Nitrate assimilation by cyanobacteria is inhibited by the presence of ammonium in the growth medium. Both nitrate uptake and transcription of the nitrate assimilatory genes are regulated. The major intracellular signal for the regulation is, however, not ammonium or glutamine, but 2-oxoglutarate (2-OG), whose concentration changes according to the change in cellular C/N balance. When nitrogen is limiting growth, accumulation of 2-OG activates the transcription factor NtcA to induce transcription of the nitrate assimilation genes. Ammonium inhibits transcription by quickly depleting the 2-OG pool through its metabolism via the glutamine synthetase/glutamate synthase cycle. The P(II) protein inhibits the ABC-type nitrate transporter, and also nitrate reductase in some strains, by an unknown mechanism(s) when the cellular 2-OG level is low. Upon nitrogen limitation, 2-OG binds to P(II) to prevent the protein from inhibiting nitrate assimilation. A pathway-specific transcriptional regulator NtcB activates the nitrate assimilation genes in response to nitrite, either added to the medium or generated intracellularly by nitrate reduction. It plays an important role in selective activation of the nitrate assimilation pathway during growth under a limited supply of nitrate. P(II) was recently shown to regulate the activity of NtcA negatively by binding to PipX, a small coactivator protein of NtcA. On the basis of accumulating genome information from a variety of cyanobacteria and the molecular genetic data obtained from the representative strains, common features and group- or species-specific characteristics of the response of cyanobacteria to nitrogen is summarized and discussed in terms of ecophysiological significance.

In Synechocystis sp. strain PCC 6803, the genes encoding the proteins involved in nitrate assimilation are organized into two transcription units, nrtABCD-narB and nirA, the expression of which was repressed by ammonium and induced by inhibition of ammonium assimilation, suggesting involvement of NtcA in the transcriptional regulation. Under inducing conditions, expression of the two transcription units was enhanced by nitrite, suggesting regulation by NtcB, the nitrite-responsive transcriptional enhancer we previously identified in Synechococcus sp. strain PCC 7942. The slr0395 gene, which encodes a protein 47% identical to Synechococcus NtcB, was identified as the Synechocystis ntcB gene, on the basis of the inability of an slr0395 mutant to rapidly accumulate the transcripts of the nitrate assimilation genes upon induction and to respond to nitrite. While Synechococcus NtcB strictly requires nitrite for its action, Synechocystis NtcB enhanced transcription significantly even in the absence of nitrite. Whereas the Synechococcus ntcB mutant expresses the nitrate assimilation genes to a significant level in an NtcA-dependent manner, the Synechocystis ntcB mutant showed only low-level expression of the nitrate assimilation genes, indicating that NtcA by itself cannot efficiently promote expression of these genes in Synechocystis. Activities of the nitrate assimilation enzymes in the Synechocystis ntcB mutant were consequently low, being 40 to 50% of the wild-type level, and the cells grew on nitrate at a rate approximately threefold lower than that of the wild-type strain. These results showed that the contribution of NtcB to the expression of nitrate assimilation capability varies considerably among different strains of cyanobacteria.In cyanobacteria, expression of the genes encoding the proteins involved in uptake and reduction of nitrate, i.e., nrtABCD or nrtP for the nitrate-nitrite transporter (NRT), narB for nitrate reductase (NR), and nirA for nitrite reductase (NiR), is negatively regulated by ammonium (3,7,19,23,26,27). These genes are usually clustered on the genome and in Synechococcus sp. strain PCC 7942 and Anabaena sp. strain PCC 7120, organized into a large operon, nirA-nrtABCD-narB (nirA operon) (3,7,22,27). Including the nitrate assimilation genes, cyanobacteria have a number of ammonium-repressible genes related to nitrogen metabolism. Expression of the ammoniumrepressible genes commonly requires a Crp-type transcriptional regulator protein, NtcA (28; see reference 11 for a review). Thus, the ammonium-promoted regulation of the nitrate assimilation genes is a part of global nitrogen control in cyanobacteria.In addition to ammonium-promoted regulation, positive regulation by nitrite of the nitrate assimilation operon has been found in Plectonema boryanum and Synechococcus sp. strain PCC 7942 (14). Studies in Synechococcus sp. strain PCC 7942 showed that the nitrite-promoted regulation is specific to the nirA operon and is mediated by a LysR family protein, NtcB (2). NtcB does not promote transcri...

Nitrite, either exogenously supplied or endogenously generated by nitrate reduction, activates transcription of the nitrate assimilation operon (nirA-nrtABCD-narB) in Synechococcus sp. strain PCC 7942 cells treated with L-methionine-DL-sulfoximine (an inhibitor of glutamine synthetase), in which there is no negative feedback resulting from fixation of the ammonium generated by nitrite reduction (Kikuchi et al., J. Bacteriol. 178:5822-5825, 1996). Other transcription units related to nitrogen assimilation, i.e., the nirB-ntcB operon, glnA, and ntcA, were not activated by nitrite. Nitrite did not activate nirA operon transcription in a mutant with a deletion of ntcB, an ammonium-repressible gene encoding a LysR-type DNA-binding protein. Introduction of plasmidborne ntcB into the ntcB deletion mutant restored the response of the cells to nitrite, indicating that NtcB activates the nirA operon in response to nitrite. Supplementation of nitrite or nitrate to nitrogen-starved cultures of the wild-type strain, but not of the ntcB deletion mutant, caused activation of the nirA operon without L-methionine-DL-sulfoximine treatment of the cells. The results suggested that the positive-regulation mechanism of nirA operon transcription plays a role in rapid adaptation of nitrogen-starved cells to changing availability of nitrate and nitrite.Transcription of the nitrate assimilation operon, nirA-nrt-ABCD-narB, of the cyanobacterium Synechococcus sp. strain PCC 7942 is repressed by ammonium (20) and activated by nitrate or nitrite in the medium (11). Ammonium represses transcription through its fixation into Gln, but Gln is not the direct regulator of transcription (20). We have proposed that cyanate, a metabolite of Gln via carbamoylphosphate, acts as the metabolic signal for the ammonium-promoted repression of the nirA operon (21). Depletion of ammonium from the medium or inhibition of ammonium fixation with L-methionine-DL-sulfoximine (MSX) derepresses the operon and induces its transcription, showing no requirement for nitrate or nitrite (20). Under the derepressing conditions, however, nitrate and nitrite further activate transcription (11). The positive effect of nitrate and nitrite is manifest in MSX-treated cells, in which there is no negative feedback by the ammonium generated internally by reduction of nitrate and nitrite (11). In the absence of MSX, the negative regulation by internally generated ammonium overrides the positive regulation, and the effects of nitrate and nitrite are marginal (11). Nitrite is the actual activator of transcription and nitrate must be reduced to nitrite to activate the operon (11).In the genomic DNA region upstream of the nirA operon are the nirB and ntcB genes, which are required for maximum nitrate assimilation and which form an operon oriented divergently from nirA (19). Since the predicted NtcB protein belongs to the LysR family of transcription factors, many members of which activate a divergently transcribed operon located upstream (7, 17), we once suspected that ntcB might be inv...

In the absence of fixation of ammonium to glutamine, nitrate and nitrite activated transcription of the nitrate assimilation (nirA-nrtABCD-narB) operon of Synechococcus sp. strain PCC 7942. In a nitrate reductasedeficient mutant, only nitrite activated transcription, indicating that nitrite is the actual activator of the operon. Nitrate and nitrite were also found to activate the transcription of a nitrate assimilation operon in the filamentous nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum.Nitrate is a major source of nitrogen for cyanobacteria (11,12). It is transported into the cells by an active transport system and reduced to ammonium by the sequential action of nitrate reductase (NR) and nitrite reductase (NiR) prior to fixation into the amide nitrogen of glutamine (Gln). As in other microorganisms capable of nitrate assimilation (3,4,6,8,19,24), expression of the nitrate assimilation system is inhibited by ammonium (12). In the unicellular non-nitrogen-fixing cyanobacterium Synechococcus sp. strain PCC 7942, the genes encoding the nitrate transporter (nrtABCD) (25-27), NR (narB) (2, 17), and NiR (nirA) (20, 32) form the nirA-nrtABCDnarB operon (32), and transcription of the operon is repressed by ammonium (32). Ammonium inhibits transcription through its fixation into Gln, but Gln is not the direct regulator of transcription (32). We have proposed that cyanate, a metabolite of Gln via carbamoylphosphate, acts as the metabolic signal for the ammonium-promoted repression of the nirA operon (34).Transcription of the nirA operon of Synechococcus sp. strain PCC 7942 is induced simply by removal of ammonium from the medium or by inhibition of ammonium fixation with L-methionine-DL-sulfoximine (MSX), showing no requirement for nitrate (32). Induction of the NiR gene is not dependent on nitrate in a filamentous, nonheterocystous nitrogen-fixing cyanobacterium, Plectonema boryanum IAM-M101, either (31). Thus, nitrate has seemingly no specific role in the transcription of the nitrate assimilation genes in the two strains of cyanobacteria. However, during studies of the expression of nitrate assimilation genes in MSX-treated cells, in which there is no negative feedback by the ammonium generated internally by reduction of nitrate and nitrite, we found that nitrate and nitrite do activate the transcription of the nitrate assimilation operons of Synechococcus sp. strain PCC 7942 and P. boryanum. By use of an NR-deficient mutant of strain PCC 7942 (⌬narB::kan) (33), the positive effect of nitrate was shown to be due to nitrite generated by reduction of nitrate.Cells of the cyanobacterial strains were grown photoautotrophically at 30ЊC under CO 2 -sufficient conditions as described previously (34). The basal medium used was a nitrogen-free medium obtained by modification of BG11 medium (30) as previously described (34). Ammonium-containing medium was prepared by adding 3.75 mM (NH 4 ) 2 SO 4 to the basal medium. Transcription of the nirA operon was induced by treatment of ammonium-grown cells with M...

An RND (resistance-nodulation-division)-type transporter having the capacity to export free fatty acids (FFAs) was identified in the cyanobacterium Synechococcus elongatus strain PCC 7942 during characterization of a mutant strain engineered to produce FFAs. The basic strategy for construction of the FFA-producing mutant was a commonly used one, involving inactivation of the endogenous acyl-acyl carrier protein synthetase gene (aas) and introduction of a foreign thioesterase gene ('tesA), but a nitrate transport mutant NA3 was used as the parental strain to achieve slow, nitrate-limited growth in batch cultures. Also, a nitrogen-regulated promoter PnirA was used to drive 'tesA to maximize thioesterase expression during the nitrate-limited growth. The resulting mutant (dAS2T) was, however, incapable of growth under the conditions of nitrate limitation, presumably due to toxicity associated with FFA overproduction. Incubation of the mutant culture under the non-permissive conditions allowed for isolation of a pseudorevertant (dAS2T-pr1) capable of growth on nitrate. Genome sequence and gene expression analyses of this strain suggested that expression of an RND-type efflux system had rescued growth on nitrate. Targeted inactivation of the RND-type transporter genes in the wild-type strain resulted in loss of tolerance to exogenously added FFAs including capric, lauric, myristic, oleic and linolenic acids. Overexpression of the genes in dAS2T, on the other hand, enhanced FFA excretion and cell growth in nitrate-containing medium, verifying that the genes encode an efflux pump for FFAs. These results demonstrate the importance of the efflux system in efficient FFA production using genetically engineered cyanobacteria.