment of all GpgS homologues revealed high se-quence conservation, and two highly conserved motifs werealso identified (9). In the conserved region F-R-Y-P/A-L-A/S-G-E-F-A, the amino acid P/A was replaced by a threonine, andin the conserved region W-G-L-E-X-G-X-L, a serine replacedthe second glycine. GpgS had 39% amino acid identity with thehomologue from M. burtonii but had higher homology (63%) withthe putative GpgS proteins from Syntrophus aciditrophicus SB(YP_462851) and Psychrobacter cryohalolentis K5 (YP_579265).

Conserved D-X-D-X-T/V-X and G-D-X-X-X-D motifs ofthe “DDDD” phosphohydrolase superfamily that are found inknown MpgPs and GpgPs were also present in the putativeGpgP protein from P. marina (9, 38). The GpgP gene contains819 bp, encoding a protein with 271 amino acids and a calcu-lated molecular mass of 31.2 kDa. Upon gel filtration, therecombinant His-tagged GpgP protein had a molecular mass ofaround 39.16 1.6 kDa, indicating that the phosphatase ismonomeric.

This protein exhibited 40% amino acid identity with thehomologue from M. burtonii but had 48 and 43% homologieswith putative GpgPs from S. aciditrophicus (YP_462853) and P.cryohalolentis (YP_579264), respectively.

Different genetic organizations of putative gpgS and gpgPgenes have been encountered previously, but the putative

operon-like organization that includes these genes in P. marinais different and more complex. An operon structure containingthe pstSCAB and phoU genes, encoding a high-affinity phos-phate (Pi)-specific transporter (Pst) system (23, 37), is locatedupstream of a second operon-like structure. This presumedoperon contains a putative phoR histidine kinase gene, which isknown to be part of the phosphate regulon (31, 37), a gpgPgene, a putative glycerate kinase gene, a putative glucosyltrans-ferase gene, and a gpgS gene (Fig. 1).

Cloning, functional overexpression of gpgS and gpgP, andpurification of recombinant enzymes. PCR amplification ofgpgS and gpgP from P. marina yielded products with the ex-pected gene sizes and sequences (AGOWA). Activity assayscarried out with GpgS- and GpgP-containing E. coli cell ex-tracts showed synthesis and dephosphorylation of GPG, re-spectively, and the negative control extracts from E. coli bear-ing the empty vector did not. GpgS and GpgP were purified tohomogeneity, as judged by SDS-PAGE (data not shown).

Catalytic properties of GpgS. Unlike the GpgS protein fromM. burtonii, which was specific for GDP-glucose, this GpgS wasmost active with UDP-glucose (94 �mol/min�mg), followed byGDP-glucose (41 �mol/min�mg), ADP-glucose (10 �mol/min�mg), and to a lesser extent, TDP-glucose (5 �mol/min�mg)(9). Activity was not observed for any other sugar donor tested,and 3-PGA was the only acceptor for these glucosyl donors.

Kinetic experiments showed that GpgS exhibited Michaelis-Menten kinetics at 70 and 90°C (Table 1). Identical Km valuesfor UDP-glucose were obtained at both temperatures. Com-paring the Km values at 70 and 90°C for 3-PGA, a 2.5-foldincrease was observed with the temperature upshift (Table 1).

GpgS was inactive at 40°C, but the activity increased dra-matically above 50°C, to a maximum at 90°C (Fig. 2A). At100°C, GpgS retained 33% of the total activity. The half-livesdetermined at 70 and 90°C were 14 and 2.3 min, respectively.Within the pH range examined, the activity of the enzyme wasmaximal near pH 8.0 (Fig. 2B). The lack of activity upon theaddition of EDTA indicated that GpgS was strictly depen-dent on divalent cations, in the following order of efficiency:Mn2� � Co2� � Mg2� � Ni2�. The specific activities ob-tained with Ni2� were similar for both the His-tagged en-zyme and the nontagged recombinant enzyme.

The maximum activation was obtained with 1.0 mM Mn2�,

FIG. 1. Genomic organization and flanking regions of gpgS and gpgP genes from P. marina strain EX-H1T. Arrows represent genes and theirdirections. The putative promoter regions upstream of pstS and phoR, predicted by using a prokaryotic promoter database (www.fruitfly.org/seq_tools/promoter.html), as well as the region between phoR and gpgP, are shown in boxes. The presumed start and stop codons are indicated in bold,and the ribosome-binding site is underlined. The putative high-affinity Pi transporter system pstSCAB contains the following genes, which encodethe indicated proteins: pstS, putative Pi binding periplasmic protein PstS; pstC, putative Pi transport permease protein PstC; pstA, putative Pitransport permease protein PstA; pstB, putative Pi transport permease protein PstB; phoU, putative Pi system regulator protein PhoU; phoR,putative PhoR histidine kinase from a phosphatase regulon; gpgP, glucosyl-3-phosphoglycerate phosphatase; gK, putative glycerate kinase; gT,putative glucosyltransferase; and gpgS, glucosyl-3-phosphoglycerate synthase.

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and 50% of the maximum activity was reached with 0.19 0.06mM Mn2�. Other cations tested, such as K�, Ca2�, Sr2�,Cu2�, Ba2�, and Zn2�, did not stimulate GpgS activity at anyconcentration (results not shown).

Catalytic properties of GpgP. Of the substrates tested, GpgPdephosphorylated both GPG and MPG, with maximal activityat 85°C (Fig. 2A). At 100°C, the enzyme still retained 15% ofthe maximum activity for both substrates (Fig. 2A). We wereunable to detect activity with MPG below 40°C; however, theenzyme retained about 20% of the maximal activity below 40°Cwhen GPG was used as a substrate (Fig. 2A). The half-livesdetermined at 70 and 85°C were 462 and 12 min, respectively.The optimum pH range for activity was near 7.0 for GPG andMPG alike, with trace activities at pH 5.0 and 9.0 (Fig. 2B).GpgP exhibited Michaelis-Menten kinetics for both substratesat 70 and 85°C. Identical Km values for GPG were obtained atboth temperatures, but the apparent Vmax at 85°C was 3.0-foldhigher. When MPG was the substrate, the Km value decreased2.2-fold from 70 to 85°C, but the Vmax values were comparable(Table 1).

GpgP was strictly dependent on divalent cations, since noactivity was detected with EDTA added to the reaction mixture

containing GPG or MPG. In fact, very small amounts of Mg2�

(0.01 mM) were sufficient to promote the dephosphorylation ofGPG by GpgP. The maximum activation was obtained with 0.1mM Mg2�, and 50% of the maximum activity was reached with0.003 0.0006 mM Mg2�. The enzyme used Co2� and, to alesser extent, Mn2� for both substrates. On the other hand,using MPG, the maximum activation was obtained with 10 mMMg2�, and 50% of the maximum activity was reached with0.79 0.28 mM Mg2�. Other cations tested, such as K�, Ca2�,Sr2�, Cu2�, Ba2�, and Zn2�, did not stimulate GpgP activity atany concentration.

DISCUSSION

Thermophiles and hyperthermophiles accumulate severalcompatible solutes that have not been found, or are rarelyencountered, in mesophilic prokaryotes, leading to the viewthat the compatible solutes of (hyper)thermophiles are specif-ically associated with life at high temperatures. These includedi-myo-inositol phosphate (DIP), MG, mannosylglyceramide,di-mannosyl-di-myo-inositol phosphate (DMDIP), and diglyc-erol phosphate (35).

The thermophilic bacterium Persephonella marina is classi-fied as a member of the class Aquificae, which along with thespecies of class Thermotogae, represents the deepest phyloge-netic branches within the domain Bacteria. Some species of thegenera Aquifex and Thermotoga have an upper temperature forgrowth of about 90°C and are slightly halophilic. Aquifexaerophilus has been shown to accumulate �-glutamate, �-glu-tamate, DIP, DMDIP, and glycerol-phosphoinositol (28). Thespecies Thermotoga maritima and Thermotoga neapolitana alsoaccumulate �-glutamate, �-glutamate, DIP, and DMDIP (30).Other thermotogales that grow at lower temperatures, such asThermosipho africanus, accumulate �-glutamate and proline,while Petrotoga miotherma accumulates primarily glycine be-taine, an unidentified compatible solute, and trehalose (30,35). The accumulation of GG was restricted to several speciesof mesophilic archaea and bacteria (8, 21, 26, 33), but thiscompatible solute was recently found to accumulate in thethermophilic bacterium P. marina (H. Santos, personal com-munication).

The synthesis of GG in P. marina proceeds via a two-steppathway, as in M. burtonii, which is common to other sugar-related compatible solutes, such as trehalose, sucrose, MG,and glucosylglycerol (9, 10, 14, 18, 22). In P. marina, gpgSand gpgP are contained in an operon-like structure in which

FIG. 2. Temperature (A) and pH (B) dependence of recombinantGpgS (■) and recombinant GpgP with GPG as the substrate (�) andwith MPG as the substrate (E) in P. marina. The data are the meanvalues for two independent experiments.

TABLE 1. Biochemical properties and kinetic parameters for the substrates of GpgS and GpgP involved in the synthesis ofglucosylglycerate in P. marinaa

Protein

Optimumfor

activityHalf-life (min)

Substrate

Km (mM) Vmax (�mol/min � mg)

Temp(°C) pH 70°C 85°C 90°C 70°C 85°C 90°C 70°C 85°C 90°C

GpgS 90 �8 14 2 2.3 UDP-Glu 1.47 0.13 1.55 0.08 1.58 0.21 66 4.3 98 3.8 106 6.03-PGA 0.09 0.04 0.25 0.06 0.3 0.02 51.5 5.2 110 2.1 128 7.3

GpgP 85 �7 462 10 12 3 GPG 0.36 0.02 0.41 0.05 0.53 0.08 97 2.0 294 4.3 288 5.6MPG 0.43 0.06 0.19 0.05 0.21 0.08 15.75 3.1 19.6 2.9 17.9 2.7

a Data are the mean values for at least three independent experiments.

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we identified genes for a glycerate kinase (gK), a glucosyl-transferase (gT), and a histidine kinase (PhoR). Glyceratekinase likely converts glycerate to 3-PGA, which is a pre-cursor of GPG (12). The putative glucosyltransferase hashomologues in Syntrophus aciditrophicus, Pyrococcus spp.,and Thermotoga maritima, but their function remains un-known. The putative histidine kinase is possibly involved inmonitoring the levels of extracellular phosphate by regulat-ing the pstSCAB operon, which is found immediately up-stream (23, 37). This operon is composed of genes of afamily of ATP-binding cassette transporters which are in-volved in phosphate uptake when it is limiting (31, 37). Theoperon-like structure immediately downstream of thepstSCAB operon contains gpgS and gpgP. The latter is lo-

cated downstream of phoR and overlaps its stop codon,suggesting that they are cotranscribed. Moreover, the single pro-moter region found upstream of phoR also leads to the idea thatphoR, gpgP, gK, gT, and gpgS are cotranscribed (Fig. 1). Wespeculate that both operons are functionally connected and thatGG synthesis may be controlled by phosphate levels in the envi-ronment. GG behaves as a compatible solute in Erwinia chrysan-themi under nitrogen-limiting conditions, replacing glutamate andglutamine, which accumulate under salt stress in the mediumwhen nitrogen is not limiting for growth (21). It is possible thatGG accumulates in P. marina during salt stress under conditionswhen Pi has to be mobilized for the synthesis of other cell com-ponents, explaining the apparent association between GG synthe-sis and the Pi uptake system.

FIG. 3. Unrooted phylogenetic tree based on available amino acid sequences of identified and putative GpgSs. Prokaryotes for which GG hasbeen detected are surrounded by black boxes, and archaeal proteins are underlined. The Clustal X program (39) was used for sequence alignment,and the MEGA3 program (27) was used to generate the phylogenetic tree. The significance of the branching order was evaluated by bootstrapanalysis of 1,000 computer-generated trees. The bootstrap values are indicated. Bar, 0.2 change/site. NCBI accession numbers are indicated in thefigure.

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GpgS and GpgP from P. marina and M. burtonii have severaldistinct biochemical and kinetic properties. The maximal ac-tivities of the P. marina enzymes occurred between 85 and 90°C(Fig. 2A), representing a thermal shift of 30°C over those of M.burtonii, as expected for a thermophilic organism. GpgS fromP. marina was rather nonspecific for glucosyl donors, usingseveral natural glucose diphosphate nucleosides, with UDP-glucose as the preferred substrate, in contrast with the totaldependence of the M. burtonii enzyme on GDP-glucose (9).This broad substrate specificity may reflect an absolute re-quirement of GG in P. marina, as observed for trehalose syn-thesis in mycobacteria (13).

The GpgPs of M. burtonii and P. marina and all MpgPsexamined dephosphorylate GPG and MPG, indicating thatthese phosphatases recognize a common determinant in thesesubstrates, probably the glycerylphosphate moiety (9). At 70°C,the optimal growth temperature for P. marina, the Km values ofGpgP for GPG and MPG were similar, but the apparent Vmax

value with GPG was more than six times higher than that withMPG. These findings strongly suggest a higher catalytic effi-ciency towards GPG. This trend was even more obvious at90°C, where the apparent Vmax towards GPG increased �15-fold compared with the Vmax for MPG considering that the Km

value for GPG was only slightly higher. The GpgP protein of P.marina appears, therefore, to be a “true” GPG phosphatasethat retains residual activity for MPG, unlike the GpgP proteinfrom M. burtonii and the MpgP protein from T. thermophilus,which have only slightly higher catalytic efficiencies for GPGover MPG at low temperatures but show no preference foreither substrate at high temperatures (9). However, the Km

value for MPG of the P. marina phosphatase decreased slightlywith increasing temperatures, even though MG was not de-tected in this organism. A similar phenomenon was also ob-served with inositol monophosphatases from Archaeoglobusfulgidus, where an 8- to 10-fold decrease in Km values wasobserved between 75 and 85°C and the binding of the sub-strates became significantly tighter at high temperatures (40).

Most GpgS sequences belong to bacteria; until now, onlyfour archaeal sequences for putative GpgSs have been identi-fied (Fig. 3). Two of these sequences, found in haloarchaea,form an outer group having significantly different amino acidsequences, while the archaeal proteins belonging to M. burtoniiand M. portucalensis have higher homologies with their bacte-rial counterparts. Putative GpgS sequences are found in manybacteria, with optimum growth temperatures ranging between8°C, for Colwellia psychrerythraea, to 70°C, for P. marina (11,20). Interestingly, the GpgS sequence from P. marina clusterswith those from the mesophilic organism Syntrophus aciditro-phicus and the psychrophilic organism Psychrobacter cryohalo-lentis (3, 24). Now that the enzymes for GG synthesis havebeen identified and characterized, research should focus on thespecific role or roles of GG in cell physiology, since they re-main elusive for psychrophiles and thermophiles.

ACKNOWLEDGMENTS

This work was supported by the European Commission, under 6thFramework Programme contract COOP-CT-2003-508644, and byFEDER and FCT, Portugal, under project no. POCI2010/010.6/A005/2005. J. Costa and N. Empadinhas acknowledge scholarships fromFCT (SFRH/BD/9681/2002 and SFRH/BPD/14828/2003).

We thank Helena Santos (ITQB, Oeiras, Portugal) for informationon the accumulation of glucosylglycerate in Persephonella marina.

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