Two strictly polyphosphate-dependent gluco(manno)kinases from diazotrophic Cyanobacteria with potential to phosphorylate hexoses from polyphosphates

1 Two strictly polyphosphate-dependent gluco(manno)kinases from diazotrophic Cyanobacteria with 1 potential to phosphorylate hexoses from polyphosphates 2 3 Tomás Albi • Aurelio Serrano* 4 Instituto de Bioquímica Vegetal y Fotosíntesis, Centro de Investigaciones Científicas Isla Cartuja, CSIC y 5 Universidad de Sevilla, Av. Américo Vespucio 49, 41092 Sevilla, SPAIN 6 7 *To whom correspondence should be addressed: Aurelio Serrano, Institute for Plant Biochemistry and 8 Photosynthesis, CSIC and University of Seville - 49 Americo Vespucio Avenue, 41092 Seville, SPAIN. 9 Phone: ++ 34 95 4489524. Fax: ++ 34 95 4460165. E-mail: [email protected] Web: 10 http://www.ibvf.csic.es/en/bioenergetics-phosphate 11 12 Keywords: Gluco(manno)kinase · Hexokinase · Cyanobacteria · PolyP-dependent ·Polyphosphate · 13 Regeneration of ATP 14 15 Abstract 16 The single-copy genes encoding putative polyphosphate-glucose phosphotransferases (PPGK, EC 17 2.7.1.63) from two nitrogen-fixing Cyanobacteria, Nostoc sp. PCC7120 and Nostoc punctiforme 18 PCC73102, were cloned and functionally characterized. In contrast to their actinobacterial counterparts, 19 the cyanobacterial PPGKs have shown the ability to phosphorylate glucose using strictly inorganic 20 polyphosphates (polyP) as phosphoryl donors. This has proven to be an economically attractive reagent in 21 contrast to the more costly ATP. Cyanobacterial PPGKs had a higher affinity for medium-long sized 22 polyP (>10 phosphoryl residues). Thus, longer polyP resulted in higher catalytic efficiency. Also in 23 contrast to most their homologs in Actinobacteria, both cyanobacterial PPGKs exhibited a modest but 24 significant polyP-mannokinase activity as well. Specific activities were in the range of 180-230 and 2-3 25 µmol min-1 mg-1 with glucose and mannose as substrates, respectively. No polyP-fructokinase activity 26 was detected. Cyanobacterial PPGKs required a divalent metal cofactor, and exhibited alkaline pH optima 27 (approx. 9.0) and a remarkable thermostability (optimum temperature, 45 ºC). The preference for Mg2+ 28 was noted with an affinity constant of 1.3 mM. Both recombinant PPGKs are homodimers with a subunit 29 molecular mass of ca. 27 kDa. Based on databases searches and experimental data from Southern blots 30 and activity assays, closely-related PPGK homologs appear to be widespread among unicellular and 31 filamentous mostly nitrogen-fixing Cyanobacteria. Overall, these findings indicate that polyP may be 32 metabolized in these photosynthetic prokaryotes to yield glucose (or mannose) 6-phosphate. They also 33 provide evidence for a novel group-specific subfamily of strictly polyP-dependent gluco(manno)kinases 34 with ancestral features and high biotechnological potential, capable of efficiently using polyP as an 35 alternative and cheap source of energy-rich phosphate instead of costly ATP. Finally, these results could 36 shed new light on the evolutionary origin of sugar kinases. 37 38 Journal: Applied Microbiology and Biotechnology (accepted 21 Oct 2014) 2 INTRODUCTION 1 Inorganic polyphosphate (polyP) is a linear polyanion composed of tens to hundreds of phosphoryl 2 residues, all of them being linked by “high-energy” phosphoanhydride bonds. Found in many diverse 3 organisms in nature, polyP has proven to be essential for the growth, response to stresses and stringencies 4 of cells (Kulaev 1979; Wood and Clark 1988; Kornberg et al. 1999; Rao et al. 2009). 5 As phosphorylated compounds with a Gibss free energy of hydrolysis similar to the ATP (-30.5 kJ mol−1), 6 inorganic pyrophosphate (diphosphate, PPi) and polyP have been suggested to be used in place of ATP in 7 diverse biological processes (Lipmann 1965). An enzyme known to hydrolyze polyP rather than ATP is 8 the polyphosphate glucokinase (PPGK, polyphosphate-glucose phosphotransferase, EC 2.7.1.63), which 9 catalyzes the phosphorylation of glucose using polyP as a phosphoryl donor to form glucose 6-phosphate 10 as follows: 11 Glucose + PolyPn → glucose 6-P + PolyPn−1. 12 PPGK was first observed in Mycobacterium phlei (Szymona 1957), and later in other Gram-positive 13 bacteria, all of them belonging to the ancient order of Actinomycetales (Szymona 1964; Szymona and 14 Widomski 1974; Szymona and Szymona 1978; Szymona and Szymona 1979; Pepin and Wood 1986; 15 Mukai et al. 2003; Tanaka et al. 2003; Lindner et al. 2010a; Hehuan et al. 2012; Koide et al. 2013). 16 However, no PPGK has been described in other sort of bacteria, archaea, fungi, algae, plants or animals to 17 date. 18 Most actinobacterial PPGKs have been validated as monomers or homodimers with a molecular mass 19 subunit of ca. 30 kDa. A remarkable feature of these enzymes is its dual substrate specificity: PPGK can 20 use both ATP and polyP as donors to phosphorylate glucose to glucose 6-phosphate. Nevertheless, a 21 PPGK from the polyP-accumulating actinobacterium Microlunatus phosphovorus, is the only PPGK 22 enzyme solely dependent on polyP as an energized phosphoryl-substrate donor described to date (Tanaka 23 2003). Concerning this matter, several studies (Hsieh et al. 1993; Phillips et al. 1999) proved that 24 although both enzymatic activities belong to the same protein, the binding site for this protein differs in 25 each phosphate donor substrate. Thus, the enzymes of most phylogenetically ancient species of the 26 Actinomycetales order seem to prefer polyP instead of ATP. As a result of this, a higher polyP-27 glucokinase/ATP-glucokinase ratio is exhibited. Compared to the rest of glucokinases, PPGKs displayed 28 a wider range of NTP as phosphoryl donors (GTP, UTP, TTP, XTP, CTP and dATP), whereas ATP-29 glucokinases from more evolved organisms are unable to use polyP, and consequently only poorly replace 30 GTP for ATP, as is the case of hexokinases (EC 2.7.1.11) from fungi and mammals, which are 31 exclusively dependent on ATP (Rao 2009). 32 PPGK belong to the ROK (Repressor ORF Kinase) superfamily (Pfam PF00480) (Finn et al. 2014), a 33 large group of mostly bacterial proteins which also include other sugar kinases and transcriptional 34 repressors, the latter with an extra h-α-h DNA binding domain. Owing to this fact, kinase enzymes within 35 this group (bacterial gluco-, fructoand manno-kinases, eukaryotic hexokinases and ADP-glucokinases) 36 reveal a significant grade of structural relationship. 37 3 PolyP should play important roles in the overcoming of nutrient and heavy-metal stresses by 1 Cyanobacteria, a group of Gram-negative oxygenic photoautotrophic prokaryotes which are among the 2 most successful and oldest forms of life (Schopf 2002) and have gained a lot of attention in recent years 3 because of their potential applications in biotechnology (Abed et al. 2009). Accumulation of polyP 4 granules has been described under various culture conditions in both unicellular (Lawry and Jensen 1979) 5 and filamentous (Jensen et al. 1982) Cyanobacteria. Moreover, induction of genes involved in polyP 6 metabolism by Pi starvation was reported in the unicellular strain Synechocystis sp. (Gómez-García et al. 7 2003). Interestingly, in the diazotrophic filamentous cyanobacterium Anabaena flos-aquae P is stored in 8 different ways depending of the nitrogen source used. Under dinitrogen fixing conditions P is stored as 9 sugar P, whereas with nitrate as the combined N source it is stored as polyP (Thompson et al. 1994). 10 However, the functional relationships between polyP metabolism and dinitrogen fixation in cyanobacteria 11 have not yet been elucidated. 12 Here, we report the first polyP-gluco(manno)kinases isolated and biochemically characterized from 13 Cyanobacteria. The single copy ppgK genes of the filamentous nitrogen-fixing strains Nostoc sp. 14 PCC7120 and Nostoc punctiforme PCC73102 were cloned and overexpressed in E. coli, and the 15 corresponding recombinant proteins, hereafter referred as NsPPGK and NpPPGK respectively, were 16 purified and characterized. As shown, these enzymes are smaller proteins and exhibit some novel 17 biochemical features compared to the previously described PPGKs. Additionally, a survey of homologous 18 closely-related PPGKs has been carried out in a wide range of diazotrophic Cyanobacteria by several 19 techniques including Southern blots, activity assays, and bioinformatic analyses. Lastly, this study could 20 also offer new evidence towards the matter of hexokinases evolution. Overall, the obtained results 21 provide indications for cyanobacterial PPGKs representing a taxonomic group-specific new subfamily of 22 strictly polyP-dependent gluco(manno)kinases with high biotechnological potential. 23 MATERIALS AND METHODS 24 Reagents and PolyP preparation 25 Restriction endonucleases and T4 DNA ligase were purchased from Takara Bio Inc (Shiga, Japan). 26 ACCUZYME™ Proofreading DNA Polymerase and the gel extraction kit were obtained from Bioline 27 Inc. (MA, USA). Primers were synthesized by Integrated DNA Technologies (Leuven, Belgium). Sodium 28 polyphosphates PPi, tripolyphosphate (P3), cyclic P3 (trimetaphosphate, P3c), tetrapolyphosphate (P4), a 29 polyphosphate mix with an average chain length of 13-18 phosphoryl residues (P13-18) and water-insoluble 30 Maddrell salt (a mixture of crystalline long-chain polyphosphates of very high molecular mass), NTPs 31 (nucleoside 5’-triphosphates), dATP and hexoses (D-glucose, D-mannose, D-fructose) were purchased 32 from Sigma Chemical Co. (St. Louis, MO, USA). Purchased substrates (polyPs and hexoses) were 33 analytical grade reagents, except the Maddrell salt which was of practical grade. P60 and P150 34 (polyphosphate mixes purified by polyacrylamide gel electrophoresis; average chain lengths 60 and 150 35 phosphoryl residues, respectively) were kindly provided by Dr. Toshikazu Shiba (RegeneTiss Co, Japan). 36 Very long chain polyPs with chain lengths of up to approximately 800 phosphoryl residues (PLC) were 37 obtained by fractionation of solubilized Maddrell salt, prepared as described by Van Wazer (1958) on a 2 38 4 % (w/v) polyacrylamide/0.8 % (w/v) agarose gel. When necessary, crystalline polyP was washed twice 1 with 70 % (v/v) ethanol, dried overnight in a vacuum dessicator, and resuspended in distilled water. 2 Otherwise stated, the polyP concentration is expressed in terms of polymer, assuming average chain 3 lengths of: 3, 4, 15, 60, 150 and 300 phosphoryl residues for P3, P4, P13-18, P60, P150 and PLC, respectively. 4 All other chemicals were of analytical grade. 5 Analytical polyacrylamide gel electrophoresis of polyP 6 Polyacrylamide slab gels (total acrylamide, 30 %, w/v; 70 x 85 mm; 1-mm thick) were prepared which 7 contained a 19.2:0.8 ratio of acrylamide to bisacrylamide. The gel was pre-electrophoresed at 100 V for 3 8 h to remove contaminating ions. The polyP samples were mixed at a ratio of 1:6 with loading buffer [100 9 mM Tris-borate buffer (pH 8.3), 30 % (v/v) glycerol and 0.25 % (w/v) bromophenol blue]. Gels were run 10 at 50 mA in TBE as electrophoresis buffer. Once electrophoresed, polyPs were fixed and stained with 11 0.05 % (w/v) Toluidine blue O, 25 % (v/v) methanol and 1 % (v/v) glycerol in water, followed by 12 destaining in an aqueous mixture containing 25 % (v/v) methanol and 5 % (v/v) glycerol. As a result, the 13 polyP stained dark blue against the colorless or lightly-blue background. 14 Bacterial strains and culture conditions 15 The cyanobacterial strains used in this work were obtained as axenic cultures from various microbial 16 culture collections of reference for Cyanobacteria (ATCC, American Type Culture Collection, Manassas, 17 VA, USA; PCC, Pasteur Culture Collection, Paris, France; UTEX, Culture Collection of Algae, 18 University of Texas, Austin, USA; the CICCartuja Biological Cultures Service, Instituto de Bioquímica 19 Vegetal y Fotosíntesis, Seville, Spain). The strains were photoautotrophically grown in BG11 liquid 20 medium without combined nitrogen source unless otherwise stated (Rippka et al. 1979), and are described 21 in Online Resource Table S1. Cultures (referred to as bubbled cultures) were supplemented with 10 mM 22 NaHCO3, and bubbled with a mixture of CO2 and air (2 %, v/v), under continuous fluorescent white light 23 (75 μE m−2 s−1). The absence of heterotrophic bacterial contamination was assessed by counts on LB 24 (Luria-Bertani) agar plates incubated in the dark. 25 Preparation of cyanobacterial cell-free extracts 26 Cyanobacterial cells were harvested by centrifugation and resuspended in 100 mM Tris-HCl (pH 9.0) 27 buffer supplemented with 5 mM MgCl2, 5 mM DTT, 0.1 mM PMSF and a 1:1000 dilution of a Protein 28 Inhibitor Cocktail for use with bacterial cell extracts (P8465; Sigma-Aldrich, USA), at a ratio of 0.2 g 29 (cells wet wt)/ml. Then cells were ultrasonically disrupted at 0-4 ºC. The cell homogenate was 30 centrifuged at 15,000 x g at 4 ºC for 20 min, and the resultant clear supernatant (cell-free extract) was 31 used for enzymatic assays. 32 DNA methodology 33 Total DNA was isolated by the following procedure: 50 ml of cyanobacterial cultures in the mid-log 34 phase of growth were harvested and resuspended in a final volume of 400 µl in a microcentrifuge tube 35 with 10 mM Tris-HCl (pH 7.5) buffer with 0.1 mM EDTA. Then, 150 µl of sterile glass beads (0.2 µm 36 5 diameter), 20 µl of 10 % (w/v) SDS, and 450 µl of phenol-chloroform-isoamyl alcohol mixture (25:24:1 1 v/v) were added. The mixture was subjected to six cycles of 1-min vigorous vortexing followed by 1-min 2 cooling on ice. The resulting suspension was centrifuged at 15,000 x g for 10 min, then the clear 3 supernatant solution was transferred to a new microcentrifuge tube and DNA was finally ethanol 4 precipitated. 5 Southern blotting 6 DNA samples isolated from a number of strains representative of the different taxonomic groups of 7 cyanobacteria were digested with appropriate restriction enzymes and loaded onto agarose gels; then 8 Southern analysis was performed (Ausubelet al. 1992) using GeneScreen Plus membranes (Dupont, 9 USA). DNA probes utilized in the hybridizations (full coding ppgK fragments) were obtained by PCR, 10 and were then labeled with [α-32P]-dCTP using the Ready-To-Go© DNA labeling kit (GE Healthcare). 11 Nucleic acid hybridization was carried out at 55 °C with gently shaking. Films were exposed for 4 days 12 and developed using a Cyclone© Storage Phosphor System (Packard, USA). 13 Construction of recombinant plasmids and gene expression in E. coli 14 The ppgK genes from Nostoc sp. PCC7120 and Nostoc punctiforme PCC73102 were PCR amplified 15 using specific primers (Online Resource Table S2) and genomic DNA as a template. The unique DNA 16 fragments of ca. 0.72 Kb obtained in both cases were initially cloned into the pGemT-Easy vector 17 (Invitrogen) for sequencing. These plasmids were then digested with BamHI and PstI, and the DNA 18 fragments carrying the native open reading frames of ppgK genes were eventually ligated into pQE-80L 19 vector (Quiagen, Germany). In this way, a His6 tag of 12 amino acid residues in total 20 (MRGSHHHHHHGS; nominal mass 1,420 Da) was added to the N-terminal end of the native proteins. E. 21 coli BL21(DE3) cells transformed with the appropriate expression plasmid were cultured at 30 °C in 1 L 22 LB liquid medium supplemented with 100 μg ml−1 ampicillin with vigorous shaking. When OD600 23 reached ca. 0.6, protein expression was induced by adding 1 mM IPTG and cultures were then incubated 24 overnight at 20 °C with shaking at 200 rpm. 25 Purification of recombinant cyanobacterial PPGKs by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity 26 chromatography 27 Cells were harvested and resuspended in buffer A (500 mM NaCl, 50 mM Na2HPO4, 10 mM imidazole, 28 pH 8.0), and then lysed by sonication at 4 °C. Cell debris were removed by centrifugation at 15,000 x g 29 for 15 min. The resultant crude extract was loaded onto a pre-equilibrated HisTrap FF Crude Ni-NTA 1-30 ml column (GE-Healthcare). Subsequently, non-target proteins were removed by washing the column 31 with buffer B (500 mM NaCl, 50 mM Na2HPO4, 50 mM imidazole, pH 8.0) until no more protein elution 32 was observed. Finally, recombinant proteins were eluted by applying a linear gradient with a target 33 concentration of 100 % of buffer C (500 mM NaCl, 50 mM Na2HPO4, 500 mM imidazole, pH 8.0). The 34 eluted PPGK proteins were dialyzed three times with 50 mM Tris-HCl (pH 9.0) to remove imidazole and 35 phosphate salts, and eventually concentrated by ultrafiltration using Amicon Ultra-3 kDa filters. 36 6 FPLC gel filtration chromatography. Estimation of molecular masses 1 Partially purified His-tagged PPGK preparations, previously concentrated by ultrafiltration, were further 2 purified by FPLC gel filtration chromatography carried out at 4 °C. The concentrated preparations (0.5-3 1.0 ml volume) were loaded on to a Superdex© 200 PG (GE Healthcare, Sweden) column equilibrated 4 with 150 mM NaCl, 20 mM KCl, 5 mM MgCl2, 50 mM Tris-HCl (pH 9.0) buffer at a flow rate of 2 ml 5 min−1 using an ÄKTA-FPLC system (GE Healthcare, Sweden). The molecular masses (Mm) of oligomeric 6 PPGK proteins were determined using the calibration plot derived from the elution volumes of a series of 7 protein standards including: thyroglobulin (Thy, 669 kDa), ferritin (Fer, 443 kDa), β-amylase (β-Amy, 8 200 kDa), alcohol dehydrogenase (ADH, 150 kDa), bovine serum albumin (BSA, 66 kDa), carbonic 9 anhydrase (CA, 29 kDa) and cytochrome c (Cyt.c, 12.4 kDa). Subunit molecular masses were determined 10 by denaturing discontinuous SDS-PAGE following the method of Laemmli (Laemmli 1970) using 12 % 11 (w/v) separating and 4 % (w/v) stacking polyacrylamide gels. Protein bands were stained with Coomassie 12 Brilliant Blue R-250. Apparent Mm of monomers under denaturing PAGE was calculated using standard 13 proteins. Absolute Mm values of purified recombinant PPGKs were confirmed by MALDI-TOF mass 14 spectrometry (see below). These purified fractions were used for the in vitro kinetics assays and 15 biochemical characterization. 16 Peptide mass fingerprinting and validation of PPGK proteins by MALDI-TOF mass spectrometry 17 Protein samples corresponding to high-purity cyanobacterial PPGKs were derived from SDS-PAGE. 18 Proteins were digested with trypsin and the resulting peptides were extracted, then loaded onto a suitable 19 MALDI matrix and eventually processed by a MALDI-TOF mass spectrometer (AutoFlex, Bruker-20 Daltonics, Proteomics Service of the Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC-University of 21 Seville) which generated peptide mass spectra in the mass range 0.8–2.5 kDa. MASCOT-Matrix Science 22 database was used to analyze the peaks lists for protein identification (Koenig et al., 2008). 23 Determination of enzymatic activities 24 Unless otherwise stated sugar-kinase enzymatic activities were determined at 40 °C and pH 9.0, using P1325 18 as a phosphoryl donor substrate. The polyP glucokinase activity was assayed spectrophotometrically by 26 monitoring the production of NADPH at 340 nm using a glucose 6-phosphate dehydrogenase coupled 27 reaction. The assay mixture (1 ml) contained of 100 mM Tris–HCl buffer (pH 9.0), 5 mM MgCl2, 5 mM 28 glucose, 1.11 mM polyP, 5 mM NADP+, and 0.5 U of yeast glucose 6-phosphate dehydrogenase (Sigma 29 Chem. Co., USA). The reaction was started by the addition 0.5-1.5 μg of purified PPGK or 10-20 µl of 30 cell-free extracts. Concentrations of polyphosphate substrates were calculated as polymers, considering 31 mean chain lengths of 15, 60 and 300 phosphate residues for P13-18, P60 and PLC, respectively. NTPs were 32 used at 2 mM concentration when assayed as alternative phosphoryl donor substrates instead of polyP. To 33 determine the dependence on pH, 1.0 μg of purified enzyme was incubated as described above in the 34 following buffers at 100 mM concentration: 2-morpholinoethanesulfonic acid (MES) (pH 5.5-7.0), MOPS 35 (pH 7.0-8.0), Tris (pH 8.0-9.0), N-cyclohexyl-2-aminoethanesulfonic acid (CHES) (pH 9.0-10.0) and 3-36 [cyclohexylamino]-1-propane sulfonic acid (CAPS) (10.0-10.5). When measuring enzymatic activity in 37 cell-free extracts or when the effects of pH, temperature, divalent metal ions, inhibitors and other factors 38 7 on glucokinase activity were examined, the assay was discontinuous and NADP+ and glucose 6-1 phosphate dehydrogenase were omitted from the assay mixture. The reaction was finished by heating the 2 test tube at 95 ºC for 5 min. Then the assay followed as described above by adding 5 mM NADP+ and 0.5 3 U of glucose 6-phosphate dehydrogenase. The polyP-mannokinase activity was assayed in a similar way, 4 but glucose was replaced by 50 mM mannose and 0.5 U of mannose 6-phosphate isomerase (from E. coli; 5 Sigma Chem. Co., USA). Finally, for fructokinase activity determinations, 50 mM fructose and 0.5 U of 6 yeast glucose 6-phosphate isomerase (Sigma Chem. Co., USA) were added in substitution of glucose. 7 Kinetic parameters (Km and kcat) were determined from initial velocity data that were fitted by the 8 nonlinear regression software Anemona.xlt (Hernández and Ruiz 1998). One unit (U) of PPGK 9 corresponds to 1 µmol of phosphorylated product per minute at 30 °C. Protein concentration was 10 determined by the Bradford method (Bradford 1976) with ovalbumin as a standard. 11 Computer-aided analysis 12 Amino acid sequence homology among the PPGK sequences was analyzed online using BLAST searches 13 (Altschul et al. 1990)against the public databases GenBank (Benson et al. 2013), DOE Joint Genome 14 Institute (JGI) (Nordberg et al. 2014) and InterPro (Hunter et al. 2011). The amino acid sequences of 15 putative PPGK orthologs from diverse bacterial strains (Online Resource Table S3) were aligned and 16 phylogenetic trees were constructed with the Evolutionary-distances (Neighbor-joining), Maximum 17 Parsimony, and Maximum Likelihood methods using the SeaView v5.2 software (Gouy et al. 2010). 18 Nucleotide sequence accession numbers 19 The nucleotide sequences of the gene constructs reported in this paper have been deposited in the 20 GenBank/EMBL/DDBJ nucleotide sequence databases under accession numbers HG764586 (ppgK of 21 Nostoc sp. PCC7120) and HG764587 (ppgK of Nostoc punctiforme PCC73102), respectively. 22 23 RESULTS 24 all1371 and Npun_R1878 genes encode functional polyP-dependent glucokinases 25 BLAST sequence similarity searches in cyanobacterial genomes (Cyanobase, Kazusa DNA Research 26 Institute) (Fujisawa et al. 2014) identified two ORFs, all1371 and Npun_R1878 of the diazotrophic 27 filamentous strains Nostoc sp. PCC7120 and Nostoc punctiforme PCC73102, respectively, with high 28 homology to the ppgK gene from Mycobacterium tuberculosis H 37Rv (Hsieh et al. 1996a). The 29 corresponding predicted proteins, thereafter named NsPPGK and NpPPGK, shared 32 % and 29% 30 identity with their mycobacterial homolog and 91% sequence identity to each other. In addition, each of 31 the genomes of Nostoc sp. PCC7120 and Nostoc punctiforme PCC73102 possessed a gene encoding a 32 putative glucokinase, alr2973 and Npun_R5075. They respectively showed 27 % and 14 % sequence 33 identity at the protein level with their corresponding PPGK homolog. Even though both sequences of 34 putative ppgK genes were available, Npun_R1878 was wrongly annotated as a transcriptional 35 regulator/sugar kinase (ROK family protein) instead of a PPGK encoding gene. The predicted NsPPGK 36 8 and NpPPGK polypeptides have 239 (nominal mass 25,919 Da) and 238 (nominal mass 25,816 Da) 1 amino acid residues, respectively. They are smaller than their actinobacterial homologs (of 260-280 2 residues) and exhibit in their primary structures the seven regions with structural motifs conserved among 3 the bacterial PolyP/ATP-dependent PPGKs (Mukai et al. 2003), as revealed by protein sequences 4 alignments. Interestingly, when other putative cyanobacterial PPGK sequences were used in the 5 alignment a high level of conservation was found within them, while when cyanobacterial PPGKs are 6 compared to their actinobacterial polyP/ATP-dependent homologs, motifs reported to be involved in 7 phosphoryl-donor and polyphosphate substrate binding (phosphate-1 and -2, connect-1) and the glucose-8 binding motif are more clearly conserved (Online Resource Fig. S1). Thus, the finding of putative ppgK 9 genes led us to investigate whether glucose 6-phosphate synthesis in Cyanobacteria could take place 10 enzymatically through a similar way to that previously described in M. tuberculosis along with other 11 Actinobacteria. To characterize NsPPGK and NpPPGK, their respective putative genes were obtained 12 from genomic DNA by PCR amplification which yielded a single product with the expected size of 0.72 13 kb in both cases (Fig. 1a). They were lastly cloned into the pQE-80L expression vector and over-14 expressed in E. coli (BL21). Protein expression was induced in early-log phase cultures by addition of 15 IPTG. The heterologous overexpression of cyanobacterial ppgK genes conferred high PPGK activity to E. 16 coli cells. Thus, crude extracts from induced E. coli cells overproducing NsPPGK or NpPPGK showed 17 fairly high glucokinase activity levels with P13-18 as a substrate, in the range of 0.15 to 0.20 µmol min-1 18 mg-1 protein, respectively. In contrast, no PPGK activity was detected in extracts from cells containing 19 the pQE-80L plasmid with no insert. Milligram quantities of the respective N-terminal His6-tagged fusion 20 proteins were subsequently isolated in ca. 95% purity after one-step affinity purification onto a HisTrap 21 FF Crude Ni-NTA column (Online Resources Figs. S2 and S3, and Table S4). Enzyme purity was further 22 enhanced by following FPLC gel-filtration chromatography, which was confirmed by electrophoresis on 23 SDS-PAGE gels (Fig. 1, Online Resource Table S4). Thus, a single protein band of ca. 27 kDa was found 24 in both purified PPGK preparations (Fig 1a), in good agreement with the nominal Mm values of 27,339 25 and 27,236 Da predicted for the recombinant NsPPGK and NpPPGK polypeptides, respectively. Besides, 26 native Mm values and oligomeric states of oligomeric states of the recombinant proteins were determined 27 by gel-filtration chromatography, and values of 49.4 ± 4 kDa and 55.1 ± 5 kDa (means + SE of three 28 independent determinations) were obtained for NsPPGK and NpPPGK, respectively (Fig. 1b). Therefore, 29 both proteins adopted a stable dimeric arrangement in solution. In accordance with these results, MALDI-30 TOF determination of absolute Mm values gave values of 27,287 Da ± 0.1% and 27,236 Da ± 0.1% for the 31 recombinant NsPPGK and NpPPGK subunits, respectively. In addition, the identities of the recombinant 32 NsPPGK and NpPPGK polypeptides were confirmed by peptide mass fingerprinting covering 33 respectively about 55 and 82 % of the natural sequences, and eventual identification by MALDI-TOF MS 34 (Online resource Fig. S4). Together, these active and high purity fractions were used for the subsequent 35 determination of their enzymatic kinetic parameters. 36 37 NsPPGK and NpPPGK are strictly polyP-dependent glucokinases with preference for long-chain 38 PolyP 39 9 The purified recombinant NsPPGK showed no activity towards ATP, CTP, GTP, TTP, or dATP as 1 compared to sorts of polyP (Fig. 2). The absolute specificity of NsPPGK for inorganic polyphosphates 2 appears to be a common property of PPGK enzymes in other heterocystous filamentous cyanobacteria, 3 since similar results were observed in the characterization of NpPPGK. The substrate specificities 4 concerning polyP as phosphoryl donor to produce glucose 6-phosphate by cyanobacterial PPGKs were 5 probed using synthetic polyP molecules of various chain lengths at saturating glucose levels (Fig. 2a). 6 The rate of sugar phosphorylation for the polyP chain lengths followed a similar trend in both 7 recombinant enzymes, longer polyP result in higher specific glucokinase activity. This indicated that 8 PPGKs from cyanobacteria bound and hydrolyzed long-chain polyP substrates most efficiently (Table 1). 9 This highlights its reasonable consistency with previous findings on polyP/ATP glucokinases of other 10 bacteria (Girbal et al. 1989; Hsieh et al. 1996b; Tanaka et al. 2003; Mukai et al. 2003; Lindneret al. 11 2010a). Noteworthy, both cyanobacterial PPGKs are also able to use short-chain polyP. With reference to 12 the sole crystal structure of a bacterial polyP/ATP glucomannokinase published to date (Mukai et al. 13 2004), it has been proposed that there is a minimal length between two phosphoryl groups consisting on a 14 putative pentapolyphosphate-binding site. However, NsPPGK and NpPPGK exhibit modest but 15 significant specific activity levels with P4 (5-7 µmol min-1 mg-1 protein) (see Table 1). Analogous 16 experiments revealed that cyanobacterial PPGKs were unable to use shorter polyP than P4, such as P3, P3c 17 or PPi. Estimation of the kinetic parameters of NsPPGK with different polyP and hexose substrates 18 revealed that there is a remarkable increase of the catalytic constant kcat (more than 30-fold) with 19 increasing polyP chain-length from P4 up to PLC while Km values remain fairly constant, which explained 20 the higher catalytic efficiency of long-chain polyP (Tables 1 and 2, Figs. S5 and S6). 21 To determine the mechanism of polyP utilization by NsPPGK, P150 at saturation concentration was used 22 as a phosphoryl substrate while the progress of the reaction was monitored by collecting sequential 23 aliquots at increasing times. PolyP were isolated and electrophoresed on a preparative polyacrylamide gel, 24 and eventually visualized with Toluidine blue O staining. As shown in Fig. 3, this medium-size polyP 25 was utilized by the cyanobacterial PPGK by an essentially non-processive mechanism, as was evidenced 26 by the non noticeable broadening of the range of polyP sizes with the reaction time. A non-processive 27 mechanism is also consistent with the observed formation of polyP of intermediate sizes from the longest 28 polyP during the reaction progress (see Fig. 3). 29 A variety of compounds which are analogs to the phosphoryl donors were also tested to assess whether or 30 not they could act as PPGK activity inhibitors (Table 3). P3 and PPi were fairly strong inhibitors, with Ki 31 values of 0.13 and 0.19 mM respectively, while ATP only modestly inhibited the activity of NsPPGK. In 32 addition, a control experiment with increasing concentrations of NaCl was conducted to determine the 33 effect of the ionic strength on the PPGK activity. Results from Table 3 indicated that NsPPGK was not as 34 severely inhibited by NaCl as by short polyP or ATP, since the observed concentrations required for 35 substantial enzyme inhibition, most probably produced by ionic strength effect, were much higher (50-36 150 mM range). An inhibitory effect on PPGK activity was also obtained with KCl being even more 37 marked than that of NaCl (Table 3), thus suggesting that electrostatic forces may be involved in the 38 interaction between polyP and the enzyme. 39 16 Koenig T, Menze BH, Kirchner M (2008) Robust prediction of the MASCOT score for an improved 1 quality assessment in mass spectrometric proteomics. J Proteome Res 7:3708–3717. doi: 2 10.1021/pr700859x 3 Koide M, Miyanaga A, Kudo F, Eguchi T (2013) Characterization of polyphosphate glucokinase 4 SCO5059 from Streptomyces coelicolor A3(2). Biosci Biotechnol Biochem 77:130498-1-3 5 Kornberg A, Rao NN, Ault-Riché D (1999) Inorganic polyphosphate: a molecule with many functions. 6 Ann Rev Biochem 68:89-125 7 Kowalczyk TH, Horn PJ, Pan WH, Phillips NFB (1996) Initial rate and equilibrium isotope exchange 8 studies on the ATP-dependent activity of polyphosphate Glucokinase from Propionibacterium shermanii. 9 Biochemistry 35:6777-6785 10 Kulaev IS (1979) The biochemistry of inorganic polyphosphates. John Wiley & Sons, Inc., New York 11 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. 12 Nature 227(5259):680-5 13 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace 14 IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) ClustalW and ClustalX version 2. 15 Bioinformatics 23: 2947-2948 16 Lawry N, Jensen T (1979) Deposition of condensed phosphate as an effect of varying sulfur deficiency in 17 the cyanobacterium Synechococcus sp. (Anacystis nidulans). Arch Microbiol 120(1):1-7 18 doi:10.1007/bf00413264 19 Lindner SN, Knebel S, Pallerla SR, Schoberth SM, Wendisch VF (2010a) Cg2091 encodes a 20 polyphosphate/ATP-dependent glucokinase of Corynebacterium glutamicum. Appl Microbiol Biotechnol 21 87:703–713. doi: 10.1007/s00253-010-2568-5 22 Lindner SN, Niederholtmeyer H, Schmitz K, Schoberth SM, Wendisch VF (2010b) Polyphosphate/ATP-23 dependent NAD kinase of Corynebacterium glutamicum: biochemical properties and impact of ppnK 24 overexpression on lysine production. Appl Microbiol Biotechnol 87:583-93. doi: 10.1007/s00253-010-25 2481-y. 26 Lipmann F (1965) The origins of prebiological system and their molecular matrices. In: Fox SW (ed) 27 Academic Press, New York, pp 259-280 28 Mukai T, Kawai, S, Matsukawa H, Matuo Y, Murata K (2003) Characterization and molecular cloning of 29 a novel enzyme, inorganic polyphosphate/ATPglucomannokinase, of Arthrobacter sp. strain KM. Appl 30 Environ Microbiol 69:3849-3857 31 Mukai T, Kawai S, Mori S, Mikami B, Murata K (2004) Crystal Structure of Bacterial Inorganic 32 Polyphosphate/ATP-glucomannokinase. Insights into kinase evolution. J Biol Chem 279:50591-50600. 33 doi: 10.1074/jbc.M408126200 34 Nordberg H, Cantor M, Dusheyko S, Hua S, Poliakov A, Shabalov I, Smirnova T, Grigoriev IV, Dubchak 35 I (2014) The genome portal of the Department of Energy Joint Genome Institute: 2014 updates. Nucleic 36 Acids Res 42(Database issue):D26-31 doi:10.1093/nar/gkt1069 37 Pepin CA, Wood HG (1986) Polyphosphate glucokinase from Propionibacterium shermanii. Kinetics and 38 demonstration that the mechanism involves both processive and nonprocessive type reactions. J Biol 39 Chem 261:4476-4480 40 17 Phillips NF B, Hsieh PC, Kowalczyk TH (1999) Polyphosphate glucokinase. Prog Mol Subcell Biol 1 23:101-125. 2 Rao NN, Gómez-García MR, Kornberg A (2009) Inorganic polyphosphate: Essential for growth and 3 survival. Annu Rev Biochem 78:605–647. doi: 10.1146/annurev.biochem.77.083007.093039 4 Rippka R, Deruelles J, Waterbury JB, Hermann M, Stainer RY (1979) Generic assignment, strains 5 histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111:1-16 6 Schopf JW (2002) The Fossil Record: Tracing the Roots of the Cyanobacterial Lineage. In: Whitton B, 7 Potts M (eds) The Ecology of Cyanobacteria. Springer Netherlands, pp 13-35 8 Serrano A, Rivas J, Losada M (1984) Purification and properties of glutathione reductase from the 9 cyanobacterium Anabaena sp. strain 7119. J Bacteriol 158(1):317-24 10 Serrano A (1992) Purification, characterization and function of dihydrolipoamide dehydrogenase from the 11 cyanobacterium Anabaena sp. strain P.C.C. 7119. Biochem J 288 ( Pt 3):823-30 12 Szymona M (1957) Utilization of inorganic polyphosphates for phosphorylation of glucose in 13 Mycobacterium phlei. Bull. Acad. Pol. Sci. Ser. Sci. Biol. 5:379-381 14 Szymona M, Ostrowski W (1964) Inorganic polyphosphate glucokinase of Mycobacterium phlei. 15 Biochim Biophys Acta 85:283-295 16 Szymona M, Widomski J (1974) A kinetic study on inorganic polyphosphate glucokinase from 17 Mycobacterium tuberculosis H37RA. Physiol Chem Phys 6:393-404 18 Szymona O, Szymona M (1978) Multiple forms of polyphosphate-glucose phosphotransferase in various 19 Mycobacterium strains. Acta Microbiol Pol 27:73-76 20 Szymona O, Szymona M (1979) Polyphosphateand ATP-glucose phosphotransferase activities of 21 Nocardia minima. Acta Microbiol Pol 28:153-160 22 Tanaka S, Lee SO, Hamaoka K, Kato J, Takiguchi N, Nakamura K, Ohtake H, Kuroda A (2003) Strictly 23 polyphosphate-dependent glucokinase in a polyphosphate-accumulating bacterium, Microlunatus 24 phosphovorus. J Bacteriol 185: 5654-5656 25 Thompson PA, Oh H-M, Rhee G-Y (1994) Storage of phosphorus in nitrogen-fixing Anabaena flos-26 aquae (Cyanophyceae). J Phycol 30:267-273 27 Van Wazer JR (1958) Phosphorus and its compounds, Vol 1. Interscience Publishers, New York 28 Wood HG, Clark JE (1988) Biological aspects of inorganic polyphosphates. Annu Rev Biochem 57:235-29 260 30 31 FIGURE CAPTIONS 32 33 Fig. 1 (a) Upper panel. PCR amplification of cyanobacterial ppgK genes. An electrophoretic analysis 34 of PCR-amplified DNA fragments corresponding to the ppgK genes of Nostoc sp. PCC7120 (lane 1) and 35 Nostoc punctiforme PCC73102 (lane 2), and DNA size markers (M), is shown. Amplification reactions 36 were performed with specific primers pairs and cyanobacterial genomic DNA as a template, as described 37 in Materials and Methods, and subsequently loaded onto 1.2 % agarose-TBE gel. As shown, a single 38 DNA band of approximately 0.72 kb was obtained in each case (arrow). Lower panel. SDS-PAGE (12 %, 39 18 w/v, polyacrylamide, 0.5 %, w/v, SDS) analysis of recombinant NsPPGK and NpPPGK purified after 1 FPLC gel filtration. Approx. 10 µg of NsPPGK (27.34 kDa, nominal subunit Mm) and NpPPGK (27.24 2 kDa, nominal subunit Mm) were applied per lane. M, protein standards. Numerals on the left indicate the 3 molecular masses (kDa). Arrow indicates the PPGK protein bands. (b) FPLC gel filtration 4 chromatography analyses of native Mm and oligomeric states of the PPGKs from Nostoc spp. Aliquots 5 (0.5 ml) of metal-chelated chromatography purified preparations of recombinant NsPPGK and NpPPGK 6 were applied to a Superdex© 200 PG column. Calibration curves with protein standards (Thy, 7 thyroglobulin; Fer, ferritine; Amy, β-amylase; ADH, alcohol dehydrogenase; BSA, bovine seroalbumin; 8 CA, carbonic anhydrase; Cyt.c, cytochrome c) are displayed on the left upper corner of the 9 chromatography elution profile figures. A SDS-PAGE analysis of selected fractions around the central 10 peak fraction (50 µl aliquots applied per lane) is also shown. Note that single elution peaks, 11 corresponding to absorbance at 280 nm (broken line) and polyP-glucokinase activity (filled circles, solid 12 line), overlapped in both cases. The asterisks indicate the fraction peaks of recombinant PPGKs as 13 determined by their enzymatic activity and absorbance at 280 nm. Native Mm values of 49.4 and 55.1 kDa 14 were estimated for NsPPK and NpPPGK, respectively. Kav, phase distribution coefficient of the analyzed 15 proteins 16 Fig. 2 Substrate specificity of cyanobacterial recombinant PPGKs. PolyP-glucokinase activity levels of 17 purified NsPPGK (black bars) and NpPPGK (white bars) were determined using polyPs of different chain 18 lengths (panel a) or diverse NTPs (panel b) as phosphoryl donor substrates. Activity levels were obtained 19 from three independent experiments and are shown as means ± S.E. Note that both cyanobacterial PPGKs 20 are strictly polyP dependent glucokinases, and long-chain polyPs are their optimal substrates. No 21 significant activity was detected with either NTPs, PPi, P3c or P3 22 Fig. 3 Non-processive utilization of P150 by NsPPGK. 2.5 mM of P150 was used as a substrate for 23 purified NsPPGK (approx. 3 µg/ml) following the standard assay conditions, as described in the Material 24 and Methods section. At different time intervals, sequential aliquots were collected and polyP was 25 isolated, electrophoresed on a preparative PAGE gel, and finally stained with Toluidine blue O. Lane 1 is 26 zero time, lanes 2 to 10 correspond to 3, 6, 8, 10, 12, 14, 16, 18 and 20 min, respectively 27 Fig. 4 Biochemical characterization of recombinant NsPPGK (black bars) and NpPPGK (white bars) 28 regarding to metal cations dependence of polyP (P60) glucokinase activity. (a) Metal cofactor specificity. 29 Several divalent metal cations were added at 5 mM concentration to the assay mixtures. No detectable 30 activity was measured with Mg2+ in the presence of 10 mM EDTA. Bars represent activity levels from 31 three independent experiments and are shown as means ± S.E. Activity is expressed in relative units (100 32 % percentage assigned to the optimum condition in each case). 100 % activity levels correspond to 33 81.7+7.4 and 95.8+12.5 µmol min-1 mg-1 for NsPPGK and NpPPGK, respectively. (b) NsPPGK activity 34 dependence on Mg2+ concentration. Each point represents the mean activity value ± S.E. of three 35 independent experiments. As shown, no activity was detected either in the absence of a divalent cation or 36 with an excess of the chelating agent EDTA 37 Fig. 5 Effect of the pH (panel a) and temperature (panel b) on the polyP (P60) glucokinase activity of 38 NsPPGK (filled circles) and NpPPGK (open circles). Data are shown as relative units (100 % percentage) 39 and were assigned to the optimum condition in each case. Activity levels were obtained from three 40 independent experiments and are shown as means ± S.E. 100 % activity values correspond to 80.6+7.3 41 and 93.5+9.2 µmol min-1 mg-1 (panel a) and 80.4+6.7 and 92.7+7.3 µmol min-1 mg-1 (panel b) for 42 NsPPGK and NpPPGK, respectively 43 Fig. 6 Experimental evidence for the widespread occurrence of homologs of Nostoc spp. ppgK genes 44 among diazotrophic Cyanobacteria. A search of putative ppgK genes was carried out by Southern blot 45 analysis with diverse cyanobacterial strains representatives of the taxonomic sections (roman numerals) in 46 the classification of Rippka et al. (1979). The strains are identified by their collection numbers.The 47 positions of EcoRI-HindIII-restricted λ DNA fragments used as standards (in the range of 21 to 2 kb) are 48 indicated on the left side. Genomic DNAs (approx. 5 µg) were digested with HindIII (left panel) or EcoRI 49 (right panel) restriction enzymes. The full coding sequence of the ppgK gene from Nostoc sp. PCC7120 50 19 was used as a probe under heterologous hybridization conditions at 55°C. As shown, no hybridization 1 band was observed only in the lanes corresponding to unicellular non-diazotrophic Cyanobacteria 2 Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942 3 Fig. 7 Molecular phylogenetic analysis of cyanobacterial PPGKs. Unrooted Neighbor-Joining (a) and 4 Maximum Parsimony (b) phylogenetic trees, obtained from amino acid sequence alignments of selected 5 bacterial orthologs, are shown. A similar topology was obtained for a Maximum Likelihood tree (not 6 shown). Numbers in selected nodes are bootstrap percentages based on 1,000 replicates. Scale bar 7 indicates number of changes per amino acid site. Most cyanobacterial strains are identified by their PCC 8 numbers. Biochemically characterized PPGKs are shown boxed, and the three strictly polyP-dependent 9 enzymes characterized so far are moreover shown in boldface. Other predicted PPGK sequences were 10 obtained from public databases (UniProtKB and IMG-JGI databases) and their details are summarized in 11 Online Resource Table S3. Note the well-defined and robust cyanobacterial cluster (shaded) which is 12 clearly divergent from the actinobacterial assembly of dual ATP/polyP-dependent homologs, as well as 13 the two deeply-branched clusters of uncharacterized putative PPGKs from αand β-proteobacteria 14 closely related to the cyanobacterial assembly 15 16 TABLES 17 18 Table 1. Kinetic parameters of purified recombinant polyP-gluco(manno)kinase from Nostoc sp. 19 PCC7120 with different polyPs and hexoses as substrates 20 Substrate (polyPn) Vmax (µmol min-1 mg-1) Km a (µM) kcat (s-1) Catalytic efficiency kcat/Km (mM-1s-1) P4 5.7 29.9 4.9 164 P13-18 31.4 37.7 27.0 717 P60 81.7 39.8 70.2 1764 PLC 176.3 49.5 151.4 3059 Glucose (PLC) 239.3 67.7 196.0 2895 Mannose (PLC) 3.1 2,360 1.6 0.7 a Km values are calculated as polyP. 21 22 Table 2. PolyP-hexokinase activities of purified recombinant NsPPGK and NpPPGK 23 Specific activity (µmol min-1 mg-1) Hexose NsPPGK NpPPGK Glucose (5 mM) 229.1±14.0 174.3±14.7 Mannose (50 mM) 3.1±0.2 2.0±0.1 Fructose (50 mM) ND a ND a ND, not detected; the minimum level of detection was ca. 0.05 nmol min-1 mg-1. 24 25 Table 3. Effect of different compounds on the activity of NsPPGK towards glucose and P13-18 as substrates 26 Chemicals Activity (%) NaCl (1 mM) 100 NaCl (5 mM) 99.7 NaCl (10 mM) 99.5 NaCl (50 mM) 84.5 20 NaCl (150 mM) 66.0 KCl (1 mM) 95.8 KCl (5 mM) 82.3 KCl (10 mM) 70.4 KCl (50 mM) 65.2 KCl (150 mM) 49.9 ATP (1 mM) 96.3 ATP (5 mM) 71.0 ATP (50 mM) 12.2 PPi (1 mM) 99.8 PPi (5 mM) 55.6 PPi (10 mM) 28.4 P3 (1 mM) 87.5 P3 (5 mM) 39.9 P3 (10 mM) 12.0 None 100 Specific activity in the absence of inhibitor (set as 100 %) was 31.8 µmol min-1 mg-1. 1 2 Table 4. PPGK specific activities levels with different polyP substrates in whole-cell extracts from 3 diverse Cyanobacteria 4 Cyanobacterial strain a P 4 P 13-18 P 60 P LC (nmol-1min-1 mg-1) Synechococcus elongatus PCC7942(I) ND b ND ND ND Synechocystis sp. PCC6803 (I) ND ND ND ND Dermocarpa sp. PCC7437 (II) 9.0±0.7 8.4±0.7 8.5±0.7 10.9±1.3 Pseudanabaena sp. PCC6903 (III) 3.6±0.4 15.1±1.3 11.1±0.9 16.3±1.4 Anabaena sp. ATCC29413 (IV) 6.8±0.7 27.4±1.7 18.2±1.5 16.0±1.8 Calothrix sp. PCC9327 (IV) 0.1±0.3 2.1±0.2 3.1±0.2 4.6±0.3 Nostoc punctiforme PCC73102 (IV) 10.5±0.9 15.4±1.2 13.7±1,1 19.0±1,4 Nostoc sp. PCC7120 (IV) 0.8±0.1 17.9±1.3 18.4±1.5 10.9±0.9 Scytonema sp. PCC7110 (IV) 0.4±0.1 5.7±0.8 3.1±0.5 12.8±1.6 Chlorogloeopsis sp. PCC6912 (V) c ND ND ND ND Fischerella muscicola UTEX1829 (V) c ND ND ND ND a ATCC (American Type Culture Collection); PCC (Pasteur Culture Collection); UTEX (University of Texas at 5 Austin Culture Collection). Roman numerals in parentheses indicate the sections of the taxonomic classification of 6 Rippka et al. (1979). The two unicellular strains of section I were grown in the presence of 2 mM NaNO3 as a 7 nitrogen source. 8 b ND, not detected activity; the minimum level of detection is ca. 0.05 nmol min-1 mg-1. 9 c The presence of large amounts of extracellular mucous material made very difficult achieving reliable 10 measurements with these colonial strains. 11 Fig. 1 4 4.5 5 5.5 6 0 0.4 0.8 log M m 55 39 kDa 28 19 * 39 28 19 55 kDa * b Absorbance at 280 nm (--) Elution volume (ml) NsPPGK Thy Fer β-Amy ADH CA Cyt.c BSA  NpPPGK 4 4.5 5 5.5 6 0 0.4 0.8 log M m  PolyP glucokinase activity (µmol min-1 ml-1) (●) kb 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 1.0 1.2 1.6 2.0 M 1 2 M a M NpPPGK NsPPGK 15 20 25 30 40 50 60 kDa 0.72 kb Thy Fer β-Amy ADH CA Cyt.c BSA 80 * Kav Kav 0 0 * 0 1 2 3 4 5 6 0.00 0.05 0.10 0.15 0.20 0.25 050 100 150 200 250 0 1 2 3 4 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 050 100 150 200 250 Figure Fig. 2 Glucokinase activity (µmol min-1 mg-1) a Phosphoryl donor PPi P3c P3 P4 P13-18 P60 PLC Phosphoryl donor Glucokinase activity (µmol min-1 mg-1) b NsPPGK NpPPGK 0 40 80 120 160 200 dATP ATP CTP GTP TTP PLC 0 40 80 120 160 200 Figure Time (min) 0 3 6 8 10 12 14 16 18 20 P150 Fig. 3 P15 Figure Fig. 4 Mg2+ (mM) a 0 5 10 15 20 25 30 0 1 2 3 4 5 6 7 Glucokinase activity (µmol min-1 mg-1) b Relative activity (%) Divalent cation NsPPGK NpPPGK NsPPGK Mg2+ Mn2+ Cu2+ Zn2+ Ca2+ Fe2+ Co2+ Mg2+ +EDTA 0 20 40 60 80 100 Figure Fig. 5 0 20 40 60 80 100 5.0 6.0 7.0 8.0 9.0 10.0 11.0 pH Relative activity (%) Relative activity (%) T (⁰C) a b 0 20 40 60 80 100 10 20 30 40 50 60 70 NsPPGK NpPPGK Figure Fig. S4. Sequence and domain structure validation of cyanobacterial PPGKs by trypticpeptide fingerprinting and MALDI-TOF mass spectrometry analysis. The Pfam domain structures of the two natural PPGKs are shown, as well as the sequences of the corresponding purified recombinant proteins in which the amino acid residues are boldcoloured accordingly, the experimentally identified peptides are underlined and the Nterminal His-tags are in lowercase. Identified peptides cover about 80 and 55 % of the predicted protein sequences of natural NsPPGK and NpPPGK, respectively. 1 mrgshhhhhh gsMVEENGSI RTLSVDIGGS GVKAMVLDIT GSPVTERARL 51 DTPQPATPGV VINAIVVLAA AQGEFHRVSV GFPGVVRCGV TETAVNLHPD 101 WIGFDLETAL LKHLNKPVRV INDADMQGFG AIAGKGVELV ITLGTGFGSA 151 LFVDGKLVPN MEMGHHPFRK GETFEQQLGR AELEKIGEKR WNRRLEKAIA 201 SLQHLFNYDY LYIGGGEAVR VNFQLPLNVK LIPNITGLLG GIALWRDEKR Mascot Search Results Probability Based Mowse Score Match to: gi|23125685 Transcriptional regulator/sugar kinase –[Nostoc punctiforme PCC 73102] –> NpPPGK Sequence coverage of natural protein: 55 % Nominal mass (Mm): 25,816 (without the N-terminal tag of 12 aa) aa: 1 12 166 238 aa: 1 12 169 239 1 mrgshhhhhh gsMVEDNGSI RTLSVDIGGS GVKALVLDIT GNPVTERARV 51 DTPQPATPEV VINAIMVLAA AQGEFHRVSV GFPGVVRAGV TETAVNLDSD 101 WIGFDLETAL SQRLHKPVRV INDADMQGFG AIKGKGVELV ITLGTGFGSA 151 LFVDGKLVPN MEMGHHPFRK GETYEEQLGR ATLDKIGQKK WNRRLEKAIA 201 SLQRLFNYDY LYIGGGEAVR VNFQLPLNVK LIPNISGLLG GIALWRDEKT 251 L Match to: gi|81772382 polyphosphate glucokinase – [Nostoc sp. PCC 7120] –> NsPPGK Sequence coverage of natural protein: 82 % Nominal mass (Mm): 25,919 (without the N-terminal tag of 12 aa) Mascot Search Results Probability Based Mowse Score Fig. S5. Determination of the kinetic parameters of the recombinant NsPPGK. Increasing concentrations of polyPs of different chain lengths were used in the range up to 300 µM to phosphorylate glucose (5 mM). Concentration dependence curves of NsPPGK activity with P4 (a), P13-18 (b), P60 (c), and PLC (d) as phosphoryl donor substrates are shown. Enzymatic reactions were carried out at optimal conditions as described in the Materials and Methods section. Points represent mean activity values + S.E. of three independent determinations. Kinetic parameters were determined by nonlinear curve fitting from the Michaelis-Menten plot using the spreadsheet Anemona.xlt (Hernández et al. 1998). 0 1 2 3 4 5 6 0 100 200 300 µmol min-1 mg-1 P4 (µM) 0 5 10 15 20 25 30 35 0 100 200 300 µmol min-1 mg-1 P13-18 (µM) a b 0 10 20 30 40 50 60 70 80 0 100 200 300 µmol min-1 mg-1 P60 (µM) c 0 20 40 60 80 100 120 140 160 0 100 200 300 µmol min-1 mg-1 PLC (µM) d Fig. S6. Determination of the kinetic parameters of the recombinant NpPPGK with different monosaccharide substrates. Concentration dependence phosphorylation activity curves with glucose (a) and mannose (b) of purified NpPPGK, using 1 mM PLC as phosphoryl donor, are shown. Enzymatic reactions were carried out at optimal conditions as described in the Materials and Methods section. Points represent mean activity values + S.E. of three independent determinations. Kinetic parameters were determined by nonlinear curve fitting from the Michaelis-Menten plot using the spreadsheet Anemona.xlt (Hernández et al. 1998). a b 0 50 100 150 200 050 100 150 200 250 300 µmol min-1 mg-1 Glucose (µM) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0 1 2 3 4 5 6 7 µmol min-1 mg-1 Mannose (mM) 0  Online Resource Table S1. List of cyanobacterial strains used in this work Strain a Description, alternative names N2-fixation conditions Group b Synechococcus elongatus PCC7942 Unicellular rod-shaped, also called Anacystis nidulans No I Synechocystis sp. PCC6803 Unicellular globular-shaped No I Dermocarpa sp. PCC7437 Colonial, also called Stanieria cyanosphaera Microaerobic II Pseudanabaena sp. PCC6903 Filamentous, non-heterocystous Microaerobic III Anabaena sp. ATCC29413 Filamentous, heterocystous, also called Anabaena variabilis Aerobic IV Anabaena sp. ATCC33047 Filamentous, heterocystous Aerobic IV Calothrix sp. PCC7601 Filamentous, heterocystous, also called Fremyella diplosiphon Unable to fix N2 (mutant strain) IV Calothrix sp. PCC9327 Filamentous, heterocystous, also called Tolypothrix sp. Aerobic IV Nodularia chucula Filamentous, heterocystous Aerobic IV Nostoc sp. PCC6719 Filamentous, heterocystous Aerobic IV Nostoc sp. PCC7107 Filamentous, heterocystous Aerobic IV Nostoc sp. PCC7120 Filamentous, heterocystous, also called Anabaena Aerobic IV Nostoc punctiforme PCC73102 Filamentous, heterocystous Aerobic IV Scytonema sp. PCC7110 Filamentous, heterocystous Aerobic IV Chlorogloeopsis sp. PCC6912 Branched filamentous, heterocystous, also called Chlorogloeopsis fritschii Aerobic V Fischerella muscicola UTEX1829 Filamentous, heterocystous Aerobic V a Culture Collection abbreviations are as described in Table 4. b With reference to the taxonomic classification of Rippka et al. (1979). Online Resource Table S2. Primers for cloning the ppgK genes from Nostoc sp. PCC7120 and Nostoc punctiforme PCC73102 Gene Primers (new restriction site, underlined) F (BamHI) 5'-GCCGGATCCATGGTGGAAGATAACGGC-3' ppgK (N.7120) ppgK (N.7120) R (PstI) 5'-TCACTGCAGCTATAGTGTTTTTTCATC-3' F (BamHI) 5'-GCCGGATCCATGGTTGAAGAAAATGGATCG-3' ppgK (N.73102) ppgK (N.73102) R (PstI) 5'-TCACTGCAGTTACCTTTTTTCATCTCGCC-3'  Online Resource Table S3. PPGK sequences used in this study Source organism Protein or Gene ID entry a Predicted protein length (aa) Taxonomy b Synechococcus sp. PCC7335* B4WQL2 228 Cyanobacteria, Chroococcales (I) Synechococcus sp. PCC7502* K9SS42 238 Cyanobacteria, Chroococcales (I) Gloeobacter kilaueensis JS1* U5QNN6 241 Cyanobacteria, Gloeobacterales (I) Pleurocapsa sp. PCC7319 2509711744 235 Cyanobacteria, Pleurocapsales (II) Dermocarpa sp. PCC7437 2503800427 237 Cyanobacteria, Pleurocapsales (II) Acaryochloris marina MBIC11017* B0C5T1 238 Cyanobacteria, Chroococcales (II) Acaryochloris sp. CCMEE5410 2514738042 238 Cyanobacteria, Chroococcales (II) Oscillatoriales sp. JSC-12 2510096246 230 Cyanobacteria, Oscillatoriales (III) Pseudanabaena biceps PCC7429 L8N8S2 234 Cyanobacteria, Oscillatoriales (III) Pseudanabaena sp. PCC6802* 2506783054 234 Cyanobacteria, Oscillatoriales (III) Coleofasciculus chthonoplastes PCC7420 B4W415 235 Cyanobacteria, Oscillatoriales (III) Crinalium epipsammum PCC9333 2504685141 235 Cyanobacteria, Oscillatoriales (III) Lyngbya majuscula 3L 2506483678 235 Cyanobacteria, Oscillatoriales (III) Microcoleus chthonoplaste PCC7420 647572171 235 Cyanobacteria, Oscillatoriales (III) Microcoleus sp. PCC7113 2509437012 235 Cyanobacteria, Oscillatoriales (III) Microcoleus vaginatus PCC9802 2505167359 235 Cyanobacteria, Oscillatoriales (III) Moorea producens 3L F4Y1Y5 235 Cyanobacteria, Oscillatoriales (III) Oscillatoria sp. PCC6407 2508875670 235 Cyanobacteria, Oscillatoriales (III) Oscillatoria sp. PCC6506* D8FXR8 235 Cyanobacteria, Oscillatoriales (III) Oscillatoria sp. PCC7112 2504089037 235 Cyanobacteria, Oscillatoriales (III) Oscillatoria acuminata PCC6304 2509419241 236 Cyanobacteria, Oscillatoriales (III) Pseudanabaena sp. PCC7367* K9SLD8 237 Cyanobacteria, Oscillatoriales (III) Lyngbya aestuarii BL J 2578016402 238 Cyanobacteria, Oscillatoriales (III) Lyngbya sp. PCC8106* A0YLE5 238 Cyanobacteria, Oscillatoriales (III) Oscillatoria sp. PCC10802 2509508264 239 Cyanobacteria, Oscillatoriales (III) Leptolyngbya sp. 2LT21S03 2509751146 243 Cyanobacteria, Oscillatoriales (III) Leptolyngbya sp. PCC7375* K9EMV6 251 Cyanobacteria, Oscillatoriales (III) Leptolyngbya sp. Heron Island J 2579003743 255 Cyanobacteria, Oscillatoriales (III) Calothrix sp. PCC6303 K9V545 234 Cyanobacteria, Microchaetaceae (IV) Calothrix desertica PCC7102 2510030452 235 Cyanobacteria, Microchaetaceae (IV) Calothrix sp. PCC7103 WP_019497362 235 Cyanobacteria, Microchaetaceae (IV) Calothrix sp. PCC7507* K9PMJ8 235 Cyanobacteria, Microchaetaceae (IV) Fremyella diplosiphon UTEX481 2501543286 235 Cyanobacteria, Microchaetaceae (IV) Microchaete sp. PCC7126 2509783891 235 Cyanobacteria, Microchaetaceae (IV) Raphidiopsis brookii D9 D4TRI7 232 Cyanobacteria, Nostocaceae (IV) Anabaena sp. PCC7108 2506493476 235 Cyanobacteria, Nostocaceae (IV) Anabaena cylindrica PCC7122 2504134473 235 Cyanobacteria, Nostocaceae (IV) Cylindrospermum stagnale PCC7417* K9WXY9 235 Cyanobacteria, Nostocaceae (IV) Nodularia spumigena CCY9414* A0ZFN0 235 Cyanobacteria, Nostocaceae (IV) Nostoc azollae (strain 0708) D7E4T8 235 Cyanobacteria, Nostocaceae (IV)  Nostoc sp. PCC7107 K9Q5V7 235 Cyanobacteria, Nostocaceae (IV) Scytonema hofmanni PCC7110 2551958472 236 Cyanobacteria, Scytonemataceae (IV) Nostoc sp. PCC7524 2509810004 237 Cyanobacteria, Nostocaceae (IV) Nostoc punctiforme PCC73102* B2J3R4 238 (NpPPGK)c Cyanobacteria, Nostocaceae (IV) Anabaena variabilis ATCC29413* Q3M5W7 239 Cyanobacteria, Nostocaceae (IV) Nostoc sp. PCC7120* Q8YX46 239 (NsPPGK)c Cyanobacteria, Nostocaceae (IV) Fischerella muscicola SAG1427-1 2550703822 234 Cyanobacteria, Stigonematales (V) Fischerella sp. PCC9605* WP_026733157 234 Cyanobacteria, Stigonematales (V) Mastigocladopsis repens MORA, PCC10914 2517243485 235 Cyanobacteria, Stigonematales (V) Fischerella sp. PCC9431 WP_026723397 235 Cyanobacteria, Stigonematales (V) Bifidobacterium longum* D6DBE0 255 Actinobacteria, Bifidobacteriaceae Corynebacterium glutamicum* Q6M4B1 250 Actinobacteria, Corynebacteriaceae Corynebacterium diphtheriae* Q6NGU6 253 Actinobacteria, Corynebacteriaceae Frankia alni* Q0RE01 289 Actinobacteria, Frankiaceae Arthrobacter sp. KM* A0JVB2 267 Actinobacteria, Micrococcaceae Arthrobacter aurescens* A1R5H5 272 Actinobacteria, Micrococcaceae Mycobacterium bovis* C1AFG1 265 Actinobacteria, Mycobacteriaceae Mycobacterium tuberculosis* P9WIN1 265 Actinobacteria, Mycobacteriaceae Rhodococcus erythropolis* C0ZYU0 273 Actinobacteria, Nocardiaceae Rhodococcus fascians* Q8VM93 274 Actinobacteria, Nocardiaceae Thermobifida fusca TM51 R9F6L8 262 Actinobacteria, Nocardiopsaceae Thermobifida fusca YX* Q47NX5 262 Actinobacteria, Nocardiopsaceae Microlunatus phosphovorus NM-1* F5XI06 253 (PPGK2) Actinobacteria, Propionibacteriaceae Propionibacterium shermanii* D7GI59 261 Actinobacteria, Propionibacteriaceae Microlunatus phosphovorus NM-1* F5XK61 266 (PPGK1)c Actinobacteria, Propionibacteriaceae Streptomyces peutecius subsp. caesius* S5DRF7 242 Actinobacteria, Streptomycetaceae Streptomyces coelicolor A3(2)* Q9ADE8 246 Actinobacteria, Streptomycetaceae Candidatus Poribacteria sp. WGA-4E d 2265139082 231 Poribacteria Agrobacterium tumefaciens C58* A9CH74 225 α-Proteobacteria Nitrobacter hamburgensis X14* Q1QLK2 229 α-Proteobacteria Mesorhizobium loti MAFF303099* Q98EJ9 240 α-Proteobacteria Bradyrhizobium sp. DFCI-1* U1H9M5 244 α-Proteobacteria Rhizobium tropici* L0LZW1 250 α-Proteobacteria Burkholderia xenovorans LB400* Q13JL2 266 β-Proteobacteria Burkholderia caribensis MBA4* W4NCD7 269 β-Proteobacteria Burkholderia phymatum DSM17167 / STM815* B2JFU9 270 β-Proteobacteria Cystobacter fuscus DSM2262 d 2538040499 255 δ-Proteobacteria Deinococcus radiodurans* Q9RW46 279 Thermus/Deinococcus group, Deinococcaceae a Sequences (mostly putatives) are referred to their corresponding UniProtKB, GeneBank or IMG-JGI databases entry codes. Those sequences used for the molecular phylogenetic analyses are indicated with an asterisk. b Taxonomy was established following both general bacteriological rules and the specific rules for Cyanobacteria (taxonomic sections denoted by roman numbers in parenthesis) of Rippka et al. (1979). c Biochemically characterized strictly polyP-dependent PPGKs.  d These two sequences of non-photosynthetic bacteria cluster into the cyanobacterial assembly and are not included in the trees for clarity purposes; they may be the results of horizontal gene transfer events. Online Resource Table S4. Purification of His-tagged NsPPGK from transformed E. coli cells Step Specific activity (µmol min-1 mg-1) Protein (mg) Recovery (%) Purification (folds) Crude supernatant 0.16 285.0 100 1 Ni-NTA 14.57 1.4 45 91 Amicon Ultra-3 kDa ultrafiltration 14.12 1.3 40 -- Superdex 200 31.40 0.5 28 196 