RESEARCH LETTER Bifunctional sucrose phosphate synthase/phosphatase is involved in the sucrose biosynthesis by Methylobacillus flagellatus KT Sergey Y. But, Valentina N. Khmelenina, Alexander S. Reshetnikov & Yuri A. Trotsenko G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Moscow, Russia Downloaded from https://academic.oup.com/femsle/article/347/1/43/531960 by guest on 29 September 2021 Correspondence: Yuri A. Trotsenko, G.K. Abstract Skryabin Institute of Biochemistry and Physiology of Microorganisms, RAS, 142290 The aerobic obligate methylotroph Methylobacillus flagellatus KT was shown to Pushchino, Moscow Region, Russia. synthesize sucrose in the presence of 0.5–2% NaCl in the growth medium. In Tel.: +7 495 925 7448; the genome of this bacterium, an open reading frame (ORF) encoding a pre- fax: +7 495 956 3370; dicted 84-kD polypeptide homologous to the plant and cyanobacterial sucrose e-mail: [email protected] phosphate synthases (SPSs) was found. Using heterologous expression of the putative sps gene in Escherichia coli, followed by affinity chromatography, pure Received 24 May 2013; revised 15 July 2013; accepted 15 July 2013. Final version recombinant protein SPS-His6 was obtained. The enzyme catalyzed two reac- published online 12 August 2013. tions: conversion of fructose 6-phosphate and UDP-glucose into sucrose 6-phosphate and hydrolysis of sucrose 6-phosphate to sucrose. The bifunctional DOI: 10.1111/1574-6968.12219 sucrose phosphate synthase/phosphatase (SPS/SPP) was a 340 kDa homotetra- 2+ meric Mg -dependent enzyme activated by fructose 1,6-phosphate2 and ATP Editor: Colin Murrell but inhibited by glucose 6-phosphate, fructose 1-phosphate, AMP and inor- ganic phosphate. The amino acid sequence of the protein had a C-terminal Keywords domain homologous to SPPs. This correlated with the absence of the spp gene sucrose phosphate synthase/phosphatase; in the M. flagellatus chromosome. The ORFs homologous to the M. flagellatus sucrose metabolism; methylo- and lithoautotrophic bacteria. SPS were found in the genomes of another obligate methylotroph Methylovorus glucosetrophus as well as the lithoautotrophic bacteria Acidithiobacillus ferrooxi- dans, 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. and Methylobacter and the methanol-utilizing bacteria of Introduction the genus Methylophaga (Khmelenina et al., 1999; Doron- Sucrose is the major product of photosynthesis in most ina et al., 2003a, b; But et al., 2013). These halotolerant plants and is known to be essential for their growth, methylotrophs can grow in the salinity range 0–2M development, carbon storage, signal transduction and (12%) NaCl and synthesize sucrose as a secondary osmo- stress protection (Winter & Huber, 2000). Sucrose has tic compound along with ectoine as the major compatible been found in cyanobacteria, and the ability to synthesize solute. Also, sucrose synthesis and accumulation in the sucrose is a widespread feature of these organisms (Kl€ahn cells of the moderately thermophilc methanotroph Methy- & Hagemann, 2011). In marine and freshwater cyanobac- localdum szegediense O-12 was first demonstrated during teria, sucrose is often synthesized in response to salt or growth at enhanced temperature (> 50 °C) and the ther- temperature stress and seems to maintain the osmotic moprotective role of this disaccharide was elucidated balance and to stabilize protein and membrane structure (Medvedkova et al., 2007). and function (Reed et al., 1986; Potts, 2004). Also, In plants and cyanobacteria, sucrose synthesis is per- sucrose can regulate metabolic pathways under conditions formed by the sequential action of sucrose phosphate MICROBIOLOGY LETTERS MICROBIOLOGY of nutritional stress (Deplats et al., 2005). synthase (SPS, UDP-glucose: D-fructose 6-phosphate Sucrose accumulation concomitant with increasing glucosyltransferase, EC 2.4.1.14) catalysing the conversion medium salinity has been observed in the aerobic of fructose 6-phosphate and UDP-glucose to sucrose methanotrophic bacteria of the genera Methylomicrobium 6-phosphate and sucrose phosphate phosphatase (SPP, FEMS Microbiol Lett 347 (2013) 43–51 ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved 44 S.Y. But et al. sucrose 6-phosphate phosphohydrolase, EC 3.1.3.24), software (ver 1.8; Thomson et al., 1997). Phylogenetic which hydrolyses sucrose 6-phosphate to sucrose (Lunn trees were generated with the MEGA 4 program using the et al., 2000; Salerno & Curatti, 2003; Cumino et al., maximum-parsimony, neighbor-joining and UPGMA meth- 2010). The SPS and SPP enzymes are related in plants and ods. There were no significant differences in architecture cyanobacteria. However, in nonphotosynthetic prokary- of the trees constructed by these methods. otes, the genes and enzymes responsible for disaccharide biosynthesis have not yet been characterized. Recently, an DNA manipulations SPS-like open reading frame (ORF) but no SPP-coding gene has been found in the genome of the obligate methy- The isolation of chromosomal and plasmid DNA, restric- lotroph Methylobacillus flagellatus KT, which uses metha- tion, ligation and transformation of E. coli cells and aga- nol or methylamine but not methane as sole carbon and rose gel electrophoresis were carried out according to Downloaded from https://academic.oup.com/femsle/article/347/1/43/531960 by guest on 29 September 2021 energy sources for growth (But et al., 2012). Sucrose syn- standard protocols (Sambrook & Russell, 2001). Restric- thesis and ability to grow at enhanced salinity were not tion enzymes, T4 DNA ligase, Taq DNA polymerase and studied in this bacterium (Govorukhina et al., 1987). dNTP mixture were purchased by Fermentas (Lithuania). Here, we report for the first time the salt- and temper- ature-dependent sucrose accumulation in cells of M. fla- Cloning of the sps gene, expression and gellatus KT and biochemical characterization of the purification of the recombinant SPS bifunctional recombinant SPS showing both SPS and SPP activities. 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 Materials and methods (Chistoserdova et al., 2007). The ORF was amplified by PCR using genomic DNA as a template with the following Bacteria and growth conditions primers: forward 5′-ATTCATATGAGCACACCTGACGA-A Methylobacillus flagellatus KT (VKM B-1610; ATCC 51484; CGCCCTATC-3′ and reverse 5′-TATAAAGCTTATGCT-C DSM 6875) was grown at 29 or 37 °C in the presence of GCTGACAGGGCTGGGCTCGAT-3′ (Restriction sites for À 0.5% methanol in minimal K medium containing (g L 1): endonucleases NdeI and HindIII respectively are under- KH2PO4 – 2, (NH4)2SO4 – 2, NaCl – 0.5, MgSO4Á7H2O – lined). The PCR mixture contained 30 lL19 PCR buffer, 0.025, FeSO4Á7H2O – 0.002, pH 7.2. Esherichia coli Rosetta 150 lM (each) deoxynucleotide triphosphates, 200 nM of (DE3; Novagen) was cultured at 37 °C in Luria–Bertani the appropriate primers, 100 ng genomic DNA and 2 U (LB) medium (Sambrook & Russell, 2001). If required, the Pfu-DNA-polymerase (Fermentas). The PCR conditions À medium was supplemented with kanamycin (50 lgmL 1) were as follows: 3 min at 96 °C, followed by 30 cycles of 20 s À and chloramphenicol (25 lgmL 1). at 94 °C, 20 s at 60 °Cand4minat72°C, and a final extensionof7minat72°C. The PCR product was purified on a Wizard column Extraction and measurement of sucrose (Promega), incubated with the endonucleases NdeI and Cells in the stationary growth phase were harvested by HindIII and ligated in the expression vector pET30(a)+ (In- centrifugation (6000 g for 15 min) and freeze-dried. vitrogen) between appropriate sites. Cells of E. coli Rosetta Then, 100 mg of dry cells was suspended in 1 mL metha- (DE3) were transformed using the resulting vector and nol and incubated for 2 h with shaking at room tempera- grown overnight at 37 °C in 20 mL LB medium containing À À ture. The cell suspension was centrifuged at 10 000 g for 50 lgmL 1 kanamycin and 25 lgmL 1 chloramphenicol, 10 min and methanol from the supernatant was evapo- and then transferred into 400 mL fresh LB medium. After rated by vacuum. The dry residue was dissolved in cultivation to an OD600 of 0.6–0.7, protein expression was 0.5 mL deionized water. The sucrose content was esti- induced by addition of 1 mM isopropyl b-D-1-thiogalacto- mated in 50-lL aliquots by using the anthrone reagent pyranoside (IPTG). After overnight incubation at 17 °C, (Van Handel, 1968). 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 Phylogenetic analysis (But et al., 2012). Fractions with SPS-His6 were combined The full-length amino acid sequences from the protein and dialysed against 50 mM Tris-HCl buffer (pH 8.0) con- databases in the National Center for Biotechnology taining 0.5 M NaCl. The purity of the recombinant enzyme Information (NCBI) were used for phylogenetic analyses. was analysed by 8% sodium dodecyl sulfate polyacrylamide Alignments of sequences were carried out using CLUSTAL X gel electrophoresis (SDS-PAGE; Laemmli, 1970). ª 2013 Federation of European Microbiological Societies. FEMS Microbiol Lett 347 (2013) 43–51 Published by John Wiley & Sons Ltd. All rights reserved Sucrose biosynthesis by Methylobacillus flagellatus 45 Enzyme assays (U) of activity was defined as the amount of the enzyme required to release 1 lmol of UDP (synthase activity) or Measurement of SPS activity inorganic phosphate (phosphatase activity) per minute. The following buffers (50 mM) were used for testing SPS activity was measured by monitoring of fructose the pH dependence of both enzyme activities: MES–KOH 6-phosphate-dependent UDP formation from UDP-glu- (pH 5.5–7.0) and Tris-HCl (pH 7.5–9.0). The following cose using pyruvate kinase (PK, EC.