1 Laboratoire de Chimie Bactérienne, IBSM, CNRS 31, chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
2 Key Laboratory of Agromicrobiology, Huazhong Agriculture University, 430070 Wuhan, Hubei, People's Republic of China
Correspondence
Cheng-Cai Zhang
cczhang{at}ibsm.cnrs-mrs.fr
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ABSTRACT |
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Present address: Department of Biology and Biochemistry, University of Houston, USA.
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INTRODUCTION |
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Heterocysts are regularly intercalated among vegetative cells, giving rise to a regular pattern along each filament (Wolk et al., 1994; Wolk, 2000
). According to one model (Wilcox et al., 1973
; Meeks & Elhai, 2002
), groups of cells may all be competent to respond to a depletion of combined nitrogen. Competition between contiguous cells is postulated to determine which one in every 1020 vegetative cells will become a heterocyst and which will regress to the original vegetative state. Heterocyst pattern would thus be set up by the interaction of different signals, some promoting heterocyst development and others inhibiting it. So far, one signal, corresponding to the product of the patS gene (Yoon & Golden, 1998
), has been identified. In a patS mutant, heterocyst frequency is increased, whereas increasing the level of the diffusible PatS peptide inhibits heterocyst differentiation. However, even in a patS null mutant, vegetative cells still represent the majority of cells on the filament (Yoon & Golden, 1998
, 2001
). This implies that, in the absence of PatS, either there are other signals that inhibit heterocyst development (Yoon & Golden, 2001
) or the signals that activate heterocyst differentiation are insufficient to turn all cells into heterocysts.
It is still not known how filaments of Anabaena sp. PCC 7120 interpret nitrogen availability in the growth medium or what is the nature of the signal(s) that reflect the depletion of combined nitrogen. The critical step for inorganic nitrogen assimilation in cyanobacteria is the set of reactions catalysed by glutamine synthetase (GS) and glutamate synthase (GOGAT), known as the GSGOGAT cycle (Wolk et al., 1994; Merrick & Edwards, 1995
; Herrero et al., 2001
). In these reactions, 2-oxoglutarate serves as the carbon skeleton for the incorporation of ammonium (
). This is particularly true in cyanobacteria, as they lack 2-oxoglutarate dehydrogenase, leaving the Krebs cycle incomplete (Stanier et al., 1977
).
Several studies suggest that 2-oxoglutarate participates in the signalling of carbon/nitrogen metabolism in both Escherichia coli and the unicellular cyanobacterium Synechococcus sp. strain PCC 7942 (Forchhammer & Hedler, 1997; Ninfa et al., 2000
; Muro-Pastor et al., 2001
). In E. coli, the intracellular concentration of glutamine constitutes the signal for nitrogen and 2-oxoglutarate the signal for carbon (Senior, 1975
; for a review, see Ninfa et al., 2000
). Measurement of metabolite pools in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 suggested that the level of 2-oxoglutarate, but not that of glutamine, responds to the nitrogen availability (Muro-Pastor et al., 2001
). Thus, 2-oxoglutarate might act as a signal in the perception of the nitrogen status. Consistent with this idea, 2-oxoglutarate stimulates the expression of the activity of nitrate (
) reductase and the transcription of two nitrogen-regulated genes, nir and amt1, in Synechococcus sp. PCC 7942 (Vazquez-Bermudez et al., 2003
). Two cyanobacterial proteins are known to interact with 2-oxoglutarate, NtcA (for a review, see Herrero et al., 2001
) and PII (Forchhammer & Hedler, 1997
). NtcA is a global control protein for nitrogen metabolism and it is a transcription factor belonging to the cAMP receptor protein family (Luque et al., 1994
; Herrero et al., 2001
). When the nitrogen source is limiting, NtcA enhances transcription of the glnA gene encoding glutamine synthetase or genes involved in the metabolism of an alternative nitrogen source, such as those involved in
acquisition and reduction (Luque et al., 1994
; Cai & Wolk, 1997
; Herrero et al., 2001
). Interestingly, recent studies demonstrate that the presence of 2-oxoglutarate increases the binding affinity of NtcA for at least some of its target promoter elements and this interaction allows the initiation of transcription (Vazquez-Bermudez et al., 2002
; Tanigawa et al., 2002
). In this case, NtcA may constitute a good candidate for sensing the depletion of combined nitrogen by interacting with 2-oxoglutarate. In heterocystous cyanobacteria, like Anabaena sp. PCC 7120, NtcA and HetR (a master control protein specific for heterocyst differentiation) form a regulatory loop to activate heterocyst development (Frias et al., 1994
; Muro-Pastor et al., 2002
). The second cyanobacterial protein known to bind 2-oxoglutarate is PII, a signalling protein well-characterized in unicellular cyanobacterial strains. PII can respond to nitrogen status through reversible phosphorylation on a serine residue in Synechococcus sp. PCC 7942 (Forchhammer & Tandeau de Marsac, 1995
). The binding of 2-oxoglutarate to PII stimulates the phosphorylation reaction of PII (Forchhammer & Tandeau de Marsac, 1995
) and inhibits its dephosphorylation reaction (Irmler et al., 1997
; Ruppert et al., 2002
). NtcA enhances the expression of the glnB gene encoding PII under nitrogen-limiting conditions (Lee et al., 1999
). Recently, it was shown that PII is required for NtcA-mediated nitrogen control under nitrogen-deprivation conditions in Synechococcus sp. PCC 7942 (Fadi Aldehni et al., 2003
; Paz-Yepes et al., 2003
). In heterocystous cyanobacteria, PII might have distinct functions, since the PII-encoding glnB gene is essential in Nostoc punctiforme ATCC 29133 (Hanson et al., 1998
). However, PII has not been functionally characterized in any filamentous strain described so far.
Because 2-oxoglutarate could be an intracellular signal for the nitrogen status in cyanobacteria, and one of its binding proteins, NtcA, is required for the initiation of heterocyst development, this metabolite could possibly exert a positive effect on heterocyst differentiation in these organisms. To test this model, we have constructed a strain of Anabaena sp. PCC 7120 that can take up 2-oxoglutarate in a controlled manner. By modulating the concentration of 2-oxoglutarate in the growth medium, we investigated the possible role of this metabolite in heterocyst development in Anabaena sp. PCC 7120 under different nitrogen regimes.
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METHODS |
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Molecular cloning of kgtP and the petE promoter.
The petE promoter from Anabaena sp. PCC 7120 (Ghassemian et al., 1994; Buikema & Haselkorn, 2001
) was amplified by PCR using primers PetE-up (5'-ctttctagaggatcctaaagcctgtgaa-3') and PetE-down (5'-cttctgcaggtagttttatttttcttattt-3'). The resulting fragment was cloned between the XbaI and PstI sites of pBluescript SK+. The kgtP gene of E. coli (Seol et al., 1991
; Vazquez-Bermudez et al., 2000
) was amplified by PCR using primers KgtP-up (5'-cttctgcagagcatcggattggcaggagac-3') and KgtP-down (5'-cttaagcttgaattcctaaagacgcatcccctt-3'). The PCR product was cloned between the PstI and HindIII sites downstream of the petE promoter in pBluescript SK-. The BamHIEcoRI fragment containing the ppetEkgtP fusion was subcloned into the shuttle vector pRL25c. pRL25c, like pAM1691, is a derivative of pDU1; the copy number of pAM1691 is about 17 per chromosome (Lee et al., 2003
). The construct was conjugated into Anabaena sp. PCC 7120, and exconjugants were selected with 50 µg neomycin ml-1.
Incubation of 2-oxoglutarate with Anabaena cultures and statistical analysis of heterocyst frequency.
Heterocyst differentiation was induced in filaments of Anabaena sp. PCC 7120, and 2-oxoglutarate was added either at the time of heterocyst induction or 1, 2 or 3 days before induction. Pre-incubation was required to observe the effect of 2-oxoglutarate on heterocyst development. Therefore, all subsequent experiments were carried out by incubating first with 2-oxoglutarate for 24 h before the induction of heterocyst differentiation. Heterocyst frequency was measured by randomly counting the number of vegetative cells between two heterocysts (heterocyst interval), as described by Yoon & Golden (1998, 2001)
. The concentration of 2-oxoglutarate was determined and used as follows: 25 mM 2-oxoglutarate for the wild-type strain, 1 mM 2-oxoglutarate for strain KGTP. The stock solution of 2-oxoglutarate was adjusted to pH 7·5.
Uptake of 2-[1-14C]oxoglutarate.
Uptake of 2-[1-14C]oxoglutarate was assayed by the method described by Vazquez-Bermudez et al. (2000). Cells were centrifuged and resuspended in half-strength BG11 medium buffered with HEPES (25 mM, pH 7·5). The optical density of the cell suspension, measured at 700 nm, was 3. Samples were then incubated at 28 °C, under 50 µE of light intensity. 2-[1-14C]Oxoglutarate (50 µCi nmol-1, 1·85 MBq nmol-1) was added at concentrations ranging from 1 to 10 µM. After 15 min incubation, cells were filtered on a 0·45 µm HA filter (Millipore) and washed with 5 ml HEPES buffer (25 mM, pH 7·5); their radioactivity was then counted.
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RESULTS |
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The petE promoter was inserted in front of the coding region of the kgtP gene encoding a 2-oxoglutarate permease in E. coli (Seol et al., 1991). This fusion was carried on the replicative plasmid pRL25c (Elhai & Wolk, 1988
) and conjugated into Anabaena sp. PCC 7120. The recombinant strain was named KGTP.
As shown in Fig. 1, when the wild-type strain of Anabaena sp. PCC 7120 was incubated with 2-[1-14C]oxoglutarate, in the presence of either a small amount of copper or 1 µM copper, only a basal level of labelled metabolites was measured in the cells. In contrast, strain KGTP containing the permease gene could take up 2-oxoglutarate, and this effect was inducible by copper. With no copper, or a low level of copper (0·075 µM), the amount of 2-oxoglutarate entering into cells was as low as in the wild-type. However, with 1 µM copper, the uptake of 2-oxoglutarate became significant (Fig. 1
). The apparent Km value was determined to be 3554 µM, comparable to that (1346 µM) reported for E. coli (Seol & Shatkin, 1992
). The 2-oxoglutarate permease encoded by kgtP under the control of copper was thus functional in Anabaena sp. PCC 7120.
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In a patS-inactivated strain, AMC451 (Yoon & Golden, 1998), the peak of heterocyst intervals was about eight vegetative cells 48 h after the transfer from
to combined-nitrogen-free medium (Fig. 4
A). When heterocyst differentiation was induced with the mutant incubated in the presence of 25 mM 2-oxoglutarate, the peak of heterocyst intervals was decreased to four vegetative cells (Fig. 4B
).
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2-Oxoglutarate advances the timing of commitment to heterocyst differentiation
If 2-oxoglutarate promotes heterocyst differentiation, it possibly also affects the timing of commitment in the process of heterocyst development. Within a few hours after induction of heterocyst differentiation, the addition of a combined-nitrogen source causes reversion of differentiating cells to their vegetative states (Yoon & Golden, 1998; also see Fig. 5
). After a certain point, however, the inhibition of heterocyst differentiation becomes less and less effective. This moment was defined as the point of commitment to heterocyst differentiation, the time at which differentiation becomes irreversible (Yoon & Golden, 2001
).
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DISCUSSION |
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The mechanism of the early perception of nitrogen depletion is possibly conserved in cyanobacteria (Luque et al., 1994; Cai & Wolk, 1997
; Herrero et al., 2001
). A signalling role for 2-oxoglutarate in carbon and nitrogen metabolism has long been suggested in Synechococcus sp. PCC 7942 (Forchhammer & Hedler, 1997
). It was also found that changes in the intracellular pool of 2-oxoglutarate correlated to the expression of genes dependent on NtcA, a global nitrogen control protein highly conserved among cyanobacteria (Muro-Pastor et al., 2001
; Vazquez-Bermudez et al., 2003
). 2-Oxoglutarate was therefore proposed as a signal reflecting the nitrogen status within cells (Muro-Pastor et al., 2001
). Furthermore, the binding affinity of NtcA towards its target DNA fragment, such as the promoter region of glnA, was enhanced by 2-oxoglutarate (Vazquez-Bermudez et al., 2002
; Tanigawa et al., 2002
). In Anabaena sp. PCC 7120, NtcA also constitutes the global regulator for nitrogen metabolism, as in unicellular strains (Luque et al., 1994
; Frias et al., 1994
; Cai & Wolk, 1997
; Herrero et al., 2001
), but it is also the first protein known so far in the regulatory cascade required for heterocyst differentiation (Wolk, 2000
; Herrero et al., 2001
). These results suggest the following model for the early signalling pathway leading to heterocyst development in heterocystous cyanobacteria. On depletion of combined nitrogen, the intracellular level of 2-oxoglurate increases rapidly to reach a threshold concentration, and binds to and activates the transcription factor NtcA. The signalling effect of 2-oxoglutarate can be further amplified through the positive autoregulation of NtcA (for a review, see Herrero et al., 2001
). As a result, the transcription of ntcA reaches the maximal level, enabling it subsequently to activate genes involved in nitrogen metabolism and heterocyst formation. This hypothesis agrees with the observation that the presence of 2-oxoglutarate leads to more heterocyst differentiation even in the presence of
(Fig. 3
). By increasing the level of 2-oxoglutarate, as we did in strain KGTP in this study, this metabolite may also help cells to reach the critical threshold concentration faster, thus leading to earlier commitment to heterocyst development. However, more experiments need to be done to confirm the hypothesis.
The signalling protein PII is also involved in the perception of the 2-oxoglutarate signal in unicellular strains and is even required for NtcA-mediated gene expression under nitrogen-deprivation conditions (Fadi Aldehni et al., 2003; Paz-Yepes et al., 2003
). However, little is known about the function of PII in filamentous cyanobacteria. In light of the results in unicellular strains, it is conceivable that PII may interact with 2-oxoglutarate and NtcA for heterocyst differentiation in heterocystous cyanobacteria.
PatS and 2-oxoglutarate may act in concert to determine heterocyst frequency. Although the increase of 2-oxoglutarate by itself led to more heterocysts, it did not produce double heterocysts. When the level of 2-oxoglutarate was increased in a PatS- mutant (Yoon & Golden, 1998; 2001
), not only was heterocyst frequency further increased, but also there was an increase in the number of double heterocysts. It seems that various signals, including 2-oxoglutarate, the products of nitrogen assimilation or fixation (Yoon & Golden, 2001
) and the inhibitor of heterocyst differentiation PatS (Yoon & Golden, 1998
), act together to determine heterocyst frequency, possibly as a means to manage the efficiency of exchanges of different metabolites between vegetative cells and heterocysts.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 6 May 2003;
revised 30 July 2003;
accepted 5 August 2003.
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