Nitrogen sources commonly used by cyanobacteria include ammonium, nitrate, nitrite, urea and atmospheric N2, and some cyanobacteria can also assimilate arginine or glutamine. ABC (ATP-binding cassette)-type permeases are involved in the uptake of nitrate/nitrite, urea and most amino acids, whereas secondary transporters take up ammonium and, in some strains, nitrate/nitrite. In cyanobacteria, nitrate and nitrite reductases are ferredoxin-dependent enzymes, arginine is catabolized by a combination of the urea cycle and arginase pathway, and urea is degraded by a Ni2+-dependent urease. These pathways provide ammonium that is incorporated into carbon skeletons through the glutamine synthetase–glutamate synthase cycle, in which 2-oxoglutarate is the final nitrogen acceptor. The expression of many nitrogen assimilation genes is subjected to regulation being activated by the nitrogen-control transcription factor NtcA, which is autoregulatory and whose activity appears to be influenced by 2-oxoglutarate and the signal transduction protein PII. In some filamentous cyanobacteria, N2 fixation takes place in specialized cells called heterocysts that differentiate from vegetative cells in a process strictly controlled by NtcA.

Introduction

The oxygenic photosynthetic bacteria, the cyanobacteria, are autotrophs that fix CO2 through the reductive pentose phosphate cycle. They make an important contribution to the Earth's nitrogen cycle by incorporating nitrogen into the biosphere through assimilatory processes. Organisms of this group frequently have the capacity to assimilate as sources of nitrogen a number of different simple N-containing compounds including ammonium, nitrate, nitrite and urea. Many cyanobacteria can also assimilate atmospheric nitrogen (a process known as N2 fixation), and still some strains have the capability to assimilate some amino acids, particularly arginine and glutamine (reviewed in [1]). The assimilation of most of these compounds provides intracellular ammonium, which itself is a preferred nitrogen source. Thus, in the presence of ammonium, the genes encoding permeases and enzymes for the assimilation of nitrogen sources alternative to ammonium are repressed, a process known as ‘nitrogen control’. Within the context of unravelling the mechanisms of nutrient incorporation in the biosphere, the aim of our research is to draw a complete picture of the mechanisms of nitrogen assimilation and its regulation in cyanobacteria.

Mechanisms of assimilation of combined nitrogen

The incorporation into the cell of nitrogen-containing compounds, which are frequently found at low concentrations (e.g. below 1 μM) in the environment, takes place through permeases that are located in the cytoplasmic membrane. Multicomponent ABC (ATP-binding cassette)-type uptake transporters have been shown to be involved in the uptake of nitrate and nitrite [2,3] or urea [4] in a number of cyanobacteria. ABC-type permeases are also required for the transport of arginine and glutamine [5]. These permeases use ATP to drive an active, concentrative transport of their substrates. On the other hand, a secondary transporter of the major facilitator superfamily has been identified as the nitrate–nitrite transporter in some marine cyanobacteria [6]. The transport of ammonium is also mediated by secondary permeases, in this case of the Amt family [7,8]. The Amt permeases can be probed with [14C]methylammonium, which has been shown to be concentrated in cells of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 to a level that suggests a membrane potential-driven transport [7].

Intracellular nitrate is sequentially reduced to nitrite and ammonium by nitrate reductase and nitrite reductase, which are the products of the narB and nir genes respectively [9,10]. Cyanobacterial nitrite reductase is homologous with ferredoxin-dependent higher-plant nitrite reductase and contains a [4Fe-4S] cluster and sirohaem as prosthetic groups [10,11]. Electrons from reduced ferredoxin are transferred to the iron–sulphur cluster and then to sirohaem, where nitrite is reduced to ammonium. Cyanobacterial nitrate reductase is homologous with Mo-containing bacterial oxidoreductases but is unique in that it uses ferredoxin as an electron donor, forming tight 1:1 complexes [12]. The Mo cofactor is of the Mo-bis-molybdopterin guanine dinucleotide type [1315], and the enzyme also contains a [4Fe-4S] cluster [16]. In this enzyme system, electrons flow from reduced ferredoxin to the iron–sulphur cluster and then to the Mo cofactor, where nitrate is reduced to nitrite. The narB and nir genes are clustered together with the nitrate/nitrite permease-encoding genes in numerous cyanobacteria forming an operon with the structure nir-permease genes-narB. The high conservation of this gene arrangement, in which the expression level is higher for the upstream than for the downstream genes in the operon [17], suggests that it ensures the production of a balanced amount of the different proteins of the pathway.

Regarding organic sources of nitrogen used by cyanobacteria, urea is degraded to ammonium and CO2 by a standard bacterial Ni2+-dependent urease [4], whereas arginine is catabolized by an unusual pathway that combines the urea cycle and the arginase pathway rendering ammonium and glutamate as final products [18]. Whatever the nitrogen source used for growth, intracellular ammonium is incorporated into carbon skeletons through the glutamine synthetase–glutamate synthase pathway (reviewed in [1]). In cyanobacteria, which lack 2-oxoglutarate dehydrogenase, the main metabolic role of 2-oxoglutarate is the incorporation of nitrogen [19]. This metabolic arrangement makes 2-oxoglutarate an indicator of the C to N ratio of the cells [20].

Figure 1 presents a scheme of the main nitrogen assimilation pathways that can be found in cyanobacteria. The scheme highlights the production of intracellular ammonium during the assimilation of different nitrogen sources and the role of 2-oxoglutarate as the C-skeleton for the incorporation of nitrogen into organic material. However, not all these pathways are present at the same time in a cyanobacterial cell, their expression being strictly regulated by the nitrogen source and also by the availability of carbon.

Main nitrogen assimilation pathways in cyanobacteria

Figure 1
Main nitrogen assimilation pathways in cyanobacteria

Combined nitrogen sources are taken up through permeases and metabolized to ammonium, which is incorporated into carbon skeletons through the glutamine synthetase–glutamate synthase pathway. Nitrogen is then distributed from glutamine or glutamate to the other nitrogen-containing organic compounds. Nrt, ABC-type nitrate/nitrite transporter; Urt, ABC-type urea transporter; Amt, ammonium permease; Nar, nitrate reductase; Nir, nitrite reductase; NifHDK, nitrogenase complex; FdxH, heterocyst-specific ferredoxin; PEP carboxylase, phosphoenolpyruvate carboxylase; 2-OG, 2-oxoglutarate; GS, glutamine synthetase; GOGAT, glutamate synthase. The urease reaction releases two molecules of ammonium and one molecule of CO2 per molecule of urea degraded (not indicated). Nitrogenase and FdxH are boxed to note that in some filamentous cyanobacteria N2 fixation takes place in heterocysts.

Figure 1
Main nitrogen assimilation pathways in cyanobacteria

Combined nitrogen sources are taken up through permeases and metabolized to ammonium, which is incorporated into carbon skeletons through the glutamine synthetase–glutamate synthase pathway. Nitrogen is then distributed from glutamine or glutamate to the other nitrogen-containing organic compounds. Nrt, ABC-type nitrate/nitrite transporter; Urt, ABC-type urea transporter; Amt, ammonium permease; Nar, nitrate reductase; Nir, nitrite reductase; NifHDK, nitrogenase complex; FdxH, heterocyst-specific ferredoxin; PEP carboxylase, phosphoenolpyruvate carboxylase; 2-OG, 2-oxoglutarate; GS, glutamine synthetase; GOGAT, glutamate synthase. The urease reaction releases two molecules of ammonium and one molecule of CO2 per molecule of urea degraded (not indicated). Nitrogenase and FdxH are boxed to note that in some filamentous cyanobacteria N2 fixation takes place in heterocysts.

Nitrogen control

When the cyanobacterial cells are incubated in the presence of a limiting concentration of ammonium, but with an adequate supply of carbon, they sense a high C to N ratio that determines expression of genes encoding permeases and enzymes required for an efficient assimilation of ammonium or for the assimilation of alternative nitrogen sources. This activation of gene expression requires NtcA [21], a transcriptional regulator that belongs to the family of bacterial transcription factors whose best-known representative is CAP (catabolite gene activator protein) of Escherichia coli [22]. Modelling of NtcA based on the CAP structure and preliminary X-ray structural data confirms that NtcA bears a structure similar to that of CAP, bearing a DNA-binding helix–turn–helix motif in its C-terminal domain [23]. NtcA binds to specific sites in the promoters of the regulated genes activating their transcription [24]. The DNA-binding site of NtcA shows the sequence signature GTAN8TAC that is usually found 22 nt upstream from a promoter −10 box [24]. This promoter structure for NtcA-activated genes, which conforms to the structure of bacterial class II promoters, has been found in most cyanobacterial nitrogen-regulated genes investigated to date (reviewed in [25]).

The ntcA gene is autoregulatory and the NtcA protein appears to be subjected to regulation being activated when the C to N ratio of the cell is high [26]. Although the precise mechanism of modulation of NtcA is still not known, two elements that influence NtcA activity have recently been identified: 2-oxoglutarate and the PII protein. Binding of NtcA to some N-regulated promoters is stimulated by 2-oxoglutarate [27,28], suggesting that this metabolite can represent an allosteric effector for NtcA. The cyanobacterial signal transduction PII protein (glnB gene product) binds ATP and 2-oxoglutarate and is phosphorylated when the cells experience a high C to N ratio, it having been suggested that it operates as a 2-oxoglutarate sensor (reviewed in [29]). The PII protein is required for the activation of NtcA-dependent genes specifically under conditions of nitrogen stress [30,31], and it appears to promote gene expression more efficiently in its phosphorylated form [30]. Therefore 2-oxoglutarate may affect NtcA activity directly and indirectly through PII. In vivo positive effects of 2-oxoglutarate on the expression of nitrogen-regulated genes have been observed in an engineered Synechococcus strain bearing the E. coli 2-oxoglutarate permease [32].

Some NtcA-dependent promoters appear to require additional regulators or transcription factors for proper operation. This is the case of the Anabaena nir operon promoter that requires binding of NtcB (a LysR-family transcription factor) in addition to binding of NtcA [33]. Use of this promoter is also dependent on CnaT, a putative glycosyl transferase that influences transcription through a mechanism that is yet to be worked out [34].

In addition to the regulation of gene expression, some elements of the nitrogen assimilation system are subjected to post-translational regulation in cyanobacteria. The nitrate/nitrite permease is a target of regulation, being inhibited when the cells sense a high N to C ratio. The non-phosphorylated form of the PII protein appears to effect specific inhibition of the permease when the cells are incubated under conditions that determine low 2-oxoglutarate levels [35,36]. Additionally, in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803, glutamine synthetase is subjected to ammonium-promoted inactivation by two small protein factors whose expression is repressed by NtcA when the cells are incubated in the absence of ammonium [37,38].

Heterocysts

Many filamentous cyanobacteria fix N2 in specialized cells called heterocysts, which differentiate from some vegetative cells when the filaments are incubated in the absence of a source of combined nitrogen. The heterocysts bear a thick cell wall, which carries extra glycolipid and polysaccharide layers and shows a low permeability to gases. The heterocysts also exhibit a modified metabolism oriented towards keeping a microaerobic environment for nitrogenase expression and towards providing ATP and electrons for nitrogenase function [39].

NtcA is strictly required for heterocyst development [40,41], apparently linking differentiation to nitrogen deficiency, and is needed for the induction of the key heterocyst development regulator HetR [40,42]. Moreover, we have shown that NtcA also participates in the activation of expression of genes whose products are required during the differentiation process or are active in the mature heterocyst. Early developmental genes such as the hetC regulatory gene [43] and genes that are expressed later during the developmental process such as devBCA, encoding a glycolipid exporter, or the two cox operons, encoding cytochrome oxidases that are essential for N2 fixation, are transcribed from NtcA-dependent promoters [44,45]. In the mature heterocyst, the glnA gene encoding glutamine synthetase, petH encoding ferredoxin:NADP+ reductase and the nifHDK operon encoding the nitrogenase complex are also expressed from NtcA-activated promoters [46]. We have recently shown that genes for the metabolism of cyanophycin, a nitrogen reserve made of aspartate and arginine that accumulates conspicuously in the heterocysts, are expressed from multiple promoters also including NtcA-dependent promoters [47]. Some of the heterocyst-related NtcA-dependent promoters do not present the structure of the standard, class II NtcA-type promoter, but rather show the structure of bacterial class I promoters with the NtcA-binding site being located approx. 90 nt upstream from the transcription start point. In summary, NtcA effects nitrogen control not only for triggering the developmental process but also for its progression and in the N2-fixing heterocyst, and does so through the use of different types of NtcA-dependent promoters.

The N2-fixing heterocyst provides the vegetative cells of the filament with fixed nitrogen, probably in the form of amino acids [48]. Glutamine is probably a transferred amino acid [49], but whether cyanophycin amino acids have a role in the nitrogen transfer from the heterocysts to the vegetative cells is currently not known. The mechanism of transfer is also not known, but mutants of some amino acid permeases of Anabaena sp. strain PCC 7120 have been shown to be impaired in diazotrophic growth [50], suggesting a role for such amino acid permeases in intercellular nitrogen transfer in the N2-fixing filament.

The 10th Nitrogen Cycle Meeting 2004: Focused Meeting held at the University of East Anglia, Norwich, U.K., 2–4 September 2004. Edited by C.S. Butler (Newcastle upon Tyne, U.K.) and D.J. Richardson (Norwich, U.K.). Sponsored by the COST (European Cooperation in the field of Scientific and Technical Research) Office and the ESF (European Science Foundation).

Abbreviations

     
  • ABC

    ATP-binding cassette

  •  
  • CAP

    catabolite gene activator protein

This work was supported by grants BMC2001-0509 and BMC2002-03902 from Ministerio de Ciencia y Tecnología and by Plan Andaluz de Investigación, group CVI-129 (Spain).

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