Cloning and Inactivation of Genes Encoding Ferredoxin- and NADH-Dependent Glutamate Synthases in the CyanobacteriumPlectonema boryanum. Imbalances in Nitrogen and Carbon Assimilations Caused by Deficiency of the Ferredoxin-Dependent Enzyme1

Abstract

Glutamate synthase (GOGAT) is a key enzyme in the assimilation of inorganic nitrogen in photosynthetic organisms. We found that, like higher plants, the facultative heterotrophic cyanobacterium Plectonema boryanum had ferredoxin (Fd)-and NADH-dependent GOGATs. The genes glsF, gltB, and gltD were cloned, and structural analyses and target mutageneses demonstrated that glsF encoded Fd-GOGAT and that gltB and gltD encoded the two subunits of NADH-GOGAT. All three mutants lacking one of the GOGAT genes were able to grow photosynthetically and heterotrophically. However, the Fd-GOGAT mutant exhibited a phenotype of marked nitrogen deficiency when grown under conditions of saturating illumination and CO 2 supply. In these conditions the rate of the ammonia uptake from the culture medium was slower in the Fd-GOGAT mutant than in the wild type or in the NADH-GOGAT mutant, but no significant differences were found in the rate of the CO 2 fixation-dependent O 2 evolution among these strains. Our results suggest that, although both Fd-and NADH-GOGATs were operative in the cells growing in light, the contribution of Fd-GOGAT, which directly utilizes photoreducing power for the catalytic reaction, is essential for balancing photosynthetic nitrogen and carbon assimilation.Inorganic nitrogen in the form of ammonia is assimilated into Gln and Glu through the combined actions of GS and GOGAT in all oxygenic photosynthetic organisms from cyanobacteria to higher plants ( Lea et al., 1990;Flores and Herrero, 1994). GS catalyzes the ATP-dependent amination of Glu to yield Gln. GOGAT catalyzes the reductive transfer of the amide group of Gln to the keto position of 2-oxoglutarate to yield two molecules of Glu. The resulting Gln and Glu serve as nitrogen donors in the biosynthesis of various nitrogen-containing compounds (Lea et al., 1989).The GS/GOGAT pathway ultimately requires ATP and reducing power generated by photosynthesis and catabolism of carbohydrates and utilizes carbon skeletons provided from intermediates of the TCA cycle, together with the downstream metabolism of Gln and Glu. This pathway is thus involved in the integration of carbon and nitrogen assimilations.In higher plants GOGAT occurs as two distinct forms, one that is Fd dependent (EC1.4.7.1) and one that is NADH dependent (EC 1.4.1.14); the two forms differ in their specificity for an electron donor and in their molecular architecture. cDNAs for both types of GOGAT have been cloned and characterized (Sakakibara et al., 1991;Gregerson et al., 1993), and they were found to be homologous to Escherichia coli NADPH-GOGAT (EC 1.4.1.13), which is composed of two different polypeptides, large and small subunits encoded by gltB and gltD, respectively (Oliver et al., 1987). Fd-GOGAT is an iron-sulfur flavoprotein composed of a single polypeptide that has a molecular mass of 160 kD and is similar to the large subunit of the E. coli enzyme (Sakakibara et al., 1991). NADH-GOGAT is also a single polypeptide but with two domains, the N-terminal, 160-kD and the C-terminal, 60-kD...

Nitrogen Control in Cyanobacteria

“…strain PCC 6803 appears to bear similar genes (57). The contribution of ferredoxin-glutamate synthase to the photoassimilation of nitrogen is dominant over that of NADH-glutamate synthase when high levels of carbon are available (91 Carbon skeleton for nitrogen incorporation. The carbon skeleton used for incorporation of ammonium through the glutamine synthetase-glutamate synthase cycle is 2-oxoglutarate, which is provided by NADP ϩ -isocitrate dehydrogenase (86,87).…”

“…The genes encoding a ferredoxin-dependent (glsF) and an NADH-dependent (gltB and gltD) glutamate synthase have been characterized in Plectonema boryanum (91). Synechocystis sp.…”

Molecular insights into a dinoflagellate bloom

“…Carbamoyl-phosphate synthase, argininosuccinate synthase and argininosuccinate lyase are three (out of five) key enzymes in the urea cycle and were also overrepresented in the bloom sample. Overrepresentation of genes in urea cycle along with those of genes involved in other nitrogen metabolisms infer high N compound cycling by blooming dinoflagellates because of high N demands (Coschigano et al, 1998;Okuhara et al, 1999;Maheswari et al, 2010;Allen et al, 2011;Kang et al, 2015). Genes involved in proline (Pro), glutamate (GOGAT) and spermidine (Spd) were also overrepresented (Figures 3 and 4).…”

“…Ammonium transporters, ferredoxindependent glutamate synthase (GOGAT2) and several components of the urea cycle were overrepresented relative to both non-bloom stations, whereas nitrite Molecular insights into a dinoflagellate bloom W Gong et al reductase was overrepresented relative to station 20 (Figure 4). GOGAT2 has a significant role in ammonium uptake and assimilation (Okuhara et al, 1999). Carbamoyl-phosphate synthase, argininosuccinate synthase and argininosuccinate lyase are three (out of five) key enzymes in the urea cycle and were also overrepresented in the bloom sample.…”

The Interaction of Ferredoxin with Ferredoxin-Dependent Enzymes

“…The key role of this enzyme in nitrogen assimilation by oxygenic higher plants has been extensively documented (Suzuki and Knaff, 2005) and so will not be reviewed here, but it is worth mentioning that the enzyme also plays a major role in nitrogen assimilation in cyanobacteria (Okuhara et al, 1999). Glutamate synthases in bacteria most commonly use NADPH as the electron donor and contain two types of subunits, one of which displays significant similarities to the single type of peptide found in Fd-dependent enzymes in terms of prosthetic group content and catalytic mechanism.…”