A new type of glutamine synthetase in cyanobacteria: the protein encoded by the glnN gene supports nitrogen assimilation in Synechocystis sp. strain PCC 6803

Abstract

A new glutamine synthetase gene, gInN, which encodes a polypeptide of 724 amino acid residues (Mf, 79,416), has been identified in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803; this is the second gene that encodes a glutamine synthetase (GS) Ammonium assimilation takes place in cyanobacteria mainly by the sequential action of glutamine synthetase (GS) and glutamate synthase (25). GS in cyanobacteria is similar to the classical prokaryotic GS type I (GSI) that has been widely studied in enterobacteria and whose structure and regulation are well known (22,46). Thus, cyanobacterial GS is composed of 12 identical subunits (Mr, about 50,000) arranged in two superimposed hexagonal rings (29,32). In contrast to the enterobacterial enzyme, cyanobacterial GS is not regulated by adenylylation in response to the nitrogen source (16,26,27). However, in Synechocystis sp. strain PCC 6803, short-term inactivation of GSI promoted by ammonium has been reported. This inactivation seems to involve a phosphorylated compound (26,27). In the N2-fixing, filamentous species Anabaena sp. strain PCC 7120 and in the unicellular species Agmenellum quadruplicatum (Synechococcus sp. strain PCC 7002), control of GS synthesis by the nitrogen source has been shown (31,44,45).The gene that encodes the classical dodecameric GS (glnA) has been cloned from several cyanobacteria, such asAnabaena, Synechocystis, Calothrix, and Agmenellum spp. (13,16,28,45). The amino acid sequences deduced from the Anabaena and Agmenellum glnA genes (44,45) show about 50% identity with the enterobacterial gene, and both are able to complement an Escherichia coli glnA mutant (16,45).The existence of more than one type of GS in prokaryotes seemed to be restricted to members of the family Rhizobiaceae, including the genera Rhizobium, Bradyrhizobium, and Agrobacterium, and to the genus Streptomyces, with two different types: one corresponding to the classical prokaryotic structure, GSI (dodecameric), and the other related to eukaryotic GS (GSII) (octameric) (3,6,10,37). Recently, a third GS homologous to prokaryotic GSI has been reported in Rhizobium spp. (9,10,14,40).Besides that, Bacteroides fragilis and Butyrivibrio fibrisolvens, two members of the family Bacteroidaceae which are obligate anaerobic bacteria that live in mammal intestines, contain a GS that differs markedly from all of the GSs previously described in subunit size (Mr, 75,000), structure (hexameric), and amino acid sequence (19,20,43).It has been recently shown that a ginA mutant of the cyanobacterium Agmenellum quadruplicatum was able to grow in the absence of glutamine, indicating that the ginA gene is nonessential for ammonium assimilation and suggesting that another enzyme is responsible for the glutamine synthesis (45).Here we describe the molecular cloning, sequence, and expression of a novel GS gene (glnN) (36). Alternatively, cultures were bubbled with 1.5% (voIvol) CO2 in air. When ammonium was used as the nitrogen source, nitrate was replaced by 10 mM NH4Cl and the me...

SPR analysis of promoter binding of Synechocystis PCC6803 transcription factors NtcA and CRP suggests cross‐talk and sheds light on regulation by effector molecules

“…To tackle these questions we use here surface plasmon resonance (SPR) to detect in a highly sensitive manner the binding of proteins to sensorchip-bound DNA. We have utilized regulator box-containing promoter fragments from three Synechocystis genes, two of them, glnA and glnN, encoding glutamine synthetases types I and III [20,21], exhibiting a canonical and a non-canonical NtcA-binding site [22], respectively, and the third one, cccS, being a well characterized SYCRP1-regulation target [9,23] (Fig. 1, bottom table).…”

Purification and Characterization of A New Type of Glutamine Synthetase from Cyanobacteria

“…However, these levels were enough to complement the glutamine auxotrophy of the E. coli glnA mutant. In Synechocystis 6803, transcription from the glnN promoter is finely regulated and the basal level of expression is very low [13], (Reyes and Florencio, unpublished results), therefore, it is not surprise that the glnN gene promoter was very inefficient in the E. coli background. To obtain a higher expression of the glnN gene, a 2.85-kb fragment, able to complement the E. coli glnA mutant, was subcloned from pBE2 into plasmid pBluescript I1 SK(+).…”

“…We have previously reported the cloning of the Syizechocystis 6803 glnN gene by complementation of an E. coli glnA mutant strain (ET6017) [13]. The plasmid pBE2, which carries a 4.5-kb insert, contained the glnN gene.…”

“…We have previously shown that in nitrate-grown Synechocystis 6803 cells, the g l d gene product (GSI) is responsible for 97% of the total GS activity, while the glnN product (GSIII) accounts for only 3 % of the activity. In contrast, under nitrogen deprivation, transcription of the glnN gene is strongly induced [13] and the total GS levels increase. However, until this work, no biochemical evidence existed to assure that part of this increase in activity corresponds [20 -231. to the GSIII encoded by the glnN gene.…”

“…Complementation experiments were performed in glucose minimal medium [24] supplemented with 5 mM glutamine and 40 yg/ml ampicillin when required. Synechocystis 6803 glnA mutant (SJCR3) and glnN mutant (SJCR6) strains, were originated by insertion of a neomycine phosphotransferase cassette in the glnA or glnN genes [13]. Both strains were grown photoautotrophically at 30°C on BGI1 medium [25] (18 mM nitrate as nitrogen source).…”