This Article Abstract Full Text (PDF) Other Versions of this Article: JB.00841-06v1 189/5/1648    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Costa, J. Articles by da Costa, M. S. Search for Related Content PubMed PubMed Citation Articles by Costa, J. Articles by da Costa, M. S.

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

Centro de Neurociências e Biologia Celular, Departamento de Zoologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal,¹ Departamento de Bioquímica, Universidade de Coimbra, 3001-401 Coimbra, Portugal²

Received 13 June 2006/ Accepted 7 December 2006

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

 
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-phosphoglycerate (GPG), which is subsequently converted to GG by a GPG phosphatase (GpgP) (9). The most common pathway for the synthesis of MG also proceeds via a phosphorylated intermediate which is dephosphorylated by mannosylglycerate-3-phosphoglycerate (MPG) phosphatase. The MPG phosphatases of Thermus thermophilus and Pyrococcus horikoshii share high amino acid identity with GpgP from M. burtonii and dephosphorylate MPG and GPG (5, 9, 14, 15). The present work describes the characterization of GpgS and GpgP, which catalyze the synthesis of GG at high temperatures in P. marina, and is the first report of the characteristics of these enzymes in a thermophilic organism.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strains, identification, cloning, and functional overexpression of gpgS and gpgP from Persephonella marina. P. marina strain EX-H1^T (DSM 14350^T) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. To identify the genes responsible for GG biosynthesis in P. marina, we used the sequences of glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from M. burtonii for BLAST searches of the P. marina genome. Preliminary sequence data were obtained from TIGR at http://www.tigr.org.

Two open reading frames with high homology to the gpgS and gpgP genes from M. burtonii were detected. The full gpgS and gpgP genes were amplified by using forward and reverse primers. Chromosomal DNA of P. marina was isolated according to the method of Rainey et al. (32). Forward primers were constructed with additional NcoI recognition sequences, reverse primers were constructed by adding XhoI sites, and PCR amplifications were carried out as previously described (9). Amplification products were visualized on 1% agarose gels, purified by band excision (Promega), and after digestion with the suitable restriction enzymes, ligated into the corresponding sites of the expression vector pET-30a (Novagen), carrying an N-terminal His tag coding sequence. Most DNA manipulations followed standard molecular techniques (34). The constructs were sequenced by AGOWA (Berlin, Germany).

Escherichia coli BL21 was used as the host for overexpression. E. coli cells carrying the plasmids were grown to the mid-exponential growth phase (optical density at 610 nm = 1.0) in LB medium at 37°C, induced with 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG), and grown for another 5 h. Kanamycin was added to a final concentration of 30 µg/ml. Cells were harvested by centrifugation (7,000 x g, 10 min, 4°C), and pellets were suspended in 20 mM sodium phosphate buffer, pH 7.4, with 0.5 M NaCl, 20 mM imidazole, and a protease inhibitor cocktail (Roche), frozen, thawed, and disrupted by sonication, followed by centrifugation (18,000 x g, 30 min, 4°C) to remove debris.

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

Synthesis of GPG and MPG. GPG was synthesized using the partially purified recombinant GpgS protein from P. marina in a mixture containing 10 mM UDP-glucose and 10 mM 3-PGA (sodium salt) in 25 mM Tris-HCl buffer (pH 8.0) with 1 mM MnCl₂. The synthesis proceeded for 30 min at 70°C. After removal of denatured host proteins by centrifugation, the GPG-containing supernatant was used for the GpgP assays. MPG was synthesized using the partially purified recombinant MpgS protein from T. thermophilus as previously described, and the MPG-containing supernatant was used for the GpgP assays (9). The concentrations of GPG and MPG were determined as previously described (9).

Enzyme assays during purification of proteins. The activity of GpgS in E. coli cell extracts and during purification was detected in reaction mixtures (50 µl) containing 15 µl cell extract, 5.0 mM (each) of UDP-glucose and 3-PGA, and 10 mM MgCl₂ in 25 mM bis-Tris-propane (BTP) buffer (pH 8.0) which were incubated at 90°C for 5 min, cooled on ice, and treated with 2 U of alkaline phosphatase (Sigma-Aldrich) for 20 min at 37°C. The products were visualized by thin-layer chromatography (TLC) (14). Cell extracts from E. coli carrying the empty 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 µl) containing 15 µl 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 used for comparative purposes (6).

Purification of recombinant GpgS. The His-tagged recombinant GpgS protein was 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 the fractions was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purest active pool was concentrated by ultracentrifugation (30-kDa cutoff) and equilibrated with 20 mM Tris-HCl, pH 8.0. The sample 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 to 1.0 M). Active fractions were concentrated by ultracentrifugation (30-kDa cutoff), equilibrated with 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl, and loaded into a Superdex 200 fast-flow column equilibrated with the same buffer. Active fractions were concentrated by ultracentrifugation (30-kDa cutoff) and equilibrated with 50 mM Tris-HCl, pH 8.0, and the purity of the fractions was determined by SDS-PAGE.

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

Purification of recombinant GpgP. The His-tagged recombinant GpgP protein was partially purified as described above. Active fractions were equilibrated with 20 mM MES, pH 6.0, loaded onto a Q-Sepharose fast-flow column equilibrated with the same buffer, and eluted by a linear gradient of NaCl (0.0 to 1.0 M). The active 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 His tag and purification of the recombinant GpgP protein were carried out as described for GpgS.

Characterization of recombinant GpgS. The substrate specificity of GpgS was determined 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 donors and glycerol, 3-PGA, D-2-phosphoglycerate, L-glycerol-3-phosphate, 2,3-diphospho-D-glycerate, and phosphoenolpyruvate as possible sugar acceptors (all from Sigma-Aldrich). Reaction mixtures (50 µl) containing 15 µl cell extract, a 2.5 mM concentration of each substrate, and 10 mM MgCl₂ in 25 mM Tris-HCl buffer (pH 8.0) were incubated at 70°C for 15 min, followed by incubation with 2 U of alkaline phosphatase for 20 min at 37°C. The products were visualized by TLC. Cell extracts from E. coli carrying the empty vector were used as negative controls.

Temperature and pH profiles of GpgS, effects of cations on the protein, and the thermostability of GpgS were determined by the addition of 0.05 µg of GpgS and an excess of MpgP (2.0 µg) from T. thermophilus HB27 to reaction mixtures containing 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, and free phosphate was quantified with the Ames test (2, 9). The specific activity for each sugar donor used was determined as described above, with a 2.5 mM concentration of each substrate at 70°C in 25 mM Tris-HCl, pH 8.0. The temperature profile for GpgS was determined between 30 and 100°C in 25 mM Tris-HCl buffer (pH 8.0) with 1.0 mM MnCl₂. The effects of pH on the activities were 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 to 9.0), and 25 mM CAPS buffer (3-[cyclohexylamino]-1-propanesulfonic acid; pH 9.0 to 11.0), with 1.0 mM MnCl₂, at 90°C for GpgS. All pH values were measured at room temperature (25°C); pH values at 85 and 90°C were calculated using a conversion factor (pK_a/T [°C]) of +0.02 for acetate buffer, –0.015 for BTP buffer, and –0.03 for Tris-HCl buffer (19, 29). The effects of cations on the activity of the enzyme were examined by incubating the sample with the appropriate substrates, with a 0.01 mM, 0.1 mM, 1 mM, 10 mM, or 100 mM concentration 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 GpgS protein, an aliquot of the enzyme was treated with enterokinase as described above, and the activity was determined at 70°C in 25 mM Tris-HCl buffer at pH 8.0, with a 0.1, 1.0, or 10 mM Ni²⁺ concentration. The thermal stability was determined at 70 and 90°C in 25 mM Tris-HCl (pH 8.0) and examined for residual activities at 50°C and 70°C, respectively.

The kinetic parameters of GpgS were determined by the dephosphorylation of GPG formed in a coupled enzyme assay where the amount of phosphate released was quantified (2). The K_m value for each substrate of GpgS was determined as follows: the reactions were initiated by the addition of 0.05 µg GpgS to 25 mM Tris-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 µg) from T. thermophilus (9). The reactions were performed at 70°C, 85°C, and 90°C and were stopped at 1-min intervals by cooling on ice-ethanol. All experiments were performed in triplicate.

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

Characterization of recombinant GpgP. Several sugar phosphates, namely, GPG, MPG, mannose-1-phosphate, mannose-6-phosphate, glucose-1-phosphate, glucose-6-phosphate, glucose-1,6-bisphosphate, fructose-1-phosphate, fructose-6-phosphate, and trehalose-6-phosphate, as well as GDP and GMP (all from Sigma-Aldrich), were examined as possible substrates for GpgP in mixtures containing a 2.5 mM concentration of each substrate, 25 mM MES buffer (pH 6.0), and 10 mM MgCl₂. The mixtures were incubated at 70°C for 10 min and cooled on ice, followed by TLC separation (14). Cell extracts from E. coli containing the plasmid without insert were used as negative controls.

To determine the temperature profile, pH dependence, and thermal stability of GpgP and the effects of cations on the protein, the activity of the enzyme was calculated based on the release of inorganic phosphate from GPG and MPG with the following protocol: reactions were initiated by the addition of 1.0 µg of GpgP to reaction mixtures containing 2.0 mM MPG or of 0.05 µg of GpgP to reaction mixtures containing 2.0 mM GPG in the appropriate buffer, with the reactions being stopped at different times by cooling on ethanol-ice. The free phosphate was quantified by the Ames reaction (2). Temperature profiles were determined between 30 and 100°C in 25 mM MES buffer (pH 6.0) with 0.01 mM MgCl₂ for both substrates. The effects of pH and cations on the activity of GpgP were determined at 85°C as described above. The thermal stability was determined at 70 and 85°C by incubation of GpgP aliquots (20 µl of a solution at 0.7 µg/µl) in 25 mM MES (pH 6.0) for the assays described above. At appropriate times, samples were withdrawn and immediately examined for residual activities at 50°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 reaction mixtures containing GPG (0.3 to 2.0 mM) in 25 mM BTP buffer (pH 7.0) with 0.1 mM MgCl₂ and 0.0125 µg of the enzyme at 85°C or 0.025 µg of the enzyme at 70°C. The kinetic parameters of GpgP using MPG as a substrate were determined in reaction mixtures containing 0.25 µg of the enzyme and MPG (0.3 to 2.0 mM) in 25 mM BTP buffer (pH 7.0) with 10 mM MgCl₂ at 70°C, 85°C, and 90°C. The protein contents of the samples were determined by the Bradford assay (7). The molecular mass of the recombinant GpgP protein was estimated by gel filtration as described for GpgS. All experiments were performed in triplicate.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of genes encoding GpgS and GpgP in P. marina. Based on the amino acid sequences of GpgS and GpgP from M. burtonii, BLAST searches were performed with the P. marina genome database, resulting in the identification of two open reading frames with high homology to gpgS and gpgP genes, which were then functionally expressed in E. coli. The GpgS gene contains 1,212 bp, coding for a polypeptide with 403 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 molecular mass of around 105.4 ± 4.3 kDa.

The alignment of all GpgS homologues revealed high sequence conservation, and two highly conserved motifs were also 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, and in the conserved region W-G-L-E-X-G-X-L, a serine replaced the second glycine. GpgS had 39% amino acid identity with the homologue from M. burtonii but had higher homology (63%) with the 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 of the "DDDD" phosphohydrolase superfamily that are found in known MpgPs and GpgPs were also present in the putative GpgP protein from P. marina (9, 38). The GpgP gene contains 819 bp, encoding a protein with 271 amino acids and a calculated molecular mass of 31.2 kDa. Upon gel filtration, the recombinant His-tagged GpgP protein had a molecular mass of around 39.16 ± 1.6 kDa, indicating that the phosphatase is monomeric.

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

Different genetic organizations of putative gpgS and gpgP genes have been encountered previously, but the putative operon-like organization that includes these genes in P. marina is different and more complex. An operon structure containing the pstSCAB and phoU genes, encoding a high-affinity phosphate (P_i)-specific transporter (Pst) system (23, 37), is located upstream of a second operon-like structure. This presumed operon contains a putative phoR histidine kinase gene, which is known to be part of the phosphate regulon (31, 37), a gpgP gene, a putative glycerate kinase gene, a putative glucosyltransferase gene, and a gpgS gene (Fig. 1).

View larger version (9K): [in this window] [in a new window]   FIG. 1. Genomic organization and flanking regions of gpgS and gpgP genes from P. marina strain EX-H1T. Arrows represent genes and their directions. 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 encode the indicated proteins: pstS, putative Pi binding periplasmic protein PstS; pstC, putative Pi transport permease protein PstC; pstA, putative Pi transport 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.

Catalytic properties of GpgS. Unlike the GpgS protein from M. burtonii, which was specific for GDP-glucose, this GpgS was most active with UDP-glucose (94 µmol/min·mg), followed by GDP-glucose (41 µmol/min·mg), ADP-glucose (10 µmol/min·mg), and to a lesser extent, TDP-glucose (5 µmol/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 K_m values for UDP-glucose were obtained at both temperatures. Comparing the K_m values at 70 and 90°C for 3-PGA, a 2.5-fold increase was observed with the temperature upshift (Table 1).

View larger version (15K): [in this window] [in a new window]   FIG. 2. Temperature (A) and pH (B) dependence of recombinant GpgS () and recombinant GpgP with GPG as the substrate () and with MPG as the substrate () in P. marina. The data are the mean values for two independent experiments.

Catalytic properties of GpgP. Of the substrates tested, GpgP dephosphorylated both GPG and MPG, with maximal activity at 85°C (Fig. 2A). At 100°C, the enzyme still retained 15% of the maximum activity for both substrates (Fig. 2A). We were unable to detect activity with MPG below 40°C; however, the enzyme retained about 20% of the maximal activity below 40°C when GPG was used as a substrate (Fig. 2A). The half-lives determined at 70 and 85°C were 462 and 12 min, respectively. The optimum pH range for activity was near 7.0 for GPG and MPG alike, with trace activities at pH 5.0 and 9.0 (Fig. 2B). GpgP exhibited Michaelis-Menten kinetics for both substrates at 70 and 85°C. Identical K_m values for GPG were obtained at both temperatures, but the apparent V_max at 85°C was 3.0-fold higher. When MPG was the substrate, the K_m value decreased 2.2-fold from 70 to 85°C, but the V_max values were comparable (Table 1).

GpgP was strictly dependent on divalent cations, since no activity 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 of GPG by GpgP. The maximum activation was obtained with 0.1 mM Mg²⁺, and 50% of the maximum activity was reached with 0.003 ± 0.0006 mM Mg²⁺. The enzyme used Co²⁺ and, to a lesser extent, Mn²⁺ for both substrates. On the other hand, using MPG, the maximum activation was obtained with 10 mM Mg²⁺, and 50% of the maximum activity was reached with 0.79 ± 0.28 mM Mg²⁺. Other cations tested, such as K⁺, Ca²⁺, Sr²⁺, Cu²⁺, Ba²⁺, and Zn²⁺, did not stimulate GpgP activity at any concentration.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thermophiles and hyperthermophiles accumulate several compatible solutes that have not been found, or are rarely encountered, in mesophilic prokaryotes, leading to the view that the compatible solutes of (hyper)thermophiles are specifically associated with life at high temperatures. These include di-myo-inositol phosphate (DIP), MG, mannosylglyceramide, di-mannosyl-di-myo-inositol phosphate (DMDIP), and diglycerol phosphate (35).

The thermophilic bacterium Persephonella marina is classified as a member of the class Aquificae, which along with the species of class Thermotogae, represents the deepest phylogenetic branches within the domain Bacteria. Some species of the genera Aquifex and Thermotoga have an upper temperature for growth of about 90°C and are slightly halophilic. Aquifex aerophilus has been shown to accumulate ß-glutamate, -glutamate, DIP, DMDIP, and glycerol-phosphoinositol (28). The species Thermotoga maritima and Thermotoga neapolitana also accumulate ß-glutamate, -glutamate, DIP, and DMDIP (30). Other thermotogales that grow at lower temperatures, such as Thermosipho africanus, accumulate -glutamate and proline, while Petrotoga miotherma accumulates primarily glycine betaine, an unidentified compatible solute, and trehalose (30, 35). The accumulation of GG was restricted to several species of mesophilic archaea and bacteria (8, 21, 26, 33), but this compatible solute was recently found to accumulate in the thermophilic bacterium P. marina (H. Santos, personal communication).

The synthesis of GG in P. marina proceeds via a two-step pathway, 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, gpgS and gpgP are contained in an operon-like structure in which we identified genes for a glycerate kinase (gK), a glucosyltransferase (gT), and a histidine kinase (PhoR). Glycerate kinase likely converts glycerate to 3-PGA, which is a precursor of GPG (12). The putative glucosyltransferase has homologues in Syntrophus aciditrophicus, Pyrococcus spp., and Thermotoga maritima, but their function remains unknown. The putative histidine kinase is possibly involved in monitoring the levels of extracellular phosphate by regulating the pstSCAB operon, which is found immediately upstream (23, 37). This operon is composed of genes of a family of ATP-binding cassette transporters which are involved in phosphate uptake when it is limiting (31, 37). The operon-like structure immediately downstream of the pstSCAB operon contains gpgS and gpgP. The latter is located downstream of phoR and overlaps its stop codon, suggesting that they are cotranscribed. Moreover, the single promoter region found upstream of phoR also leads to the idea that phoR, gpgP, gK, gT, and gpgS are cotranscribed (Fig. 1). We speculate that both operons are functionally connected and that GG synthesis may be controlled by phosphate levels in the environment. GG behaves as a compatible solute in Erwinia chrysanthemi under nitrogen-limiting conditions, replacing glutamate and glutamine, which accumulate under salt stress in the medium when nitrogen is not limiting for growth (21). It is possible that GG accumulates in P. marina during salt stress under conditions when P_i has to be mobilized for the synthesis of other cell components, explaining the apparent association between GG synthesis and the P_i uptake system.

GpgS and GpgP from P. marina and M. burtonii have several distinct biochemical and kinetic properties. The maximal activities 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 from P. marina was rather nonspecific for glucosyl donors, using several natural glucose diphosphate nucleosides, with UDP-glucose as the preferred substrate, in contrast with the total dependence of the M. burtonii enzyme on GDP-glucose (9). This broad substrate specificity may reflect an absolute requirement of GG in P. marina, as observed for trehalose synthesis in mycobacteria (13).

The GpgPs of M. burtonii and P. marina and all MpgPs examined dephosphorylate GPG and MPG, indicating that these phosphatases recognize a common determinant in these substrates, probably the glycerylphosphate moiety (9). At 70°C, the optimal growth temperature for P. marina, the K_m values of GpgP for GPG and MPG were similar, but the apparent V_max value with GPG was more than six times higher than that with MPG. These findings strongly suggest a higher catalytic efficiency towards GPG. This trend was even more obvious at 90°C, where the apparent V_max towards GPG increased >15-fold compared with the V_max for MPG considering that the K_m value for GPG was only slightly higher. The GpgP protein of P. marina appears, therefore, to be a "true" GPG phosphatase that retains residual activity for MPG, unlike the GpgP protein from M. burtonii and the MpgP protein from T. thermophilus, which have only slightly higher catalytic efficiencies for GPG over MPG at low temperatures but show no preference for either substrate at high temperatures (9). However, the K_m value for MPG of the P. marina phosphatase decreased slightly with increasing temperatures, even though MG was not detected in this organism. A similar phenomenon was also observed with inositol monophosphatases from Archaeoglobus fulgidus, where an 8- to 10-fold decrease in K_m values was observed between 75 and 85°C and the binding of the substrates became significantly tighter at high temperatures (40).

Most GpgS sequences belong to bacteria; until now, only four archaeal sequences for putative GpgSs have been identified (Fig. 3). Two of these sequences, found in haloarchaea, form an outer group having significantly different amino acid sequences, while the archaeal proteins belonging to M. burtonii and M. portucalensis have higher homologies with their bacterial counterparts. Putative GpgS sequences are found in many bacteria, with optimum growth temperatures ranging between 8°C, for Colwellia psychrerythraea, to 70°C, for P. marina (11, 20). Interestingly, the GpgS sequence from P. marina clusters with those from the mesophilic organism Syntrophus aciditrophicus and the psychrophilic organism Psychrobacter cryohalolentis (3, 24). Now that the enzymes for GG synthesis have been identified and characterized, research should focus on the specific role or roles of GG in cell physiology, since they remain elusive for psychrophiles and thermophiles.

View larger version (29K): [in this window] [in a new window]   FIG. 3. Unrooted phylogenetic tree based on available amino acid sequences of identified and putative GpgSs. Prokaryotes for which GG has been 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 bootstrap analysis of 1,000 computer-generated trees. The bootstrap values are indicated. Bar, 0.2 change/site. NCBI accession numbers are indicated in the figure.

	ACKNOWLEDGMENTS

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

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

	FOOTNOTES

 
* Corresponding author. Mailing address: Departamento de Bioquímica, Universidade de Coimbra, 3001-401 Coimbra, Portugal. Phone: 351-239824024. Fax: 351-239826798. E-mail: milton{at}ci.uc.pt.

Published ahead of print on 22 December 2006.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Alarico, S., N. Empadinhas, C. Simões, Z. Silva, A. Henne, A. Mingote, H. Santos, and M. S. da Costa. 2005. Distribution of genes for the synthesis of trehalose and mannosylglycerate in Thermus spp. and direct correlation with halotolerance. Appl. Environ. Microbiol. 71:2460-2466.[Abstract/Free Full Text]
2. Ames, B. N. 1966. Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol. 8:115-118.
3. Bakermans, C., A. I. Tsapin, V. Souza-Egipsy, D. A. Gilichinsky, and K. H. Nealson. 2003. Reproduction and metabolism at –10°C of bacteria isolated from Siberian permafrost. Environ. Microbiol. 5:321-326.[CrossRef][Medline]
4. Battistoni, A., F. Pacello, A. P. Mazzetti, C. Capo, J. S. Kroll, P. R. Langford, A. Sansone, G. Donnarumma, P. Valenti, and G. Rotilio. 2001. A histidine-rich metal binding domain at the N terminus of Cu, Zn-superoxide dismutases from pathogenic bacteria. J. Biol. Chem. 276:30315-30325.[Abstract/Free Full Text]
5. Borges, N., J. D. Marugg, N. Empadinhas, M. S. da Costa, and H. Santos. 2004. Specialized roles of the two pathways for the synthesis of mannosylglycerate in osmoadaptation and thermoadaptation of Rhodothermus marinus. J. Biol. Chem. 279:9892-9898.[Abstract/Free Full Text]
6. Brabban, A. D., E. N. Orcutt, and S. H. Zinder. 1999. Interactions between nitrogen fixation and osmoregulation in the methanogenic archaeon Methanosarcina barkeri 227. Appl. Environ. Microbiol. 65:1222-1227.[Abstract/Free Full Text]
7. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254.[CrossRef][Medline]
8. Cánovas, D., N. Borges, C. Vargas, A. Ventosa, J. J. Nieto, and H. Santos. 1999. Role of N--acetyldiaminobutyrate as an enzyme stabilizer and an intermediate in the biosynthesis of hydroxyectoine. Appl. Environ. Microbiol. 65:3774-3779.[Abstract/Free Full Text]
9. Costa, J., N. Empadinhas, L. Gonçalves, P. Lamosa, H. Santos, and M. S. da Costa. 2006. Characterization of the biosynthetic pathway of glucosylglycerate in the archaeon Methanococcoides burtonii. J. Bacteriol. 188:1022-1030.[Abstract/Free Full Text]
10. Curatti, L., E. Folco, P. Desplats, G. Abratti, V. Limones, L. Herrera-Estrella, and G. Salerno. 1998. Sucrose-phosphate synthase from Synechocystis sp. strain PCC 6803: identification of the spsA gene and characterization of the enzyme expressed in Escherichia coli. J. Bacteriol. 180:6776-6779.[Abstract/Free Full Text]
11. D'Aoust, J. Y., and D. J. Kushner. 1972. Vibrio psychroerythrus sp. n.: classification of the psychrophilic marine bacterium, NRC 1004. J. Bacteriol. 111:340-342.[Abstract/Free Full Text]
12. Doughty, C. C., J. A. Hayashi, and H. L. Guenther. 1966. Purification and properties of D-glycerate 3-kinase from Escherichia coli. J. Biol. Chem. 241:568-572.[Abstract/Free Full Text]
13. Elbein, A. D., Y. T. Pan, I. Pastuszak, and D. Carroll. 2003. New insights on trehalose: a multifunctional molecule. Glycobiology 13:17R-27R.[Abstract/Free Full Text]
14. Empadinhas, N., J. D. Marugg, N. Borges, H. Santos, and M. S. da Costa. 2001. Pathway for the synthesis of mannosylglycerate in the hyperthermophilic archaeon Pyrococcus horikoshii. Biochemical and genetic characterization of key enzymes. J. Biol. Chem. 276:43580-43588.[Abstract/Free Full Text]
15. Empadinhas, N., L. Albuquerque, A. Henne, H. Santos, and M. S. da Costa. 2003. The bacterium Thermus thermophilus, like hyperthermophilic archaea, uses a two-step pathway for the synthesis of mannosylglycerate. Appl. Environ. Microbiol. 69:3272-3279.[Abstract/Free Full Text]
16. Empadinhas, N., L. Albuquerque, J. Costa, S. H. Zinder, M. A. S. Santos, H. Santos, and M. S. da Costa. 2004. A gene from the mesophilic bacterium Dehalococcoides ethenogenes encodes a novel mannosylglycerate synthase. J. Bacteriol. 186:4075-4084.[Abstract/Free Full Text]
17. Ge, R., R. M. Watt, X. Sun, J. A. Tanner, Q.-Y. He, J.-D. Huang, and H. Sun. 2006. Expression and characterization of a histidine-rich protein, Hpn: potential for Ni²⁺ storage in Helicobacter pylori. Biochem. J. 393:285-293.[CrossRef][Medline]
18. Giæver, H. M., O. B. Styrvold, I. Kaasen, and A. R. Strom. 1988. Biochemical and genetic characterization of osmoregulatory trehalose synthesis in Escherichia coli. J. Bacteriol. 170:2841-2849.[Abstract/Free Full Text]
19. Good, N. E., G. D. Winget, W. Winter, T. N. Connolly, S. Izawa, and R. M. Singh. 1966. Hydrogen ion buffers for biological research. Biochemistry 5:467-477.[CrossRef][Medline]
20. Gotz, D., A. Banta, T. J. Beveridge, A. I. Rushdi, B. R. Simoneit, and A.-L. Reysenbach. 2002. Persephonella marina gen. nov., sp. nov. and Persephonella guaymasensis sp. nov., two novel, thermophilic, hydrogen-oxidizing microaerophiles from deep-sea hydrothermal vents. Int. J. Syst. Evol. Microbiol. 52:1349-1359.[Abstract]
21. Goude, R., S. Renaud, S. Bonnassie, T. Bernard, and C. Blanco. 2004. Glutamine, glutamate, and -glucosylglycerate are the major osmotic solutes accumulated by Erwinia chrysanthemi strain 3937. Appl. Environ. Microbiol. 70:6535-6541.[Abstract/Free Full Text]
22. Hagemann, M., U. Effmert, T. Kerstan, A. Schoor, and N. Erdmann. 2001. Biochemical characterization of glucosylglycerol-phosphate synthase of Synechocystis sp. strain PCC 6803: comparison of crude, purified, and recombinant enzymes. Curr. Microbiol. 43:278-283.[CrossRef][Medline]
23. Hirani, T. A., I. Suzuki, N. Murata, H. Hayashi, and J. J. Eaton-Rye. 2001. Characterization of a two-component signal transduction system involved in the induction of alkaline phosphatase under phosphate-limiting conditions in Synechocystis sp. PCC 6803. Plant Mol. Biol. 45:133-144.[CrossRef][Medline]
24. Jackson, B. E., V. K. Bhupathiraju, R. S. Tanner, C. R. Woese, and M. J. McInerney. 1999. Syntrophus aciditrophicus sp. nov., a new anaerobic bacterium that degrades fatty acids and benzoate in syntrophic association with hydrogen-using microorganisms. Arch. Microbiol. 171:107-114.[CrossRef][Medline]
25. Karsten, U., J. A. West, G. C. Zuccarello, R. Engbrodt, A. Yokoyama, Y. Hara, and J. Brodie. 2003. Low molecular weight carbohydrates of the Bangiophycidae (Rhodophyta). J. Phycol. 39:584-589.[CrossRef]
26. Kollman, V. H., J. L. Hanners, R. E. London, E. G. Adame, and T. E. Walker. 1979. Photosynthetic preparation and characterization of ¹³C-labeled carbohydrates in Agmenellum quadruplicatum. Carbohydr. Res. 73:193-202.[CrossRef]
27. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5:150-163.[Abstract/Free Full Text]
28. Lamosa, P., G. L. Gonçalves, M. W. Rodrigues, L. O. Martins, N. D. Raven, and H. Santos. 2006. Occurrence of 1-glyceryl-1-myo-inosityl phosphate in hyperthermophiles. Appl. Environ. Microbiol. 72:6169-6173.[Abstract/Free Full Text]
29. Mana-Capelli, S., A. K. Mandal, and J. M. Arguello. 2003. Archaeoglobus fulgidus CopB is a thermophilic Cu²⁺-ATPase. J. Biol. Chem. 278:40534-40541.[Abstract/Free Full Text]
30. Martins, L. O., L. S. Carreto, M. S. da Costa, and H. Santos. 1996. New compatible solutes related to di-myo-inositol-phosphate in members of the order Thermotogales. J. Bacteriol. 178:5644-5651.[Abstract/Free Full Text]
31. Oganesyan, V., N. Oganesyan, P. D. Adams, J. Jancarik, H. A. Yokota, R. Kim, and S.-H. Kim. 2005. Crystal structure of the "PhoU-like" phosphate uptake regulator from Aquifex aeolicus. J. Bacteriol. 187:4238-4244.[Abstract/Free Full Text]
32. Rainey, F. A., N. Ward-Rainey, R. M. Kroppenstedt, and E. Stackebrandt. 1996. The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. Int. J. Syst. Bacteriol. 46:1088-1092.[CrossRef][Medline]
33. Robertson, D. E., M. Lai, R. P. Gunsalus, and M. F. Roberts. 1992. Composition, variation, and dynamics of major osmotic solutes in Methanohalophilus strain FDF1. Appl. Environ. Microbiol. 58:2438-2443.[Abstract/Free Full Text]
34. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
35. Santos, H., and M. S. da Costa. 2002. Compatible solutes of organisms that live in hot saline environments. Environ. Microbiol. 4:501-509.[CrossRef][Medline]
36. Silva, Z., S. Alarico, A. Nobre, R. Horlacher, J. Marugg, W. Boos, A. I. Mingote, and M. S. da Costa. 2003. Osmotic adaptation of Thermus thermophilus RQ-1: lesson from a mutant deficient in synthesis of trehalose. J. Bacteriol. 185:5943-5952.[Abstract/Free Full Text]
37. Steed, P. M., and B. L. Wanner. 1993. Use of the rep technique for allele replacement to construct mutants with deletions of the pstSCAB-phoU operon: evidence of a new role for the PhoU protein in the phosphate regulon. J. Bacteriol. 175:6797-6809.[Abstract/Free Full Text]
38. Thaller, M. C., S. Schippa, and G. M. Rossolini. 1998. Conserved sequence motifs among bacterial, eukaryotic, and archaeal phosphatases that define a new phosphohydrolase superfamily. Protein Sci. 7:1647-1652.[Abstract]
39. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 24:4876-4882.
40. Wang, Y., A. Morgan, K. Stieglitz, B. Stec, B. Thompson, S. Miller, and M. F. Roberts. 2006. The temperature dependence of the inositol monophosphatase K_m correlates with accumulation of di-myo-inositol 1,1'-phosphate in Archaeoglobus fulgidus. Biochemistry 45:3307-3314.[CrossRef][Medline]

This article has been cited by other articles:

• Alarico, S., da Costa, M. S., Empadinhas, N. (2008). Molecular and Physiological Role of the Trehalose-Hydrolyzing {alpha}-Glucosidase from Thermus thermophilus HB27. J. Bacteriol. 190: 2298-2305 [Abstract] [Full Text]  
• Fernandes, C., Empadinhas, N., da Costa, M. S. (2007). Single-Step Pathway for Synthesis of Glucosylglycerate in Persephonella marina. J. Bacteriol. 189: 4014-4019 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Other Versions of this Article: JB.00841-06v1 189/5/1648    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Costa, J. Articles by da Costa, M. S. Search for Related Content PubMed PubMed Citation Articles by Costa, J. Articles by da Costa, M. S.