Nitrogen Assimilation In Cyanobacteria Research Articles

A glutamine riboswitch is a key element for the regulation of glutamine synthetase in cyanobacteria.

As the key enzyme of bacterial nitrogen assimilation, glutamine synthetase (GS) is tightly regulated. In cyanobacteria, GS activity is controlled by the interaction with inactivating protein factors IF7 and IF17 encoded by the genes gifA and gifB, respectively. We show that a glutamine-binding aptamer within the gifB 5′ UTR of Synechocystis sp. PCC 6803 is critical for the expression of IF17. Binding of glutamine induced structural re-arrangements in this RNA element leading to enhanced protein synthesis in vivo and characterizing it as a riboswitch. Mutagenesis showed the riboswitch mechanism to contribute at least as much to the control of gene expression as the promoter-mediated transcriptional regulation. We suggest this and a structurally related but distinct element, to be designated type 1 and type 2 glutamine riboswitches. Extended biocomputational searches revealed that glutamine riboswitches are exclusively but frequently found in cyanobacterial genomes, where they are primarily associated with gifB homologs. Hence, this RNA-based sensing mechanism is common in cyanobacteria and establishes a regulatory feedback loop that couples the IF17-mediated GS inactivation to the intracellular glutamine levels. Together with the previously described sRNA NsiR4, these results show that non-coding RNA is an indispensable component in the control of nitrogen assimilation in cyanobacteria.

Efficient Gene Induction and Endogenous Gene Repression Systems for the Filamentous Cyanobacterium Anabaena sp. PCC 7120.

In the last decade, many studies have been conducted to employ genetically engineered cyanobacteria in the production of various metabolites. However, the lack of a strict gene regulation system in cyanobacteria has hampered these attempts. The filamentous cyanobacterium Anabaena sp. PCC 7120 performs both nitrogen and carbon fixation and is, therefore, a good candidate organism for such production. To employ Anabaena cells for this purpose, we intended to develop artificial gene regulation systems to alter the cell metabolic pathways efficiently. We introduced into Anabaena a transcriptional repressor TetR, widely used in diverse organisms, and green fluorescent protein (GFP) as a reporter. We found that anhydrotetracycline (aTc) substantially induced GFP fluorescence in a concentration-dependent manner. By expressing tetR under the nitrate-specific promoter nirA, we successfully reduced the concentration of aTc required for the induction of gfp under nitrogen fixation conditions (to 10% of the concentration needed under nitrate-replete conditions). Further, we succeeded in the overexpression of GFP by depletion of nitrate without the inducer by means of promoter engineering of the nirA promoter. Moreover, we applied these gene regulation systems to a metabolic enzyme in Anabaena and successfully repressed glnA, the gene encoding glutamine synthetase that is essential for nitrogen assimilation in cyanobacteria, by expressing the small antisense RNA for glnA. Consequently, the ammonium production of an ammonium-excreting Anabaena mutant was significantly enhanced. We therefore conclude that the gene regulation systems developed in this study are useful tools for the regulation of metabolic enzymes and will help to increase the production of desired substances in Anabaena.

Global Proteomics Reveal an Atypical Strategy for Carbon/Nitrogen Assimilation by a Cyanobacterium Under Diverse Environmental Perturbations

Cyanobacteria, the only prokaryotes capable of oxygenic photosynthesis, are present in diverse ecological niches and play crucial roles in global carbon and nitrogen cycles. To proliferate in nature, cyanobacteria utilize a host of stress responses to accommodate periodic changes in environmental conditions. A detailed knowledge of the composition of, as well as the dynamic changes in, the proteome is necessary to gain fundamental insights into such stress responses. Toward this goal, we have performed a large-scale proteomic analysis of the widely studied model cyanobacterium Synechocystis sp. PCC 6803 under 33 different environmental conditions. The resulting high-quality dataset consists of 22,318 unique peptides corresponding to 1955 proteins, a coverage of 53% of the predicted proteome. Quantitative determination of protein abundances has led to the identification of 1198 differentially regulated proteins. Notably, our analysis revealed that a common stress response under various environmental perturbations, irrespective of amplitude and duration, is the activation of atypical pathways for the acquisition of carbon and nitrogen from urea and arginine. In particular, arginine is catabolized via putrescine to produce succinate and glutamate, sources of carbon and nitrogen, respectively. This study provides the most comprehensive functional and quantitative analysis of the Synechocystis proteome to date, and shows that a significant stress response of cyanobacteria involves an uncommon mode of acquisition of carbon and nitrogen.

Expression of Genes Involved in Nitrogen Assimilation and the C/N Balance Sensing in <I>Prochlorococcus</I> sp. Strain SS120

The expression of five genes involved in nitrogen assimilation in cyanobacteria, namely glnA, glsF, icd, ntcA, and glnB, encoding three key enzymes from that pathway (glutamine synthetase, glutamate synthase, isocitrate dehydrogenase) and two regulatory proteins (NtcA and PII), was studied in this work. Their changes under different conditions were analyzed by quantitative real-time RT-PCR. Nutrient limitation induced clear modifications on the expression of most studied genes: lack of nitrogen provoked an initial increase, followed by a marked decrease; in the cases of phosphorus and iron starvation, a general, stronger expression decrease was observed, particularly striking in the case of iron. Darkness and addition of the photosynthethic inhibitors DCMU and DBMIB also had a strong effect on gene expression. Methionine sulfoximine and azaserine, inhibitors of glutamine synthetase and glutamate synthase, respectively, provoked a sharp increase in icd expression. These results, together with previous studies, suggest that 2-oxoglutarate could be the molecule utilized by Prochlorococcus to sense the C/N balance. Besides, our results confirm the different regulation of nitrogen assimilation in Prochlorococcus with regard to other cyanobacteria.

Coevolution of metal availability and nitrogen assimilation in cyanobacteria and algae

Marine primary producers adapted over eons to the changing chemistry of the oceans. Because a number of metalloenzymes are necessary for N assimilation, changes in the availability of transition metals posed a particular challenge to the supply of this critical nutrient that regulates marine biomass and productivity. Integrating recently developed geochemical, biochemical, and genetic evidence, we infer that the use of metals in N assimilation - particularly Fe and Mo - can be understood in terms of the history of metal availability through time. Anoxic, Fe-rich Archean oceans were conducive to the evolution of Fe-using enzymes that assimilate abiogenic NH(4)(+) and NO(2)(-). The N demands of an expanding biosphere were satisfied by the evolution of biological N(2) fixation, possibly utilizing only Fe. Trace O(2) in late Archean environments, and the eventual 'Great Oxidation Event' c. 2.3 Ga, mobilized metals such as Mo, enabling the evolution of Mo (or V)-based N(2) fixation and the Mo-dependent enzymes for NO(3)(-) assimilation and denitrification by prokaryotes. However, the subsequent onset of deep-sea euxinia, an increasingly-accepted idea, may have kept ocean Mo inventories low and depressed Fe, limiting the rate of N(2) fixation and the supply of fixed N. Eukaryotic ecosystems may have been particularly disadvantaged by N scarcity and the high Mo requirement of eukaryotic NO(3)(-) assimilation. Thorough ocean oxygenation in the Neoproterozoic led to Mo-rich oceans, possibly contributing to the proliferation of eukaryotes and thus the Cambrian explosion of metazoan life. These ideas can be tested by more intensive study of the metal requirements in N assimilation and the biological strategies for metal uptake, regulation, and storage.

All4312, an NtcA-regulated two-component response regulator inAnabaenasp. strain PCC 7120

All4312, encoded by open reading frame all4312 in the genome of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, exhibits a CheY-like receiver domain and an output domain similar to that of OmpR, characteristic of two-component response regulators. Expression of all4312 was directly regulated by NtcA, the global transcriptional regulator of nitrogen assimilation in cyanobacteria. Features characteristic of NtcA-activated promoters were also found upstream from genes encoding All4312 homologues in several other cyanobacterial genomes. Expression of all4312 was however unaffected in a mutant of hetR, which encodes a regulator triggering heterocyst development. The function of All4312 may be related to the cellular response to nitrogen deprivation.

Growth Phase-dependent Activation of Nitrogen-related Genes by a Control Network of Group 1 and Group 2 σ Factors in a Cyanobacterium

It has been reported that an RNA polymerase sigma factor, SigC, mainly contributes to specific transcription from the promoter PglnB-54,-53 under nitrogen-deprived conditions during the stationary phase of cell growth in the cyanobacterium Synechocystis sp. strain PCC 6803 (Asayama, M., Imamura, S., Yoshihara, S., Miyazaki, A., Yoshida, N., Sazuka, T., Kaneko, T., Ohara, O., Tabata, S., Osanai, T., Tanaka, K., Takahashi, H., and Shirai, M. (2004) Biosci. Biotechnol. Biochem. 68, 477-487). In this study, we further examined the functions of group 2 sigma factors of RNA polymerase in NtcA-dependent nitrogen-related gene expression in PCC 6803. Results indicated that SigB and SigC contribute to the transcription from PglnB-54,-53 with a sigma factor replaced in a growth phase-dependent manner. We also confirmed the contribution of SigB and SigC to the transcription of other NtcA-dependent genes, glnA, sigE, and amt1, as in the case of glnB. On the other hand, the transcription of glnN was dependent on SigB and SigE. In the SigB and SigC-based regulation, the level of SigB increased, but that of SigC was constant under conditions of nitrogen deprivation. Furthermore, it was found that SigC negatively and positively regulates the level of SigB in the log and stationary phase, respectively. SigC also had a positive effect on the level of sigB transcript during the stationary phase. In contrast, SigB acts positively on SigC levels in both growth phases. These results and previous findings indicated that multiple group 2 sigma factors take part in the control of NtcA-dependent nitrogen-related gene expression in cooperation with a group 1 sigma factor, SigA.

Transcriptional activation of NtcA-dependent promoters of Synechococcus sp. PCC 7942 by 2-oxoglutarate in vitro.

The transcription factor NtcA is a global regulator of nitrogen homeostasis in cyanobacteria. It thus positively regulates the expression of genes related to nitrogen assimilation such as glnA (which encodes glutamine synthetase) and ntcA itself in response to nitrogen shortage or depletion. The binding of NtcA to the glnA and ntcA promoters of Synechococcus sp. PCC 7942 in vitro now has been shown to be enhanced by 2-oxoglutarate. In vitro analysis of gene transcription also revealed that the interaction of NtcA with its promoter element was not sufficient for activation of transcription, and 2-oxoglutarate was required for transcriptional initiation by NtcA. Given that the intracellular concentration of 2-oxoglutarate is inversely related to nitrogen availability, it is proposed that this metabolite functions as a signaling molecule that transmits information on cellular nitrogen status to NtcA and thereby regulates the transcription of genes related to nitrogen assimilation in cyanobacteria.

Photosynthetic electron transport controls nitrogen assimilation in cyanobacteria by means of posttranslational modification of the glnB gene product.

A glnB gene is identified in the cyanobacterium Synechococcus sp. PCC 7942, and its gene product is found to be covalently modified as a result of imbalance in electron transfer in photosynthesis, where photosystem II is favored over photosystem I. The gene was cloned and sequenced and found to encode a polypeptide of 112 amino acid residues, whose sequence shows a high degree of similarity to the Escherichia coli regulatory protein, PII. In E. coli, PII is involved in signal transduction in transcriptional and post-translational regulation of nitrogen assimilation. Increase in ammonium ion concentration is shown to decrease covalent modification of the Synechococcus PII protein, as in enteric bacteria. We therefore propose that the photosynthetic electron transport chain may regulate the pathway of nitrogen assimilation in cyanobacteria by means of posttranslational, covalent modification of the glnB gene product. The existence of the glnB gene in different strains of cyanobacteria is demonstrated and its implications are discussed.