Laboratory of Molecular Plant Physiology, Graduate School of Bioagricultural Sciences,

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1 Plant and Cell Physiology Advance Access published July 18, 2006 Running title: Regulation of nitrate reductase in cyanobacteria *Corresponding author: Tatsuo Omata Laboratory of Molecular Plant Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya , Japan Phone: Fax: Subject area (2) Environmental and stress responses (4) Proteins, enzymes and metabolism Number of black and white figures: 2 Total number of words including the abstract:

2 Title: Regulation of Nitrate Reductase by Non-Modifiable Derivatives of PII in the Cells of Synechococcus elongatus Strain PCC 7942 Authors: Nobuyuki Takatani, Masaki Kobayashi 1, Shin-ichi Maeda, and Tatsuo Omata* Laboratory of Molecular Plant Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya , Japan Abbreviations: NAGK, N-acetyl-L-glutamate kinase; NR, nitrate reductase; NRT, nitrate/nitrite transporter; 2-OG, 2-oxoglutarate. Footnote: 1 Present address: Biological Research Laboratories, Nissan Chemical Industries, Saitama , Japan 2

3 Abstract In Synechococcus elongatus, the PII protein inhibits both transport and reduction of nitrate when ammonium is present in the medium. Using a transporter mutant having ammoniumresistant nitrate transport activity as a genetic background, we analyzed specific effects of PII on in vivo nitrate reductase activity by measuring uptake of nitrate from medium. The results showed that the regulation of nitrate reductase does not require changes in the electric charge or size of the side chain at the phosphorylation site of PII. Phosphorylation of PII is thus unlikely to play a role in the regulation of nitrate reductase. Key words: Cyanobacterium Nitrate assimilation Nitrate reductase PII protein Post translational regulation Synechococcus elongatus strain PCC

4 The PII protein is a signal transducer that is conserved in archaea, bacteria, and the phototrophic eukaryotes (Ninfa and Atkinson 2000, Arcondéguy et al. 2001, Forchhammer 2004). Although knowledge about the role of PII is limited in algae and plants (Sugiyama et al. 2004, Chen et al. 2006, Ferrario-Méry et al. 2006), its role in the regulation of various aspects of nitrogen assimilation has been established in prokaryotic organisms, including cyanobacteria (Ninfa and Atkinson 2000, Arcondéguy et al. 2001, Forchhammer 2004). The cyanobacterial PII protein (a homotrimer of the glnb gene product) is similar to its counterparts in proteobacteria in its ability to bind 2-oxoglutarate (2-OG) and ATP with high affinity (Forchhammer and Hedler 1997). However, the cyanobacterial PII protein is distinct from proteobacterial PII, because it is modified by phosphorylation at Ser 49 rather than by uridylylation at Tyr 51 (Forchhammer and Tandeau de Marsac 1994, Forchhammer and Tandeau de Marsac 1995b). Because of the absence of 2-OG dehydrogenase, the tricarboxylic acid cycle is blocked at the 2-OG oxidation step in cyanobacteria (Smith et al. 1967, Stanier and Cohen-Bazire 1977), hence 2-OG serves solely as the acceptor of the newly fixed nitrogen in the glutamine synthetase-glutamate synthase cycle. Cellular 2-OG content in cyanobacteria therefore responds to the balance between the rates of nitrogen and carbon assimilation. Under CO 2 -fixing conditions, the intracellular concentration of 2-OG is high in nitrogen-starved cells and low in nitrogen-replete cells (i.e., in ammonium-grown cells) (Muro-Pastor et al. 2001). In the presence of physiological concentrations of ATP, 2- OG promotes phosphorylation of PII and inhibits its dephosphorylation, presumably by binding to PII (Irmler et al. 1997). As a consequence, the phosphorylation state of the GlnB trimer changes from fully dephosphorylated in ammonium-grown cells to highly phosphorylated in nitrogen-starved cells (Forchhammer and Tandeau de Marsac 1994, Forchhammer and Tandeau de Marsac 1995a). The cyanobacterial PII protein thus senses the cellular nitrogen status by binding 2-OG and changing its phosphorylation state accordingly. The targets of PII in cyanobacteria include the ABC-type nitrate and nitrite bispecific transporter (NRT) (Lee et al. 1998, Lee et al. 2000, Kobayashi et al. 2005), nitrate reductase (NR) (Lee et al. 1998, Lee et al. 2000), N-acetyl-L-glutamate kinase (NAGK) (Burillo et al. 2004, Heinrich et al. 2004), and a transmembrane protein PamA (Osanai et al. 2005). NRT 4

5 has been shown to be regulated by PII in both Synechococcus elongatus strain PCC 7942 and Synechocystis sp. strain PCC 6803, whereas NR is regulated only in the former species (Lee et al. 1998, Lee et al. 2000, Kobayashi et al. 2005). NAGK has been shown to be regulated in both of the two cyanobacterial species (Heinrich et al. 2004, Maheswaran et al. 2006). PamA is a PII-binding protein found only in the Synechocystis strain (Osanai et al. 2005). Thus, some of the PII targets seem to be common to different cyanobacterial species, while others are limited to certain species. When ammonium is added to cultures of S. elongatus strain PCC 7942 cells assimilating nitrate, NRT and NR are inhibited (Kobayashi et al. 1997) whereas NAGK is activated (Heinrich et al. 2004). The regulation of NAGK activity has been shown to involve phosphorylation and dephosphorylation of PII (Heinrich et al. 2004); the wild-type PII protein with non-phosphorylated Ser 49 activates NAGK in vitro whereas the S49A derivative of PII only weakly activates NAGK, indicating the critical role of the hydroxyl group of Ser 49 (Heinrich et al. 2004). Also, the S49E derivative of PII, which has a negative charge at amino acid position 49, does not activate NAGK in vitro or in vivo (Heinrich et al. 2004). Conversely, the PII derivatives that have Ala or Glu in place of Ser at position 49 inhibit NRT activity in vivo in an ammonium-responsive manner like the wild-type PII protein in both S. elongatus strain PCC 7942 and Synechocystis sp. strain PCC 6803, indicating that the hydroxyl group at amino acid position 49, or changes in the electric charge or size of the side chain at the phosphorylation site, are not required for the regulation of NRT (Kobayashi et al. 2005). PII phosphorylation is thus unlikely to play a role in the regulation of NRT. In vitro interaction of Synechocystis PII and PamA is not affected by the phosphorylation status of PII, either (Osanai et al. 2005). In the present study, we examined whether the regulation of NR involves phosphorylation/dephosphorylation of PII. To analyze NR regulation separately from the NRT regulation, we used the NC2 mutant of S. elongatus strain PCC 7942 as the parental strain for the construction of PII mutants. In this NC2 parental strain, NRT has ammoniumresistant activity due to the lack of the regulatory domain of the NrtC subunit (Kobayashi et al. 1997); NR is therefore the only step in the nitrate assimilation pathway that is inhibited by 5

6 ammonium. A PII-null derivative of NC2 thus constructed was designated PD2. Southern hybridization analysis of the PstI digest of genomic DNA from the PD2 mutant confirmed the absence of the wild-type copy of glnb (Fig. 1A). Additionally, PD2 grew as rapidly as the wild-type strain and the NC2 strain (Fig. 1B), confirming that glnb is not an essential gene in S. elongatus. We have previously shown that nitrate assimilation by NC2 is completely inhibited by ammonium because of the inhibition of NR (Kobayashi et al. 1997). By contrast, nitrate assimilation by PD2 was insensitive to ammonium (Fig. 2A, a), verifying the role of PII in the ammonium-promoted inhibition of NR. To further analyze the structure-function relationship of PII in the regulation of NR, the expression plasmids pglnbs, pglnba and pglnbe (Kobayashi et al. 2005) were introduced into PD2 to construct the PD2S, PD2A, and PD2E strains, respectively. pglnbs encodes a GlnB derivative with a K2E amino acid substitution introduced for the purpose of cloning into the shuttle expression vector pse1. In addition to the K2E substitution, the GlnB derivatives encoded by pglnba and pglnbe carried S49A and S49E amino acid substitutions, respectively. It has been shown that the K2E amino acid substitution does not affect the nitrogen-responsive phosphorylation of PII or the ammonium-responsive inhibition of NRT by PII (Kobayashi et al. 2005). The PD2S strain showed ammonium-responsive inhibition of nitrate assimilation (Fig. 2A, b) like that of the NC2 parental strain, confirming the involvement of PII in the inhibition of NR. The result also indicated that the K2E amino acid substitution did not affect the activity of PII to regulate NR in an ammonium-responsive manner. In the PD2A and PD2E strains expressing the non-modifiable GlnB derivatives with S49A and S49E amino acid substitutions, respectively, nitrate assimilation proceeded normally in the absence of ammonium and was inhibited in the presence of ammonium (Fig. 2A, c and d) as in NC2 (Kobayashi et al. 1997) and PD2S (Fig. 2A, b). Uptake of low concentrations of nitrite, which represents the activity of NRT (Kobayashi et al. 1997, Maeda and Omata 1997), proceeded in the presence of ammonium in the PD2, PD2S, PD2A, and PD2E strains (Fig. 2B), verifying the ammonium resistance of NRT in the NC2 derivatives. The ammonium inhibition of nitrate assimilation in PD2A and PD2E (Fig. 2A, c and d) thus 6

7 indicated that the GlnB derivatives carrying S49A and S49E mutations are active in sensing the cellular nitrogen status and in the regulation of NR activity. Lee et al. (2000) previously reported that nitrate assimilation of S. elongatus strain PCC 7942 is inhibited by PII(S49A) irrespective of the nitrogen conditions of the cell, which implies the permanent inhibition of either NRT or NR. Since NRT was regulated in an ammonium-responsive manner by the PII derivatives with Ala in place of Ser 49 in S. elongatus strain PCC 7942 and in Synechocystis sp. strain PCC 6803, we previously inferred that PII(S49A) permanently inhibits NR (Kobayashi et al. 2005). However, this was clearly not the case; in the absence of ammonium, nitrate assimilation was not inhibited by the PII derivative carrying the S49A amino acid substitution even under the condition of overexpression (Fig. 2A, c). Since the substitution of Ala or Glu for Ser 49 has no significant effect on the binding of 2-OG to PII (Lee et al. 2000), it is not surprising that the PII derivatives are active in sensing the cellular nitrogen status. However, the nitrogenresponsive regulation by the non-modifiable PII derivatives indicates that transmission of the 2-OG signal to NR does not require changes in the electric charge or size of the side chain at amino acid position 49. Thus, phosphorylation and dephosphorylation of PII are unlikely to play a principal role in the regulation of NR. In Escherichia coli, binding of 2-OG to PII induces a conformational change in PII and changes its interaction with target proteins (Jiang and Ninfa 1999). In S. elongatus, 2-OG and ATP synergistically inhibit the action of PII phosphatase on phospho-pii but not that on phosphocasein, suggesting that the effector binding may also result in a conformational change in the cyanobacterial PII (Irmler et al. 1997). We therefore infer that the conformational change of PII, induced by the binding of 2-OG, controls the ability of the PII protein to inhibit NR and NRT. Whether PII interacts directly with NR is yet to be determined by in vitro experiments using purified NR, PII, and ferredoxin. The presumed occurrence of the two distinct modes of PII-mediated signal transduction in cyanobacteria, one dependant on the covalent modification of the protein and the other dependant on conformational changes caused by binding of 2-OG, accounts for the difference in the speed at which the regulation of NRT/NR and NAGK occurs. The inhibition of nitrate 7

8 assimilation is a rapid process, which takes place upon addition of ammonium to medium (Flores et al. 1980, Kobayashi et al. 1997). Reactivation of nitrate assimilation is also rapid, taking place upon consumption of ammonium in medium (Flores et al. 1980, Kobayashi et al. 1997). The fast regulation of NRT and NR activities presumably represents the rapid changes occurring at the 2-OG level in the cell. By contrast, changes in the phosphorylation state of PII are slower, taking >30 minutes for the transition between the fully dephosphorylated state and the highly phosphorylated state (Forchhammer and Tandeau de Marsac 1994, Forchhammer and Tandeau de Marsac 1995a). In accordance with the slow phosphorylation of PII (Forchhammer and Tandeau de Marsac 1995a), inactivation of NAGK is slow, taking hours after nitrogen step-down (Heinrich et al. 2004). We therefore infer that the rapid regulation of NR and NRT enables the cells to adapt promptly to the changing availability of inorganic nitrogen sources, whereas the slow regulation of NAGK ensures that allocation of glutamate for arginine biosynthesis is optimized according to the average rate of nitrogen assimilation over a longer period of time, i.e., 30 to 60 min. It should be noted that in Synechocystis, binding of PII to PamA is inhibited in the presence of ATP and 2-OG but is not affected by the phosphorylation status of PII (Osanai et al. 2005), suggesting that binding of 2-OG to PII inhibits the interaction of PII with PamA. Formation and dissociation of the PII-PamA complex are therefore likely to mediate a rapid and yet unknown response of the cell to changing availability of inorganic nitrogen source. Because 2-OG promotes phosphorylation of PII, presumably by binding to the protein (Forchhammer and Tandeau de Marsac 1995b, Irmler et al. 1997), the metabolic information transmitted to PII by the 2-OG binding is qualitatively the same as that transmitted by the covalent modification of the protein, i.e., nitrogen shortage relative to carbon assimilation. However, as discussed above, the two modes of signal transmission enable PII to adopt different time resolutions for monitoring cellular nitrogen status and for regulating different targets accordingly. This contrasts with E. coli, in which the physiological significance of the two modes of signal transduction by PII seem to reside in integration of two different metabolic signals; the PII uridylylating enzyme responds to the nitrogen signal, glutamine, 8

9 while the binding of 2-OG to PII acts as a carbon signal, allowing PII to regulate a target protein according to the N/C balance of the cell (Ninfa and Atkinson 2000). Maerials and Methods The NC2 mutant of Synechococcus elongatus strain PCC 7942 (Kobayashi et al. 1997) was the parental strain of PD2 and its derivatives. The PII-deficient strain PD2 was constructed from the NC2 strain by insertionally inactivating the glnb gene, as described previously for the construction of the PD1 mutant from the wild-type strain (Kobayashi et al. 2005). The plasmids pglnbs, pglnba and pglnbe (Kobayashi et al. 2005), which are the derivatives of the shuttle expression vector pse1 and carry glnb derivatives transcriptionally fused with the Ptrc promoter, were introduced into the PD2 mutant to yield the strains PD2S, PD2A and PD2E, respectively. Cyanobacterial cells were grown photoautotrophically at 30 C under CO 2 -sufficient conditions in a nitrate-containing medium as described previously (Suzuki et al. 1995). Spectinomycin and kanamycin were added to the medium at 15 µg/ml when appropriate. For Southern hybridization analysis, PstI-digested genomic DNA samples (2 µg/lane) were fractionated on a 1% agarose gel, transferred to positively charged nylon membrane (Hybond N+; Amersham Biosciences, Piscataway, NJ, USA), and hybridized with a 343-bp PCR-amplified glnb fragment labeled with 32 P (Kobayashi et al. 2005). The uptake of nitrate and nitrite by the cells of PD2, PD2S, PD2A, and PD2E was measured at ph 9.6 by monitoring the decreases in the extracellular concentrations of nitrate and nitrite, respectively, as described previously (Kobayashi et al. 1997). Acknowledgments This work was supported by a Grant-in-aid for Scientific Research in Priority Areas ( ) and in part by a Grant-in-Aid for Specially Promoted Research (13CE2005) and The 21st Century COE Program from Ministry of Education, Culture, Sports, Science and Technology of Japan. 9

10 References Arcondéguy, T., Jack, R., and Merrick, M. (2001) P II signal transduction proteins, pivotal players in microbial nitrogen control. Microbiol. Mol. Biol. Rev. 65: Burillo, S., Luque, I., Fuentes, I., and Contreras, A. (2004) Interactions between the nitrogen signal transduction protein PII and N-acetyl glutamate kinase in organisms that perform oxygenic photosynthesis. J. Bacteriol. 186: Chen, Y.M., Ferrar, T.S., Lohmeir-Vogel, E., Morrice, N., Mizuno, Y., Berenger, B., Ng, K.K.S., Muench, D.G., and Moorhead, G.B.G. (2006) The PII signal transduction protein of Arabidopsis thaliana forms an arginine-regulated complex with plastid N-acetyl glutamate kinase. J. Biol. Chem. 281: Ferrario-Méry, S., Besin, E., Pichon, O., Meyer, C., and Hodges, M. (2006) The regulatory PII protein controls arginine biosynthesis in Arabidopsis. FEBS Lett. 580: Flores, E., Guerrero, M.G., and Losada, M. (1980) Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria. Arch. Microbiol. 128: Forchhammer, K. (2004) Global carbon/nitrogen control by P II signal transduction in cyanobacteria: from signals to targets. FEMS Microbiol. Rev. 28: Forchhammer, K., and Hedler, A. (1997) Phosphoprotein P II from cyanobacteria: analysis of functional conservation with the P II signal-transduction protein from Escherichia coli. Eur. J. Biochem. 244:

11 Forchhammer, K., and Tandeau de Marsac, N. (1994) The P II protein in the cyanobacterium Synechococcus sp. strain PCC 7942 is modified by serine phosphorylation and signals the cellular N-status. J. Bacteriol. 176: Forchhammer, K., and Tandeau de Marsac, N. (1995a) Functional analysis of the phosphoprotein P II (glnb gene product) in the cyanobacterium Synechococcus sp. strain PCC J. Bacteriol. 177: Forchhammer, K., and Tandeau de Marsac, N. (1995b) Phosphorylation of the P II protein (glnb gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942: analysis of in vitro kinase activity. J. Bacteriol. 177: Heinrich, A., Maheswaran, M., Ruppert, U., and Forchhammer, K. (2004) The Synechococcus elongatus P II signal transduction protein controls arginine synthesis by complex formation with N-acetyl-L-glutamate kinase. Mol. Microbiol. 52: Irmler, A., Sanner, S., Dierks, H., and Forchhammer, K. (1997) Dephosphorylation of the phosphoprotein P II in Synechococcus PCC 7942: identification of an ATP and 2- oxoglutarate-regulated phosphatase activity. Mol. Microbiol. 26: Jiang, P., and Ninfa, A.J. (1999) Regulation of autophosphorylation of Escherichia coli nitrogen regulator II by the PII signal transduction protein. J. Bacteriol. 181: Kobayashi, M., Rodríguez, R., Lara, C., and Omata, T. (1997) Involvement of the C- terminal domain of an ATP-binding subunit in the regulation of the ABC-type nitrate/nitrite transporter of the cyanobacterium Synechococcus sp. strain PCC J. Biol. Chem. 272:

12 Kobayashi, M., Takatani, N., Tanigawa, M., and Omata, T. (2005) Posttranslational regulation of nitrate assimilation in the cyanobacterium Synechocystis sp. strain PCC J. Bacteriol. 187: Lee, H.-M., Flores, E., Forchhammer, K., Herrero, A., and Tandeau de Marsac, N. (2000) Phosphorylation of the signal transducer P II protein and an additional effector are required for the P II -mediated regulation of nitrate and nitrite uptake in the cyanobacterium Synechococcus sp. PCC Eur. J. Biochem. 267: Lee, H.-M., Flores, E., Herrero, A., Houmard, J., and Tandeau de Marsac, N. (1998) A role for the signal transduction protein P II in the control of nitrate/nitrite uptake in a cyanobacterium. FEBS Lett. 427: Maeda, S., and Omata, T. (1997) Substrate-binding lipoprotein of the cyanobacterium Synechococcus sp. strain PCC 7942 involved in the transport of nitrate and nitrite. J. Biol. Chem. 272: Maheswaran, M., Ziegler, K., Lockau, W., Hagemann, M., and Forchhammer, K. (2006) PII-regulated arginine synthesis controls accumulation of cyanophycin in Synechocystis sp. strain PCC J. Bacteriol. 188: Muro-Pastor, M.I., Reyes, J.C., and Florencio, F.J. (2001) Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. J. Biol. Chem. 276: Ninfa, A.J., and Atkinson, M.R. (2000) PII signal transduction proteins. Trends Microbiol. 8:

13 Osanai, T., Sato, S., Tabata, S., and Tanaka, K. (2005) Identification of PamA as a PIIbinding membrane protein important in nitrogen-related and sugar-catabolic gene expression in Synechocystis sp. PCC J. Biol. Chem. 280: Smith, A.J., London, J., and Stanier, R.Y. (1967) Biochemical basis of obligate autotrophy in blue-green algae and Thiobacilli. J. Bacteriol. 94: Stanier, R.Y., and Cohen-Bazire, G. (1977) Phototrophic prokaryotes: the cyanobacteria. Annu. Rev. Microbiol. 31: Sugiyama, K., Hayakawa, T., Kudo, T., Ito, T., and Yamaya, T. (2004) Interaction of N- acetylglutamate kinase with a PII-like protein in rice. Plant Cell Physiol. 45: Suzuki, I., Kikuchi, H., Nakanishi, S., Fujita, Y., Sugiyama, T., and Omata, T. (1995) A novel nitrite reductase gene from the cyanobacterium Plectonema boryanum. J. Bacteriol. 177:

14 Legends to Figures Fig. 1 Construction of the Synechococcus elongatus glnb mutant PD2. (A) Southern hybridization analysis of PstI-digested genomic DNA samples (2 µg per lane) from the wildtype strain and the glnb insertional mutants PD1 and PD2, using the entire glnb coding region as a probe. (B) Growth of the wild-type strain and mutants lacking the regulatory domain of NrtC and/or the PII protein in nitrate-containing medium (2 mm). Open circles, the wild-type strain; closed circles, NC2; open triangles, PD1; closed triangles, PD2. Fig. 2 Effects of ammonium on the uptake of nitrate (A) and nitrite (B) by PD2 (panel a), PD2S (panel b), PD2A (panel c) and PD2E (panel d) strains. Cells of PD2S, PD2A and PD2E were treated with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 16 h prior to the measurement to induce expression of the non-modifiable GlnB derivatives. Nitrate (A) or nitrite (B) was added at time zero to cell suspensions containing 5 µg of chlorophyll per ml, and ammonium (500 µm) was added immediately after the addition of the nitrate or nitrite. Changes in concentration of nitrate (closed symbols) and nitrite (open symbols) in the medium are shown. Circles, control; triangles, suspensions with added ammonium. Representative of the three sets of essentially the same results obtained with independent cultures is shown. 14

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