Antonia Herrero

Nitrogen Control in Cyanobacteria

Nitrogen is a quantitatively important bioelement which is incorporated into the biosphere through assimilatory processes carried out by microorganisms and plants. Numerous nitrogencontaining compounds can be used by different organisms as sources of nitrogen. These include, for instance, inorganic ions like nitrate or ammonium and simple organic compounds like urea, amino acids, and some nitrogen-containing bases. Additionally, many bacteria are capable of fixing N 2. Nitrogen control is a phenomenon that occurs widely among microorganisms and consists of repression of the pathways of assimilation of some nitrogen sources when some other, more easily assimilated source of nitrogen is available to the cells. Ammonium is the preferred nitrogen source for most bacteria, but glutamine is also a very good source of nitrogen for many microorganisms. Two thoroughly investigated nitrogen control systems are the NtrB-NtrC two-component regulatory system found in enterics and some other proteobacteria (80) and the GATA family global nitrogen control transcription factors of yeast and some fungi (75). Novel nitrogen control systems have, however, been identified in bacteria other than the proteobacteria, like Bacillus subtilis (26), Corynebacterium glutamicum (52), and the cyanobacteria. The cyanobacterial system is the subject of this review. The cyanobacteria are prokaryotes that belong to the Bacteria domain and are characterized by the ability to perform oxygenic photosynthesis. Cyanobacteria have a wide ecological distribution, and they occupy a range of habitats, which includes vast oceanic areas, temperate soils, and freshwater lakes, and even extreme habitats like arid deserts, frigid lakes, or hot springs. Photoautotrophy, fixing CO 2 through the Calvin cycle, is the dominant mode of growth of these organisms (109). A salient feature of the intermediary metabolism of cyanobacteria is their lack of 2-oxoglutarate dehydrogenase (109). As a consequence, they use 2-oxoglutarate mainly as a substrate for the incorporation of nitrogen, a metabolic arrangement that may have regulatory consequences. Notwithstanding their rather homogeneous metabolism, cyanobacteria exhibit remarkable morphological diversity, being found as either unicellular or filamentous forms and exhibiting a number of cell differentiation processes, some of which take place in response to defined environmental cues, as is the case for the differentiation of N 2-fixing heterocysts (109). Nitrogen control in cyanobacteria is mediated by NtcA, a transcriptional regulator which belongs to the CAP (the catabolite gene activator or cyclic AMP [cAMP] receptor protein) family and is therefore different from the well-characterized Ntr system. Interestingly, however, the signal transduction P II protein, which plays a key role in Ntr regulation, is found in cyanobacteria but with characteristics which differentiate it from proteobacterial P II. In the following paragraphs, we shall first briefly summarize our current knowledge of the cyanobacterial nitrogen assimilation pathways and of what is known about their regulation at the protein level. This description will introduce most of the known cyanobacterial nitrogen assimilation genes. We shall then describe the ntcA gene and the NtcA protein themselves to finally discuss NtcA function through a survey of the NtcA-regulated genes which participate in simple nitrogen assimilation pathways or in heterocyst differentiation and function.

Compartmentalized function through cell differentiation in filamentous cyanobacteria

Within the wide biodiversity that is found in the bacterial world, Cyanobacteria represents a unique phylogenetic group that is responsible for a key metabolic process in the biosphere — oxygenic photosynthesis — and that includes representatives exhibiting complex morphologies. Many cyanobacteria are multicellular, growing as filaments of cells in which some cells can differentiate to carry out specialized functions. These differentiated cells include resistance and dispersal forms as well as a metabolically specialized form that is devoted to N2 fixation, known as the heterocyst. In this Review we address cyanobacterial intercellular communication, the supracellular structure of the cyanobacterial filament and the basic principles that govern the process of heterocyst differentiation.

Assimilatory Nitrogen Metabolism and Its Regulation

The element nitrogen (N) constitutes about 5–10% of the dry weight of a cyanobacterial cell. The purpose of this chapter is to review the assimilatory pathways which in free-living cyanobacteria lead from different extracellular N-sources to cellular N-containing components. Inorganic nitrogen in the form of ammonium is incorporated into glutamine and glutamate via the glutamine synthetase/glutamate synthase cycle. The glnA gene, encoding glutamine synthetase, has been characterized in a number of cyanobacteria. Glutamate (and glutamine) distribute N to other organic compounds by means of transaminases, and glutamate is itself a precursor of some other nitrogenous metabolites. Ammonium can be taken up from the external medium by the cyanobacterial cell, but it can also be derived from other nutrients, essentially N2, nitrate and urea. Many cyanobacteria are able to fix N2 under aerobic conditions. Strategies for protecting nitrogenase from O2 in cyanobacteria include the temporal separation of nitrogenase activity and photosynthetic O2 evolution, and in some filamentous cyanobacteria, the differentiation of heterocysts (cells specialized in N2 fixation). A detailed characterization of nif genes has only been performed in a heterocyst-forming cyanobacterium. Nitrate reduction has been found to use photosynthetically reduced ferredoxin as an electron donor, and genes encoding nitrate transport and reduction proteins have been identified and shown to constitute an operon. Some amino acids like arginine and glutamine can also contribute N to some cyanobacteria; however, urea and amino acid utilization have been poorly investigated thus far. Pathways of N assimilation in cyanobacteria are induced upon ammonium deprivation, ammonium being the preferred N source. A gene, ntcA, encoding a transcriptional regulator required for expression of proteins subjected to nitrogen control has been identified. A major theme for future research is how information about the N status of the cell is sensed and transduced to the protein(s) effecting regulation of gene expression.

Requirement of the regulatory protein NtcA for the expression of nitrogen assimilation and heterocyst development genes in the cyanobacterium Anabaena sp. PCC7120

The cyanobacterial ntcA gene encodes a DNA‐binding protein that belongs to the Crp family of bacterial transcriptional regulators. In this work, we describe the isolation of an ntcA insertional mutant of the dinitrogen‐fixing, heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120. The Anabaena ntcA mutant was able to use ammonium as a source of nitrogen for growth, but was unable to assimilate atmospheric nitrogen (dinitrogen) or nitrate. Nitrogenase and enzymes of the nitrate reduction system were not synthesized in the ntcA mutant under derepressing conditions, and glutamine synthetase levels were lower in the mutant than in the wild‐type strain. In the ntcA mutant, in response to removal of ammonium, accumulation of mRNA of the genes encoding nitrogenase (nifHDK), nitrite reductase (nir, the first gene of the nitrate assimilation operon), and glutamine synthetase (glnA) was not observed. A transcription start point of the Anabaena glnA gene (corresponding to RNA1), that has been shown to be used preferentially after nitrogen step‐down, was not used in the ntcA insertional mutant. Heterocyst development (which is necessary for the aerobic fixation of dinitrogen) and induction of hetR (a regulatory gene that is required for heterocyst development) were also impaired in the ntcA mutant. These results showed that the ntcA gene product, NtcA, is required in Anabaena sp. PCC 7120 for the expression of genes encoding proteins involved in the assimilation of nitrogen sources alternative to ammonium including dinitrogen and nitrate, and that the process of heterocyst development is also controlled by NtcA.

2‐Oxoglutarate increases the binding affinity of the NtcA (nitrogen control) transcription factor for the Synechococcus glnA promoter

The cyanobacterial NtcA global nitrogen regulator belongs to the catabolite activator protein (CAP) family and activates transcription of nitrogen assimilation genes in response to nitrogen step‐down. The binding affinity of NtcA towards a DNA fragment carrying the promoter of the glnA gene from Synechococcus sp. PCC 7942, analyzed in vitro by band‐shift assay, was increased five‐fold by 2‐oxoglutarate in the presence of Mg2+ ions. The 2‐oxoglutarate effect peaked at about 0.6 mM, a rather physiological concentration for this compound under nitrogen‐limiting conditions, and could be partially reproduced by 3‐oxoglutarate but not by oxaloacetate or glutamate. These results suggest 2‐oxoglutarate as a signal of the C to N balance of the cells to regulate NtcA activity and provide a new example of regulation in the versatile CAP family of proteins.

NtcA, a global nitrogen regulator from the cyanobacterium Synechococcus that belongs to the Crp family of bacterial regulators

The gene ntcA is required for full expression of proteins subject to ammonium repression in the cyanobacterium Synechococcus. A 3.1 kb DNA fragment able to complement an ntcA mutant was digested with exonuclease III, and deleted fragments of different sizes were tested for complementation of that mutant, allowing the localization of its mutation within a BamHI‐HindIII genomic fragment of c. 0.4 kb. Insertion of a chloramphenicol‐resistance‐encoding gene cassette into both the BamHI and the HindiIII sites of wild‐type Synechococcus resulted in a pleiotropic, nitrogen‐assimilation‐minus phenotype, corroborating the presence of the ntcA gene in that genomic region. Sequencing of DNA in this region showed the presence of an open reading frame that included both the BamHI and the HindIII sites. The ntcA gene product, NtcA, is a protein of 24817 Da which belongs to a family of bacterial transcriptional activators that, among others, includes Crp and Fnr from Escherichia coli. Of special biological significance, it appears, is the presence of a conserved helix‐turn‐helix motif in the sequence close to the C‐terminal end of all the proteins in the family. The gene ntcA is proposed to encode a transcriptional activator of genes subject to nitrogen control in Synechococcus.

Photosynthetic nitrate assimilation in cyanobacteria.

Nitrate uptake and reduction to nitrite and ammonium are driven in cyanobacteria by photosynthetically generated assimilatory power, i.e., ATP and reduced ferredoxin. High-affinity nitrate and nitrite uptake takes place in different cyanobacteria through either an ABC-type transporter or a permease from the major facilitator superfamily (MFS). Nitrate reductase and nitrite reductase are ferredoxin-dependent metalloenzymes that carry as prosthetic groups a [4Fe–4S] center and Mo-bis-molybdopterin guanine dinucleotide (nitrate reductase) and [4Fe–4S] and siroheme centers (nitrite reductase). Nitrate assimilation genes are commonly found forming an operon with the structure: nir (nitrite reductase)-permease gene(s)-narB (nitrate reductase). When the cells perceive a high C to N ratio, this operon is transcribed from a complex promoter that includes binding sites for NtcA, a global nitrogen-control regulator that belongs to the CAP family of bacterial transcription factors, and NtcB, a pathway-specific regulator that belongs to the LysR family of bacterial transcription factors. Transcription is also affected by other factors such as CnaT, a putative glycosyl transferase, and the signal transduction protein PII. The latter is also a key factor for regulation of the activity of the ABC-type nitrate/nitrite transporter, which is inhibited when the cells are incubated in the presence of ammonium or in the absence of CO2. Notwithstanding significant advance in understanding the regulation of nitrate assimilation in cyanobacteria, further post-transcriptional regulatory mechanisms are likely to be discovered.

Mutual dependence of the expression of the cell differentiation regulatory protein HetR and the global nitrogen regulator NtcA during heterocyst development

Heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120 depends on both the global nitrogen regulator NtcA and the cell differentiation regulatory protein HetR, and induction of hetR upon nitrogen step‐down depends on NtcA. The use of two out of the four transcription start points (tsps) described for the hetR gene (those located at positions –728 and –271) was found to be dependent on NtcA, and the use of the tsp located at position –271 was also dependent on HetR. Thus, autoregulation of hetR could take place via the activation of transcription from this tsp. Expression of ntcA in nitrogen‐fixing cultures was higher than in cells growing in the presence of ammonium or nitrate, and high expression of ntcA under nitrogen deficiency resulted from an increased use of tsps located at positions –180 and –49. The induction of the use of these tsps did not take place in ntcA or hetR mutant strains. These results indicate a mutual dependency in the induction of the regulatory genes hetR and ntcA that takes place in response to nitrogen step‐down in Anabaena cells. Expression of the hetC gene, which is also involved in the early steps of heterocyst differentiation, from its NtcA‐dependent tsp was, however, not dependent on HetR.

Mechanism of intercellular molecular exchange in heterocyst-forming cyanobacteria

Heterocyst‐forming filamentous cyanobacteria are true multicellular prokaryotes, in which heterocysts and vegetative cells have complementary metabolism and are mutually dependent. The mechanism for metabolite exchange between cells has remained unclear. To gain insight into the mechanism and kinetics of metabolite exchange, we introduced calcein, a 623‐Da fluorophore, into the Anabaena cytoplasm. We used fluorescence recovery after photobleaching to quantify rapid diffusion of this molecule between the cytoplasms of all the cells in the filament. This indicates nonspecific intercellular channels allowing the movement of molecules from cytoplasm to cytoplasm. We quantify rates of molecular exchange as filaments adapt to diazotrophic growth. Exchange among vegetative cells becomes faster as filaments differentiate, becoming considerably faster than exchange with heterocysts. Slower exchange is probably a price paid to maintain a microaerobic environment in the heterocyst. We show that the slower exchange is partly due to the presence of cyanophycin polar nodules in heterocysts. The phenotype of a null mutant identifies FraG (SepJ), a membrane protein localised at the cell–cell interface, as a strong candidate for the channel‐forming protein.

Ammonium/Methylammonium Permeases of a Cyanobacterium IDENTIFICATION AND ANALYSIS OF THREE NITROGEN-REGULATEDamt GENES IN SYNECHOCYSTIS sp. PCC 6803

Ammonium is an important nitrogen source for many microorganisms and plants. Ammonium transporters whose activity can be probed with [14C]methylammonium have been described in several organisms including some cyanobacteria, and amtgenes encoding ammonium/methylammonium permeases have been recently identified in yeast, Arabidopsis thaliana, and some bacteria. The unicellular cyanobacterium Synechocystis sp. PCC 6803 exhibited a [14C]methylammonium uptake activity that was inhibited by externally added ammonium. Three putativeamt genes that are found in the recently published complete sequence of the chromosome of strain PCC 6803 were inactivated by insertion of antibiotic resistance-encoding gene-cassettes. The corresponding mutant strains were impaired in uptake of [14C]methylammonium. Open reading framesll0108 (amt1) was responsible for a high affinity uptake activity (K s for methylammonium, 2.7 μm), whereas open reading frames sll1017(amt2) and sll0537 (amt3) made minor contributions to uptake at low substrate concentrations. Expression of the three amt genes was higher in nitrogen-starved cells than in cells incubated in the presence of a source of nitrogen (either ammonium or nitrate), but amt1was expressed at higher levels than the other two amtgenes. Transcription of amt1 was found to take place from a promoter bearing the structure of the cyanobacterial promoters activated by the nitrogen control transcription factor, NtcA.