Accessory Proteins of the Nitrogenase Assembly, NifW, NifX/NafY, and NifZ, Are Essential for Diazotrophic Growth in the Nonheterocystous Cyanobacterium Leptolyngbya boryana.

Since nitrogenase is extremely vulnerable to oxygen, aerobic or micro-aerobic nitrogen-fixing organisms need to create anaerobic microenvironments in the cells for diazotrophic growth, which would be one of the major barriers to express active nitrogenase in plants in efforts to create nitrogen-fixing plants. Numerous cyanobacteria are able to fix nitrogen with nitrogenase by coping with the endogenous oxygen production by photosynthesis. Understanding of the molecular mechanisms enabling to the coexistence of nitrogen fixation and photosynthesis in nonheterocystous cyanobacteria could offer valuable insights for the transfer of nitrogen fixation capacity into plants. We previously identified the cnfR gene encoding the master regulator for the nitrogen fixation (nif) gene cluster in the genome of a nonheterocystous cyanobacterium Leptolyngbya boryana, in addition to initial characterization of the nif gene cluster. Here we isolated nine mutants, in which the nif and nif-related genes were individually knocked out in L. boryana to investigate the individual functions of (1) accessory proteins (NifW, NifX/NafY, and NifZ) in the biosynthesis of nitrogenase metallocenters, (2) serine acetyltransferase (NifP) in cysteine supply for iron-sulfur clusters, (3) pyruvate formate lyase in anaerobic metabolism, and (4) NifT and HesAB proteins. ΔnifW, ΔnifXnafY, and ΔnifZ exhibited the most severe phenotype characterized by low nitrogenase activity (<10%) and loss of diazotrophic growth ability. The phenotypes of ΔnifX, ΔnafY, and ΔnifXnafY suggested that the functions of the homologous proteins NifX and NafY partially overlap. ΔnifP exhibited significantly slower diazotrophic growth than the wild type, with lower nitrogenase activity (22%). The other four mutants (ΔpflB, ΔnifT, ΔhesA, and ΔhesB) grew diazotrophically similar to the wild type. Western blot analysis revealed a high correlation between nitrogenase activity and NifD contents, suggesting that NifD is more susceptible to proteolytic degradation than NifK in L. boryana. The phenotype of the mutants lacking the accessory proteins was more severe than that observed in heterotrophic bacteria such as Azotobacter vinelandii, which suggests that the functions of NifW, NifX/NafY, and NifZ are critical for diazotrophic growth of oxygenic photosynthetic cells. L. boryana provides a promising model for studying the molecular mechanisms that produce active nitrogenase, to facilitate the creation of nitrogen-fixing plants.

Functional expression of an oxygen-labile nitrogenase in an oxygenic photosynthetic organism.

Transfer of nitrogen fixation ability to plants, especially crops, is a promising approach to mitigate dependence on chemical nitrogen fertilizer and alleviate environmental pollution caused by nitrogen fertilizer run-off. However, the need to transfer a large number of nitrogen fixation (nif) genes and the extreme vulnerability of nitrogenase to oxygen constitute major obstacles for transfer of nitrogen-fixing ability to plants. Here we demonstrate functional expression of a cyanobacterial nitrogenase in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803). A 20.8-kb chromosomal fragment containing 25 nif and nif-related genes of the diazotrophic cyanobacterium Leptolyngbya boryana was integrated into a neutral genome site of Synechocystis 6803 by five-step homologous recombination together with the cnfR gene encoding the transcriptional activator of the nif genes to isolate CN1. In addition, two other transformants CN2 and CN3 carrying additional one and four genes, respectively, were isolated from CN1. Low but significant nitrogenase activity was detected in all transformants. This is the first example of nitrogenase activity detected in non-diazotrophic photosynthetic organisms. These strains provide valuable platforms to investigate unknown factors that enable nitrogen-fixing growth of non-diazotrophic photosynthetic organisms, including plants.

Identification of a cis-acting element in nitrogen fixation genes recognized by CnfR in the nonheterocystous nitrogen-fixing cyanobacterium Leptolyngbya boryana.

The filamentous cyanobacterium Leptolyngbya boryana has the ability to fix nitrogen without any heterocysts under microoxic conditions. Previously, we identified the cnfR gene for a master transcriptional activator for nitrogen fixation (nif) genes in a 50-kb gene cluster containing nif and nif-related genes in L. boryana. We showed that CnfR activates the transcription of nif genes in response to low oxygen conditions, which allows the oxygen-vulnerable enzyme nitrogenase to function. However, the regulatory mechanism that underlies regulation by CnfR remains unknown. In this study, we identified a conserved cis-acting element that is recognized by CnfR. We established a reporter system in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 using luciferase genes (luxAB). Reporter analysis was performed with a series of truncated and modified upstream regulatory regions of nifB and nifP. The cis-element can be divided into nine motifs I-IX, and it is located 76 bp upstream of the transcriptional start sites of nifB and nifP. Six motifs of them are essential for transcriptional activation by CnfR. This cis-acting element is conserved in the upstream regions of nif genes in all diazotrophic cyanobacteria, including Anabaena and Cyanothece, thereby suggesting that the transcriptional regulation by CnfR is widespread in nitrogen-fixing cyanobacteria.

Evolutionary Aspects and Regulation of Tetrapyrrole Biosynthesis in Cyanobacteria under Aerobic and Anaerobic Environments.

Chlorophyll a (Chl) is a light-absorbing tetrapyrrole pigment that is essential for photosynthesis. The molecule is produced from glutamate via a complex biosynthetic pathway comprised of at least 15 enzymatic steps. The first half of the Chl pathway is shared with heme biosynthesis, and the latter half, called the Mg-branch, is specific to Mg-containing Chl a. Bilin pigments, such as phycocyanobilin, are additionally produced from heme, so these light-harvesting pigments also share many common biosynthetic steps with Chl biosynthesis. Some of these common steps in the biosynthetic pathways of heme, Chl and bilins require molecular oxygen for catalysis, such as oxygen-dependent coproporphyrinogen III oxidase. Cyanobacteria thrive in diverse environments in terms of oxygen levels. To cope with Chl deficiency caused by low-oxygen conditions, cyanobacteria have developed elaborate mechanisms to maintain Chl production, even under microoxic environments. The use of enzymes specialized for low-oxygen conditions, such as oxygen-independent coproporphyrinogen III oxidase, constitutes part of a mechanism adapted to low-oxygen conditions. Another mechanism adaptive to hypoxic conditions is mediated by the transcriptional regulator ChlR that senses low oxygen and subsequently activates the transcription of genes encoding enzymes that work under low-oxygen tension. In diazotrophic cyanobacteria, this multilayered regulation also contributes in Chl biosynthesis by supporting energy production for nitrogen fixation that also requires low-oxygen conditions. We will also discuss the evolutionary implications of cyanobacterial tetrapyrrole biosynthesis and regulation, because low oxygen-type enzymes also appear to be evolutionarily older than oxygen-dependent enzymes.

Loss of cytochrome cM stimulates cyanobacterial heterotrophic growth in the dark.

Although cyanobacteria are photoautotrophs, they have the capability for heterotrophic metabolism that enables them to survive in their natural habitat. However, cyanobacterial species that grow heterotrophically in the dark are rare. It remains largely unknown how cyanobacteria regulate heterotrophic activity. The cyanobacterium Leptolyngbya boryana grows heterotrophically with glucose in the dark. A dark-adapted variant dg5 isolated from the wild type (WT) exhibits enhanced heterotrophic growth in the dark. We sequenced the genomes of dg5 and the WT to identify the mutation(s) of dg5. The WT genome consists of a circular chromosome (6,176,364 bp), a circular plasmid pLBA (77,793 bp) and two linear plasmids pLBX (504,942 bp) and pLBY (44,369 bp). Genome comparison revealed three mutation sites. Phenotype analysis of mutants isolated from the WT by introducing these mutations individually revealed that the relevant mutation is a single adenine insertion causing a frameshift of cytM encoding Cyt c(M). The respiratory oxygen consumption of the cytM-lacking mutant grown in the dark was significantly higher than that of the WT. We isolated a cytM-lacking mutant, ΔcytM, from another cyanobacterium Synechocystis sp. PCC 6803, and ΔcytM grew in the dark with a doubling time of 33 h in contrast to no growth of the WT. The respiratory oxygen consumption of ΔcytM grown in the dark was about 2-fold higher than that of the WT. These results suggest a suppressive role(s) for Cyt cM in regulation of heterotrophic activity.

Transcriptional regulators ChlR and CnfR are essential for diazotrophic growth in nonheterocystous cyanobacteria.

Leptolyngbya boryana (Plectonema boryanum) is a diazotrophic cyanobacterium lacking heterocysts. How nitrogen fixation is regulated in filamentous nonheterocystous cyanobacteria remains unclear. Here we describe a large 50-kb nitrogen fixation (nif) gene cluster in L. boryana containing 50 genes. This gene cluster contains 14 nif genes (nifBSUHDKVZT and nifPENXW), two genes encoding transcriptional regulators showing high similarity to ChlR (chlorophyll regulator) and PatB, three genes encoding ferredoxin, three genes encoding cytochrome oxidase subunits, and 28 genes encoding nif-related proteins and proteins with putative or unknown functions. Eleven mutants lacking one gene or a subset of genes were isolated. Five of them did not grow under diazotrophic conditions, including two mutants lacking the transcriptional regulators. Although the chlR homolog-lacking mutant showed a normal level of nitrogenase activity, various intermediates of chlorophyll biosynthesis were accumulated under micro-oxic conditions. The phenotype suggested that ChlR activates the expression of the genes responsible for anaerobic chlorophyll biosynthesis to support energy supply for nitrogen fixation. In another mutant lacking the patB homolog, no transcripts of any nif genes were detected under nitrogen fixation conditions, which was consistent with no activity. Constitutive expression of patB in a shuttle vector resulted in low but significant nitrogenase activity even under nitrate-replete conditions, suggesting that the PatB homolog is the master regulator of nitrogen fixation. We propose to rename the patB homolog as cnfR, after cyanobacterial nitrogen fixation regulator.

A nitrate-inducible GARP family gene encodes an auto-repressible transcriptional repressor in rice.

Nitrogen is the most important macronutrient in plants and its supply induces responses in gene expression, metabolism and developmental processes. However, the molecular mechanisms underlying the nitrogen responses remain poorly understood. Here we show that the supply of nitrate but not ammonium immediately induces the expression of a transcriptional repressor gene in rice, designated NIGT1 (Nitrate-Inducible, GARP-type Transcriptional Repressor 1). The results of DNA-binding site selection experiments and electrophoretic mobility shift assays indicated that NIGT1 binds to DNA containing either of two consensus sequences, GAATC or GAATATTC. In transient reporter assays, NIGT1 was found to repress transcription from the promoters containing the identified NIGT1-binding sequences in vivo. Furthermore, NIGT1 repressed the activity of its own promoter, suggesting an autorepression mechanism. Consistently, nitrate-induced NIGT1 expression was found to be down-regulated after a transient peak during nitrate treatment, and the nitrate-induced expression of NIGT1 decreased in transgenic rice plants in which this gene was constitutively overexpressed. Furthermore, the chlorophyll content that could be a marker of nitrogen utilization was found to be decreased in NIGT1 overexpressors of rice grown with nitrate medium but not with ammonium medium. Thus, we propose NIGT1 as a nitrate-inducible and autorepressible transcriptional repressor that may play a role in the nitrogen response in rice. Taken together with the fact that the NIGT1-binding sites are conserved in promoter sequences of Arabidopsis NIGT1 homologs, our findings imply the presence of a time-dependent complex system for nitrate-responsive transcriptional regulation that is conserved in both monocots and dicots.

Distinct roles of nitrate and nitrite in regulation of expression of the nitrate transport genes in the moss Physcomitrella patens.

Five NRT2 genes and three Nar2 genes, encoding putative high-affinity nitrate transporters, and the respective cDNAs were identified and characterized in Physcomitrella patens. The deduced moss NRT2 and NAR2 proteins were more similar to the corresponding proteins of higher plants than to those of the green alga Chlamydomonas reinhardtii. Expression of all the genes was inhibited by ammonium added to the medium. The regulation by ammonium was abolished by an inhibitor of glutamine synthetase, but the effect of this inhibitor was counteracted by an inhibitor of glutamate synthase. Negative correlation was observed between the glutamine content of protonemata and the transcript levels of PpNRT2 and PpNar2. These results indicated that glutamine is the signal for repression of the genes. All the genes except PpNRT2;5 showed transient expression stimulated by nitrate but not by nitrite, peaking at 2-4 h after the medium was deprived of ammonium. When the glutamine synthetase inhibitor was used to inhibit assimilation of the ammonium generated intracellularly from nitrate or nitrite, the second phase of activation of genes became manifest at approximately 8 h after the medium was deprived of ammonium. Surprisingly, both nitrate and nitrite stimulated gene expression at this stage. PpNRT2;5 was distinct from the other genes in that its expression is sharply induced by nitrite, is strictly dependent on nitrite or nitrate, and is much less susceptible to the feedback regulation, retaining a constant level in nitrate-containing medium. These results indicated that P. patens has multiple mechanisms for sensing nitrate and nitrite.