Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität Giessen, 35392 Giessen, Germany
Correspondence
Karl Forchhammer
Karl.Forchhammer{at}mikro.bio.uni-giessen.de
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ABSTRACT |
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INTRODUCTION |
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PII signalling proteins are the most widely distributed signal transduction proteins in nature, and homologues are found in bacteria, archaea and plants. PII proteins play a central role in the regulation of anabolic nitrogen metabolism, but despite their high conservation at the structural level, the regulatory targets are quite diverse in different phylogenetic lineages (for reviews see Ninfa & Atkinson, 2000; Arcondeguy et al., 2001
; Forchhammer 2004
). The hallmark of PII signalling in the cyanobacteria Synechococcus and Synechocystis is reversible phosphorylation at a seryl residue (S49) (Forchhammer & Tandeau de Marsac, 1995a
). The phosphorylation state of PII is an indicator of the cellular C/N status. The highest level of PII phosphorylation is observed in nitrogen-starved cells and intermediate degrees of PII phosphorylation are observed in the presence of nitrate. Carbon limitation and the presence of ammonium stimulate dephosphorylation of PII (reviewed by Forchhammer, 2004
). PII regulates various cellular processes such as nitrate/nitrite and CO2 uptake (Lee et al., 1998
; Hisbergues et al., 1999
) or NtcA-dependent gene expression under conditions of nitrogen deprivation (Aldehni et al., 2003
; Paz-Yepez et al., 2003
). Recently, the first molecular target of PII regulation was identified in cyanobacteria: the non-phosphorylated form of PII forms a tight complex with N-acetylglutamate kinase (NAGK), the key enzyme of arginine biosynthesis (Heinrich et al., 2004
; Burillo et al., 2004
). PII stimulates NAGK activity by an order of magnitude, and thus subjects arginine synthesis to global C/N control through the dependence on the phosphorylation state of PII (Heinrich et al., 2004
; Maheswaran et al., 2004
).
Insights into the mechanism of the PII phosphorylation/dephosphorylation cycle in Synechococcus elongatus could be obtained by biochemical and physiological studies (reviewed by Forchhammer, 1999, 2004
). The S. elongatus PII protein binds the effector molecules 2-oxoglutarate and ATP in a synergistic manner (Forchhammer & Hedler, 1997
). In the ATP and 2-oxoglutarate bound state, PII is phosphorylated by an ATP-dependent kinase activity (Forchhammer & Tandeau de Marsac, 1995a
). Dephosphorylation of PII-P is catalysed by the PP2C homologue PphA. In vitro, PphA reactivity towards PII-P was strongly affected by the addition of ATP, ADP and 2-oxoglutarate. It was suggested that these effector molecules modulate the molecular recognition of PII-P by PphA through binding to PII (Ruppert et al., 2002
). Inhibition of PII-P dephosphorylation by ATP was strongly enhanced by 2-oxoglutarate and to a lower extent by oxaloacetate. In the presence of physiological levels of the effector molecules, PII-P dephosphorylation mainly responded to the 2-oxoglutarate levels (Forchhammer et al., 2004
).
Despite the detailed knowledge about in vitro PII dephosphorylation by PphA, the physiological context of PII dephosphorylation has been poorly investigated. Only the short-term response of PII-P dephosphorylation has been reported in a PphA-deficient mutant of Synechocystis PCC 6803 (MPphA) (Irmler & Forchhammer, 2001; Forchhammer et al., 2004
). Within a period of 30 min, the mutant was unable to dephosphorylate PII-P in response to various external stimuli, suggesting that PphA was the major PII-P phosphatase in these cells. However, the contribution of PphA to total phosphatase activity in Synechocystis cells was not known, nor has the long-term acclimatization of the PII-P phosphorylation state to various C/N conditions been investigated in the PphA-deficient mutant. Also, the abundance and cellular localization of PphA under various growth conditions was not known. The present investigation was performed to clarify these points, which are crucial to understanding the in vivo specificity of PIIPphA recognition and to gain deeper insights into the in vivo conditions of PphA-mediated PII dephosphorylation in Synechocystis PCC 6803.
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METHODS |
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Determination of the modification state of PII.
The phosphorylation state of PII in vivo and in vitro was analysed by non-denaturing PAGE followed by immunoblot analysis of PII as described by Forchhammer & Tandeau de Marsac (1994). In the figures, PII0, PII1, PII2 and PII3 represent isoforms of trimeric PII carrying no, one, two or three phosphorylated subunits, respectively.
Enzyme assays
p-Nitrophenyl phosphate (pNPP) phosphatase activity.
Phosphatase activity with pNPP as substrate was assayed in cell-free extracts according to Mackintosh (1993). Cultures of Synechocystis PCC 6803 wild-type and MPphA were grown in BG11N medium to an OD750 of 0·8. The cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris/HCl pH 7·4, 2 mM MgCl2, 50 mM KCl, 0·5 mM EDTA and 1 mM benzamidine) and broken by sonification. Cell debris and insoluble material was removed by two consecutive centrifugation steps (10 min at 10 000 g and 1 h at 100 000 g). The final supernatants were dialysed against a buffer without divalent cations (20 mM Tris/HCl pH 7·5, 50 mM NaCl, 1 mM DTT and 0·5 mM EDTA). In a 1 ml standard assay, 250 µg protein of the supernatants was reacted with 3 mM pNPP in a buffer (20 mM Tris/HCl pH 8·3, 10 mM NaCl and 0·2 mM DTT) in the presence or absence of 0·4 mM MnCl2. The increase in A400 was recorded in an Ultrospec 3000 spectrophotometer (Amersham-Pharmacia) against a blank reaction in which pNPP was omitted.
In situ glutamine synthetase (GS) assay.
GS activity was determined by the formation of -glutamylhydroxamate (transferase assay) as described previously (Forchhammer & Tandeau de Marsac, 1995b
).
In vitro PII dephosphorylation in cell extracts.
Cultures of the wild-type and MPphA strain of Synechocystis PCC 6803, grown in liquid BG11N medium to mid-exponential phase, were treated with 0·5 mM DON (6-diazo-5-oxo-L-norleucine) for 1 h prior to harvest, to maximize the phosphorylation state of PII. Extracts of these cells were prepared in a Tris/HCl pH 7·4, 4 mM EDTA buffer by grinding with glass beads in a RiboLyser (Hybaid) apparatus as described by Heinrich et al. (2004). After removal of small effector molecules through a protein-desalting spin column (Pierce) the extracts were concentrated by ultrafiltration using a 5 kDa cut-off membrane (Nanosept, Pall Life Science). The extracts (approx. 8 µg protein µl1) were diluted in reaction buffer (10 mM Tris/HCl pH 7·4, 1 mM DTT, 50 mM NaCl and 5 mM benzamidine) with or without 20 mM MgCl2 to a final concentration of 1 µg µl1 and incubated at 37 °C. After different times, samples (each 15 µg protein) were removed and the phosphorylation state of PII was analysed by non-denaturing PAGE followed by immunoblot analysis.
PphA expression under different growth conditions.
To analyse nitrogen-dependent expression of PphA in Synechocystis sp. PCC 6803, a pre-culture was grown in BG11N or in modified BG11A medium, in which molybdenum was replaced by tungsten (4·8 µM), as indicated. At the mid-exponential phase of growth, the cells were harvested by centrifugation and washed in medium free of combined nitrogen (BG110). The cells were resuspended in BG110 or in modified BG110 medium (containing tungsten) to an OD750 of approx. 0·4 and distributed to culture flasks. NH4Cl (5 mM final concentration), NaNO3 or NaNO2 (concentrations as indicated) were added, the cultures were incubated for 24 h as described above and then samples were removed for PphA analysis. For the analysis of PphA expression in the presence of NaNO2 and MSX (L-methionine-DL-sulfoximine), BG11N-grown cells were washed in BG110 medium and resuspended in nitrogen-free medium to an OD750 of 0·5. One half of the culture was treated with MSX (0·2 mM final concentration) as a control, while the other half was treated with MSX and NaNO2 (10 mM final concentration). Samples were removed after 4 h and 8 h incubation.
To determine PphA abundance in the various samples as described above, the cells from a sample equivalent to 1 ml with an OD750 of 1 were harvested by centrifugation. Cell pellets were resuspended in 80 µl SDS sample buffer (75 mM Tris/HCl pH 6·8, 100 mM DTT, 70 mM SDS, 10 %, v/v, glycerol and bromophenol blue) and were lysed by heating for 5 min at 95 °C. After removing the cell debris by centrifugation, 20 µl aliquots were subjected to electrophoresis in a 12·5 % SDS-polyacrylamide gel. Following electrophoresis, PphA was revealed by immunoblot analysis, using polyclonal antibodies (Pineda, Berlin) raised against purified PphA (Irmler & Forchhammer, 2001). The amount of PphA was quantified using a Bio-Rad FluorS Imager together with the Quantity One software (Bio-Rad).
RNA isolation, Northern blot hybridization and RT-PCR analysis.
For analysis of pphA mRNA abundance under different growth conditions, wild-type cells were grown as described above and 50 ml aliquots were removed from the cultures for RNA extraction. The samples were rapidly chilled on ice, centrifuged, and the cells were stored at 80 °C. For analysis of gif mRNA abundances, wild-type and MPphA cells were grown in BG11N medium to an OD750 of 0·7. A 50 ml sample was removed from each culture for RNA analysis at time zero and then the cultures were treated with NH4Cl (5 mM final concentration). At 15 min after ammonium addition, a second 50 ml sample was removed. RNA was isolated by using the RiboPure-Bacteria kit (Ambion) according to the manufacturer's description; 5 µg and 15 µg of total RNA was used for Northern blot analysis of the gif (IF7, IF17) and pphA genes, respectively, according to the method of Sambrook et al. (1989). RNA probes were denatured by treatment with formamide. The samples were loaded and separated in a 1·5 % or 1·2 % (w/v) agarose gel containing formaldehyde and were transferred to nylon membrane (Roti-Nylon plus, Roth). DNA probes internal to IF7 (GS inactivating factor 7), IF17 (GS inactivating factor 17) and rnpB (RNase P subunit B) were obtained by PCR amplification (Table 1
). The pphA probe was a DNA fragment corresponding to nucleotides 317 to 765 of the pphA (sll1771) coding region generated by restriction of plasmid pT7-7pphA (Irmler & Forchhammer, 2001
) with KpnI and SmaI.
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The RT-PCR analysis of pphA mRNA abundance under different growth conditions was performed by using the Qiagen OneStep RT-PCR kit (Qiagen) according to the manufacturer's description. The primers used are shown in Table 1. PCR products were separated on a 2 % (w/v) agarose gel. Quantification was performed using the Bio-Rad Quantity One software.
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RESULTS AND DISCUSSION |
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To gain further insight into the residual PII-P dephosphorylating activity, crude extracts from wild-type and PphA-deficient cells were tested for their ability to dephosphorylate endogenous PII-P protein. As shown in Fig. 2, PII-P was rapidly dephosphorylated in wild-type extracts in a Mg2+-dependent manner, whereas no dephosphorylation was detected in the absence of divalent cations. By contrast, Mg2+-dependent dephosphorylation of PII was almost completely absent in MPphA extracts. Although PphA contributes only a minor proportion of total PP2C activity (see above), in vitro and in vivo dephosphorylation of PII-P is strongly impaired in the PphA-deficient mutant, implying that PII-P is a poor substrate for the other protein phosphatases. The mechanistic basis of its specificity towards PphA deserves further investigation.
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Effect of PphA deficiency on short-term inhibition of GS
The PphA-deficient strain was not impaired in acclimatizing to different nitrogen sources, as far as cell growth was concerned. Since it was shown recently that NtcA-dependent gene expression depends on PII signalling under conditions of nitrogen deprivation (Aldehni et al., 2003; Paz-Yepez et al., 2003
), we wanted to investigate whether impaired PII-P dephosphorylation affects an NtcA-dependent response. In Synechocystis PCC 6803, GS activity is down-regulated by the inactivating factors IF7 and IF17, whose expression depends on NtcA (Garcia-Dominguez et al., 2000
). In the absence of ammonium, active NtcA represses transcription of the IF-encoding gif genes. The addition of ammonium leads to inactivation of NtcA (Herrero et al., 2001
), and thereby to the loss of gif repression. Synthesis of IF7/IF17 then depresses GS activity. Down-regulation of GS activity can therefore be used as a means to determine in vivo inactivation of NtcA following ammonium addition, a strategy which has been used successfully in previous studies (Muro-Pastor et al., 2001
). To investigate whether the PphA-deficient mutant was able to rapidly acclimatize to ammonium with respect to the regulation of GS activity, nitrate-grown cells of the wild-type and MPphA strain were challenged with 5 mM NH4Cl, and after different times, aliquots were removed and the in situ GS (transferase) activity was determined. As shown in Fig. 7(A)
, GS activity declined in the mutant even faster than in the wild-type, indicating that the lack of PphA (and thereby the lack of PII-P dephosphorylation) does not affect the rapid in vivo inactivation of GS. The mechanistic basis of the accelerated GS inactivation in MPphA requires further investigation. To confirm that GS inactivation was indeed due to ammonium-prompted gif gene expression, the levels of gifA and gifB mRNA from cells that were challenged with ammonium was analysed by Northern blotting (Fig. 7B
). The same expression pattern as reported in previous studies of ammonium-treated Synechocystis wild-type cells was observed. The transcript of the tightly controlled gifB gene increased strongly and that of gifA partially (Muro-Pastor et al., 2001
) in both the wild-type and MPphA strains, confirming that gif induction is independent of PII dephosphorylation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 17 November 2004;
revised 14 January 2005;
accepted 17 January 2005.
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