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Keywords:

  • sucrose phosphate synthase/phosphatase;
  • sucrose metabolism;
  • methylo- and lithoautotrophic bacteria

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

The aerobic obligate methylotroph Methylobacillus flagellatus KT was shown to synthesize sucrose in the presence of 0.5–2% NaCl in the growth medium. In the genome of this bacterium, an open reading frame (ORF) encoding a predicted 84-kD polypeptide homologous to the plant and cyanobacterial sucrose phosphate synthases (SPSs) was found. Using heterologous expression of the putative sps gene in Escherichia coli, followed by affinity chromatography, pure recombinant protein SPS-His6 was obtained. The enzyme catalyzed two reactions: conversion of fructose 6-phosphate and UDP-glucose into sucrose 6-phosphate and hydrolysis of sucrose 6-phosphate to sucrose. The bifunctional sucrose phosphate synthase/phosphatase (SPS/SPP) was a 340 kDa homotetrameric Mg2+-dependent enzyme activated by fructose 1,6-phosphate2 and ATP but inhibited by glucose 6-phosphate, fructose 1-phosphate, AMP and inorganic phosphate. The amino acid sequence of the protein had a C-terminal domain homologous to SPPs. This correlated with the absence of the spp gene in the M. flagellatus chromosome. The ORFs homologous to the M. flagellatus SPS were found in the genomes of another obligate methylotroph Methylovorus glucosetrophus as well as the lithoautotrophic bacteria Acidithiobacillus ferrooxidans, Nitrosomonas europaea and Nitrosospira multiformis whose genomes lacked the spp genes. Thus, data extending the knowledge of biochemical properties of bacterial SPSs have been obtained.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Sucrose is the major product of photosynthesis in most plants and is known to be essential for their growth, development, carbon storage, signal transduction and stress protection (Winter & Huber, 2000). Sucrose has been found in cyanobacteria, and the ability to synthesize sucrose is a widespread feature of these organisms (Klähn & Hagemann, 2011). In marine and freshwater cyanobacteria, sucrose is often synthesized in response to salt or temperature stress and seems to maintain the osmotic balance and to stabilize protein and membrane structure and function (Reed et al., 1986; Potts, 2004). Also, sucrose can regulate metabolic pathways under conditions of nutritional stress (Deplats et al., 2005).

Sucrose accumulation concomitant with increasing medium salinity has been observed in the aerobic methanotrophic bacteria of the genera Methylomicrobium and Methylobacter and the methanol-utilizing bacteria of the genus Methylophaga (Khmelenina et al., 1999; Doronina et al., 2003a, b; But et al., 2013). These halotolerant methylotrophs can grow in the salinity range 0–2 M (12%) NaCl and synthesize sucrose as a secondary osmotic compound along with ectoine as the major compatible solute. Also, sucrose synthesis and accumulation in the cells of the moderately thermophilc methanotroph Methylocaldum szegediense O-12 was first demonstrated during growth at enhanced temperature (> 50 °C) and the thermoprotective role of this disaccharide was elucidated (Medvedkova et al., 2007).

In plants and cyanobacteria, sucrose synthesis is performed by the sequential action of sucrose phosphate synthase (SPS, UDP-glucose: d-fructose 6-phosphate glucosyltransferase, EC 2.4.1.14) catalysing the conversion of fructose 6-phosphate and UDP-glucose to sucrose 6-phosphate and sucrose phosphate phosphatase (SPP, sucrose 6-phosphate phosphohydrolase, EC 3.1.3.24), which hydrolyses sucrose 6-phosphate to sucrose (Lunn et al., 2000; Salerno & Curatti, 2003; Cumino et al., 2010). The SPS and SPP enzymes are related in plants and cyanobacteria. However, in nonphotosynthetic prokaryotes, the genes and enzymes responsible for disaccharide biosynthesis have not yet been characterized. Recently, an SPS-like open reading frame (ORF) but no SPP-coding gene has been found in the genome of the obligate methylotroph Methylobacillus flagellatus KT, which uses methanol or methylamine but not methane as sole carbon and energy sources for growth (But et al., 2012). Sucrose synthesis and ability to grow at enhanced salinity were not studied in this bacterium (Govorukhina et al., 1987).

Here, we report for the first time the salt- and temperature-dependent sucrose accumulation in cells of M. flagellatus KT and biochemical characterization of the bifunctional recombinant SPS showing both SPS and SPP activities.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacteria and growth conditions

Methylobacillus flagellatus KT (VKM B-1610; ATCC 51484; DSM 6875) was grown at 29 or 37 °C in the presence of 0.5% methanol in minimal K medium containing (g L−1): KH2PO4 – 2, (NH4)2SO4 – 2, NaCl – 0.5, MgSO4·7H2O – 0.025, FeSO4·7H2O – 0.002, pH 7.2. Esherichia coli Rosetta (DE3; Novagen) was cultured at 37 °C in Luria–Bertani (LB) medium (Sambrook & Russell, 2001). If required, the medium was supplemented with kanamycin (50 μg mL−1) and chloramphenicol (25 μg mL−1).

Extraction and measurement of sucrose

Cells in the stationary growth phase were harvested by centrifugation (6000 g for 15 min) and freeze-dried. Then, 100 mg of dry cells was suspended in 1 mL methanol and incubated for 2 h with shaking at room temperature. The cell suspension was centrifuged at 10 000 g for 10 min and methanol from the supernatant was evaporated by vacuum. The dry residue was dissolved in 0.5 mL deionized water. The sucrose content was estimated in 50-μL aliquots by using the anthrone reagent (Van Handel, 1968).

Phylogenetic analysis

The full-length amino acid sequences from the protein databases in the National Center for Biotechnology Information (NCBI) were used for phylogenetic analyses. Alignments of sequences were carried out using clustal x software (ver 1.8; Thomson et al., 1997). Phylogenetic trees were generated with the mega 4 program using the maximum-parsimony, neighbor-joining and upgma methods. There were no significant differences in architecture of the trees constructed by these methods.

DNA manipulations

The isolation of chromosomal and plasmid DNA, restriction, ligation and transformation of E. coli cells and agarose gel electrophoresis were carried out according to standard protocols (Sambrook & Russell, 2001). Restriction enzymes, T4 DNA ligase, Taq DNA polymerase and dNTP mixture were purchased by Fermentas (Lithuania).

Cloning of the sps gene, expression and purification of the recombinant SPS

The full sequence of a putative sps gene (chromosome sequence GenBank accession number: ABE50874.1) was obtained from the sequenced genome of M. flagellatus KT (Chistoserdova et al., 2007). The ORF was amplified by PCR using genomic DNA as a template with the following primers: forward 5′-ATTCATATGAGCACACCTGACGA-ACGCCCTATC-3′ and reverse 5′-TATAAAGCTTATGCT-CGCTGACAGGGCTGGGCTCGAT-3′ (Restriction sites for endonucleases NdeI and HindIII respectively are underlined). The PCR mixture contained 30 μL 1× PCR buffer, 150 μM (each) deoxynucleotide triphosphates, 200 nM of the appropriate primers, 100 ng genomic DNA and 2 U Pfu-DNA-polymerase (Fermentas). The PCR conditions were as follows: 3 min at 96 °C, followed by 30 cycles of 20 s at 94 °C, 20 s at 60 °C and 4 min at 72 °C, and a final extension of 7 min at 72 °C.

The PCR product was purified on a Wizard column (Promega), incubated with the endonucleases NdeI and HindIII and ligated in the expression vector pET30(a)+ (Invitrogen) between appropriate sites. Cells of E. coli Rosetta (DE3) were transformed using the resulting vector and grown overnight at 37 °C in 20 mL LB medium containing 50 μg mL−1 kanamycin and 25 μg mL−1 chloramphenicol, and then transferred into 400 mL fresh LB medium. After cultivation to an OD600 of 0.6–0.7, protein expression was induced by addition of 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). After overnight incubation at 17 °C, cells were harvested by centrifugation at 6000 g for 20 min (4 °C). The His6-tagged protein was purified by affinity chromatography on an Ni2+-NTA column as described (But et al., 2012). Fractions with SPS-His6 were combined and dialysed against 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl. The purity of the recombinant enzyme was analysed by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, 1970).

Enzyme assays

Measurement of SPS activity

SPS activity was measured by monitoring of fructose 6-phosphate-dependent UDP formation from UDP-glucose using pyruvate kinase (PK, EC. 2.7.1.40) and lactate dehydrogenase (LDH, EC. 1.1.1.27) as coupling enzymes. PK is known as an enzyme of wide substrate specificity towards nucleoside diphosphates (ADP, UDP) forming pyruvate from phosphoenolpyruvate. The reaction was performed at 45 °C in 1 mL of 50 mM 2-(N-morpholino)ethanesulfonate (MES)–KOH buffer (pH 6.5), 0.1 mM NADH, 10 mM UDP-glucose, 10 mM fructose 6-phosphate, 5 mM phosphoenolpyruvate, 17.5 mM MgCl2, 1 U LDH, 5 U PK and 0.07 mg mL−1 purified SPS. NADH oxidation coupled with reduction of pyruvate by LDH was monitored at 340 nm on a Shimadzu UV-1700 spectrophotometer (Japan). For measurement of Km and Vmax, 0.1–20 mM of the substrate and the saturated concentration of another (10 mM) was used.

Measurement of SPP activity

Enzyme activity was measured by using two methods. (1) Two hundred microliters of the reaction mixture containing 50 mM MES–KOH buffer, pH 6.5, 17.5 mM MgCl2 and 400 μM of sucrose 6-phosphate was incubated at 45 °C. Orthophosphate released from sucrose 6-phosphate was determined by the Bencini method (Bencini et al., 1983). This procedure was used to follow dependence of the enzyme activity on Mg2+, pH, temperature and effectors. (2) To study the enzyme kinetics, the coupling enzyme pyrophosphate-dependent 6-phosphofructokinase (PFK, EC. 2.7.1.90) catalysing inorganic phosphate (Pi)-dependent formation of fructose 6-phosphate from fructose 1,6-phosphate2 was used for activity assay (Rozova et al., 2010). Glucose 6-phosphate formed from fructose 6-phosphate by phosphoglucose isomerase (PGI, EC. 5.3.1.9) is then oxidized by NADP+-dependent glucose 6-phosphate dehydrogenase (GPDH, EC. 1.1.1.49). Activity was measured in 1 mL of the reaction mixture containing 50 mM MES buffer (pH 6.5), 40–800 μM sucrose 6-phosphate, 1 mM fructose-1,6-phosphate2, 17.5 mM MgCl2, 0.5 mM NADP+, 0.25 U PGI, 2 U GPDH and 2 U PFK-His6 obtained from Methylomicrobium alcaliphilum 20Z (Rozova et al., 2010). The reduction of NADP+ was registered at 340 nm.

Enzyme kinetics

The Enzyme Kinetics Module of the sigmaplot 11 software was used for calculation of Vmax and Km. One unit (U) of activity was defined as the amount of the enzyme required to release 1 μmol of UDP (synthase activity) or inorganic phosphate (phosphatase activity) per minute.

The following buffers (50 mM) were used for testing the pH dependence of both enzyme activities: MES–KOH (pH 5.5–7.0) and Tris-HCl (pH 7.5–9.0). The following compounds (5 mM) were tested as potential effectors for the enzyme: d-glucose, d-fructose, sucrose, fructose 1-phosphate, fructose-1,6-phosphate2, glucose 1-phosphate, ribose 1-phosphate, AMP, ATP and Pi. Protein concentration was determined by the Lowry method using bovine serum albumin (BSA) as a standard (Shacterle & Pollack, 1973).

Determination of molecular mass of the enzyme

The quaternary structure of the enzyme was determined by gel filtration on a calibrated column with Ultrogel AcA 34 (Pharmacia, Sweden) equilibrated by 50 mM Tris-HCl buffer, pH 8.0, containing 1 м NaCl. Thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa) and BSA (66 kDa) from Sigma were used as molecular mass markers.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Accumulation of sucrose in M. flagellatus KT

Methylobacillus flagellatus KT was grown on methanol at salinities of 0.01–0.34 M (0.05–2%) NaCl at 30 or 37 °C but it was unable to grow with ≥0.5 M (3%) NaCl. Sucrose was not detectable in the cells grown at both temperatures in the basal medium containing 0.01 M NaCl, although it was found in cells growing in the presence of 0.1, 0.17 or 0.34 M NaCl. Intracellular sucrose content increased upon increasing salt concentration in the growth medium and was higher if the culture was grown at the same salinities at 37 °C in comparison with 30 °C (Fig. 1). This implies that in this bacterium, sucrose serves as an osmolyte and a thermoprotective compound, although we cannot exclude that sucrose biosynthesis may be related to some other stress responses.

image

Figure 1. Accumulation of sucrose by Methylobacillus flagellatus cells grown at different temperatures and medium salinity (mean value of three independent experiment ± SE are represented).

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Until now, among nonphotosynthetic microorganisms, only gammaproteobacterial, moderately halotolerant methylotrophs utilizing methane or methanol as the carbon and energy source have been shown to synthesize sucrose along with ectoine as a major osmoprotectant (Khmelenina et al., 1999; Doronina et al., 2003a,b; But et al., 2013). In contrast, in M. flagellatus, belonging to the Betaproteobacteria, intracellular sucrose accumulation provided only low salt tolerance (at no more than 0.34 M NaCl). The osmo- and thermoprotective functions of sucrose were previously demonstrated in many cyanobacterial species, including freshwater species where it supported a relatively higher halotolerance (up to 0.6 M NaCl; Reed et al., 1984; Hagemann, 2011).

Cloning of the sps gene and purification of the recombinant enzyme

Sequence analysis of the M. flagellatus genome revealed the presence of an ORF (Mfla_2610), which shared 53.1% and 47.5% identities, respectively, with SPSs from Synecochoccus sp. 7002 and Synechocystis sp. 6803, the functionality of which is known (Lunn et al., 1999; Cumino et al., 2010). To clarify if this ORF encodes a protein with SPS activity, the gene Mfla_2610 was amplified by PCR. The amplicon was cloned into vector pET30(a)+ designed for expression of the C-terminal His6-tagged fusion proteins under control of the T7 promoter. Escherichia coli Rosetta (DE3) cells transformed with the recombinant plasmid expressed protein upon addition of IPTG in the early log phase of the culture. An electrophoretically homogeneous preparation of SPS-His6 was obtained by metal-chelating affinity chromatography on Ni2+-NTA-agarose. The molecular mass of the enzyme subunit as determined by SDS-PAGE was in agreement with the theoretical mass (84.132 kDa) deduced from the amino-acid sequence (Fig. S1, Supporting information). According to gel filtration, the native molecular mass of the enzyme was about 340 kDa, indicating that the enzyme is a tetramer (data not shown). Tetrameric organization was also shown for plant SPSs, but all prokaryotic SPSs studied so far are monomeric enzymes (Table 1).

Table 1. Properties of some SPSs and SPPs
  Methylobacillus flagellatus Synechocystis sp. PCC 6803Anabaena sp. PCC 7120 Oriza sativa Pisum sativum
Substrate
SPS/SPPSPSSPPSPS-ASPPSPSSPPSPSSPP
UDP-GlucF6PS6PUDP-GlucF6PS6PUDP-GlucF6PS6PUDP-GlucF6PS6PUDP-GlucF6PS6P
  1. F6P, fructose 6-phosphate; S6P, sucrose 6-phosphate; –, not tested.

  2. a

    In the presence of 10 mM F6P.

  3. b

    In the presence of 10 mM UDP-Gluc.

Characteristic
Vmax (U mg−1)13.31117462.815.662.912504.22468
Km (mM)1.3a1.2b0.192.20.20.00751.30.40.353.63.50.0653.630.25
Vmax/Km10.211.157.97.78561332.2744.70.80.819 2301.21.41872
Temperature optimum (°C)45
pH optimum6.58.56.56.576.8
Native molecular mass (kDa) (subunit structure)336 (84 × 4)85 (85 × 1)46 (46 × 1)28 (28 × 1)464 (116 × 4)100 (50 × 2)456 (–)110 (55 × 2)
ReferencesThis workCuratti et al. (1998) and Lunn et al. (1999)Lunn (2002)Cumino et al. (2007)Cumino et al. (2001)Salerno et al. (1998), Pagnussat et al. (2000)Lunn et al. (2000)Lunn & Ap Rees (1990)Whitaker (1984)

Properties of the recombinant SPS

The recombinant SPS-His6 purified from M. flagellatus catalysed fructose 6-phosphate-dependent UDP formation from UDP-glucose and orthophosphate release from sucrose 6-phosphate with the specific activities of 13.3 and 11 U mg−1 of the protein, respectively. Thus, the enzyme could be represented as bifunctional sucrose phosphate synthase/phosphatase (SPS/SPP). Both enzyme activities were strongly dependent on Mg2+ with an optimal ion concentration of 17.5 mM (data not shown). Both reactions were maximal at 45 °C and pH 6.5, although the dephosphorylating activity occurred across broader temperature and pH ranges than those of the synthase reaction (Fig. 2a,b). The SPS activity was stimulated by adding up to 50 mM NaCl in the reaction mixture and was inhibited by ≥ 100 mM NaCl, whereas SPP activity was maximal at 200 mM NaCl and decreased only slightly with increasing salt concentration (Fig. 2c). These enzyme properties are in accordance with the thermo- and osmoprotective functions of sucrose.

image

Figure 2. Temperature (a), pH (b) and NaCl concentration (c) dependence of the recombinant SPS/SPP activity.

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The dependence of velocities of both reactions on substrate concentrations showed that the enzyme obeyed Michaelis–Menten kinetics (Fig. S2). The apparent Km was 1.3 ± 0.1 mM and 1.2 ± 0.1 mM for UDP-glucose and fructose 6-phosphate, respectively. These values are comparable with those recorded for plant SPSs (Table 1). As the Km value for sucrose 6-phosphate was much lower (0.19 ± 0.03 mM), the catalytic efficacy of the hydrolytic reaction, Vmax/Km, was five-fold higher than that of the synthetic reaction (Table 1), thus indicating that the formation of sucrose 6-phosphate may be a limiting step in the sucrose biosynthesis pathway. In contrast, the catalytic efficacy of the most characterized SPSs is much lower than that of SPP enzymes (Table 1). Therefore, it is not improbable that sucrose synthesis in M. flagellatus might be regulated in both reactions.

The action of the potential allosteric effectors on the enzyme activities was assayed. The following compounds had similar influence on synthetic and hydrolytic reactions at the concentration of 5 mM: fructose 1,6-phosphate2 and ATP activated the enzyme but ribose 5-phosphate, glucose 6-phosphate, fructose 1-phosphate and AMP had an inhibitory effect (Table 2). Orthophosphate at 5 mM caused only a 10% reduction in SPS activity, while sucrose and fructose had no marked effect on either activity. Rather different regulatory properties were shown for the plant SPS: glucose 6-phosphate and fructose 1,6-phosphate2 activated whereas Pi inhibited the enzyme (Lunn & Ap Rees, 1990; Huber & Huber, 1996; Salerno et al., 1998). By comparison, glucose 6-phosphate had no effect and orthophosphate caused only a 17% reduction of the activity of SPS from the filamentous heterocystous cyanobacterium Anabaena sp. PCC 7119 (Porchia & Salerno, 1996) and the unicellular Synechocystis sp. PCC 6803 (Curatti et al., 1998). Thus, the biochemical properties of M. flagellatus SPS/SPP were strikingly different from those of plant and cyanobacterial SPSs.

Table 2. Relative activity of the recombinant SPS/SPP in the presence of different compounds (% of control without effector)
Effector (5 mM)SPSSPP
100100
Glucose 6-phosphate7667
Glucose 1-phosphate102104
Pi90
Fructose 1-phosphate8762
Ribose 5-phosphate679
Fructose 1,6-phosphate2132124
Fructose9798
Sucrose9599
AMP3568
ATP132148

Phylogenetic and sequence analysis of SPS/SPP

blast analysis revealed SPS-like ORFs in the genomes of methylotrophic and chemoautotrophic bacteria (Fig. 3). The M. flagellatus SPS shares highest similarity with the putative enzymes from the methylotroph Methylovorus glucosetrophus SIP3-4 (69.6%) and the chemoautotrophs Nitrosomonas sp. AL212 (65.8%), Nitrosomonas europaea ATCC 19718 (65.4%), Nitrosospira multiformis ATCC 25196 (62.6%) and Acidithiobacillus ferrooxidans (YP002425979.1 (49.6%). Remarkably, in the genomes of the above bacteria, no separate SPP-like ORFs coding for the enzyme catalysing the final step of sucrose biosynthesis was found. At the same time, like the plant and most cyanobacterial SPSs, the enzymes from these bacteria are proteins with a modular architecture containing a glucosyltransferase domain (GTD) and a phosphohydrolase domain (PHD), the latter being coincidental with the PHD domain of SPP (Salerno & Curatti, 2003). The PHD of M. flagellatus SPS has conserved amino acid residues, particularly invariant aspartate, which is a functional nucleophile in the SPP active site (Fig. 4).

image

Figure 3. Phylogenetic tree constructed from the amino acid sequences of various putative and characterized bacterial and plants SPSs. The characterized enzymes are in bold and the amino acid accession numbers are in brackets.

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image

Figure 4. Multiple alignment of the deduced amino acid sequences of the conserved PHD domain of some SPS and SPP. The GenBank accession numbers of the sequences are as follows: Methylobacillus flagellatus KT SPS, YP546715.1; Methylovorus glucosetrophus SIP3-4 SPS, YP003052527.1; Nitrosomonas europaea ATCC 19718 SPS, NP 841268.1; Acidithiobacillus ferrooxidans ATCC 23270 SPS (YP002425979.1); Methylomicrobium alcaliphilum 20Z SPS (YP004915906.1); Methylomicrobium alcaliphilum 20Z SPP, YP004915907.1; Methylomicrobium album BG8 SPS, ZP09864641.1; Methylophaga aminisulfidivorans MP SPS, ZP08536075.1; Methylophaga aminisulfidivorans MP SPP, ZP08536074.1; Synechococcus sp. PCC 7002 SPS, YP001734148.1; Synechococcus sp. PCC 7002 SPP, YP001734147.1; Synechocystis sp. PCC 6803 SPS, NP442711.1; Synechocystis sp. PCC 6803, SPS NP_441739.1. Asterisk indicates the invariant Asp which is functional nucleophile in the SPP active site.

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Phosphatase activity was earlier proposed for the A-family SPSs from spinach and several legumes, because their carboxyterminal region showed 35% similarity to the catalytic PHD domain containing the complete conserved motif DXDX(T/V) in the active center (Lunn, 2002). Although SPSs from Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 are two-domain proteins, they do not show SPP activity. In these enzymes, Asp9 is replaced by Tyr. Interestingly, the halotelerant methylotrophs Methylomicrobium alcaliphilum 20Z and Methylophaga aminisulfidivorans MP possess putative SPSs with the functional PHD domain along with the separate SPP encoding genes in their chromosomes (Fig. 4). Presumably, in some methylo- and autotrophic proteobacteria, the bifunctional enzyme SPS/SPP may also be involved in sucrose synthesis.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

We have demonstrated by the expression in E. coli that the ORF Mfla_2610 from M. flagellatus encodes a two-domain protein with SPS and SPP activities. This is the first characterization of bifunctional SPS enzyme from prokaryotic organisms. This SPS has some distinct structural and regulatory properties in comparison with those of plant and cyanobacterial SPSs. As follows from the phylogenetic tree (Fig. 3), the genes of sucrose synthesis are almost ubiquitous in Proteobacteria assimilating reduced C1 compounds either via the ribulose monophosphate cycle (methylotrophs) or fixing CO2 via the Calvin cycle (lithoautotrophs). However, no sucrose biosynthesis genes were found in the genomes of methylotrophic bacteria realizing the serine pathway of C1 assimilation. We have also shown that in M. flagellatus sucrose synthesis is associated with responses to salt and temperature stresses.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

The work was supported by grants of the Russian Foundation for Basic Research (12-04-32122-a and RFBR 13-04-01119-a).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Conclusions
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
fml12219-sup-0001-FigS1-S2.docWord document211K

Fig. S1. SDS-PAGE of the purified recombinant SPS. Lane 1, purified SPS; lane 2, prestained protein molecular weight markers (Fermentas).

Fig. S2. Dependence of the recombinant SPS/SPP activity on concentration of UDP-glucose (a), fructose 6-phosphate (b) and sucrose 6-phosphate (c).

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