Cloning of a third nitrate reductase gene from the cyanobacterium Anacystis nidulans R2 using a shuttle cosmid library

Kuhlemeier, C.J.; Teeuwsen, Vera; Janssen, Marlieke; Arkel, G.A. van

doi:10.1016/0378-1119(84)90200-2

Cited by 33 publications

(24 citation statements)

References 15 publications

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“…In accordance with the view that the chloroplast originated from a prokaryotic endosymbiont similar to cyanobacteria (10,45), the cyanobacterial and plant NiRs are both ferredoxin-dependent enzymes and are homologous to each other (27,42). By contrast, the ferredoxin-dependent NR of cyanobacteria (3,22) is distinct from the pyridine nucleotide-dependent NRs of plants and eukaryotic algae (7).…”

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confidence: 87%

“…strain PCC7942, the gene encoding NiR (nirA) (27,42) is cotranscribed with the genes encoding the nitrate transporter (nrtABCD) (33)(34)(35) and NR (narB) (3,22) as an nirA-nrtABCD-narB operon (42). Transcription from the operon is activated upon removal of ammonium from the medium or upon inhibition of ammonium fixation, showing no requirements for the presence of nitrate (42).…”

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A novel nitrite reductase gene from the cyanobacterium Plectonema boryanum

et al. 1995

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The gene (nirA) for nitrite reductase was cloned from the nonheterocystous, filamentous cyanobacterium Plectonema boryanum. The predicted protein consists of 654 amino acids and has a calculated molecular weight of 72,135. The deduced amino acid sequence from positions 1 to 511 is strongly similar to the entire sequence of the ferredoxin-dependent nitrite reductases from other phototrophs, while the remainder of the protein is unique to the Plectonema nitrite reductase. The C-terminal portion of the protein (amino acids 584 to 654) is 30 to 35% identical to [2Fe-2S] ferredoxins from higher plants and cyanobacteria, with all of the four Cys residues involved in binding of the [2Fe-2S] cluster in the ferredoxins being conserved. Immunoblotting analysis of the extracts of P. boryanum cells showed that the NirA polypeptide has an apparent molecular mass of 75 kDa. An insertional mutant of nirA lacked the 75-kDa polypeptide, had no nitrite reductase activity, and failed to grow on nitrate and nitrite, indicating that the novel nirA is the sole nitrite reductase gene in P. boryanum and that the NirA polypeptide with the ferredoxin-like domain is the apoprotein of the functional nitrite reductase. As in Synechococcus sp. strain PCC7942, nirA is the first gene of a large transcription unit (>7 kb in size) and is repressed by ammonium and derepressed simply by deprivation of ammonium from the medium. The development of nitrite reductase activity was, however, found to require the presence of nitrate in the medium.Nitrate is the major source of nitrogen for photosynthetic organisms such as plants, algae, and cyanobacteria (15, 16). It is transported into the cells by an active transport system and reduced to nitrite by nitrate reductase (NR). Nitrite is further reduced to ammonium by nitrite reductase (NiR), and the resulting ammonium is fixed as the amide group of Gln by glutamine synthetase. In plant cells, NR and NiR are located in the cytoplasm and the chloroplast, respectively (7), while in cyanobacteria, NR and NiR are located in the same compartment, the cytoplasm of the prokaryotic cell (15). In accordance with the view that the chloroplast originated from a prokaryotic endosymbiont similar to cyanobacteria (10, 45), the cyanobacterial and plant NiRs are both ferredoxin-dependent enzymes and are homologous to each other (27,42). By contrast, the ferredoxin-dependent NR of cyanobacteria (3, 22) is distinct from the pyridine nucleotide-dependent NRs of plants and eukaryotic algae (7).In the unicellular non-nitrogen-fixing cyanobacterium Synechococcus sp. strain PCC7942, the gene encoding NiR (nirA) (27, 42) is cotranscribed with the genes encoding the nitrate transporter (nrtABCD) (33-35) and NR (narB) (3, 22) as an nirA-nrtABCD-narB operon (42). Transcription from the operon is activated upon removal of ammonium from the medium or upon inhibition of ammonium fixation, showing no requirements for the presence of nitrate (42). In filamentous nitrogen-fixing cyanobacteria such as Nostoc sp. strain PCC6719, Anabaena sp...

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A novel nitrite reductase gene from the cyanobacterium Plectonema boryanum

et al. 1995

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“…PCC7942, narB (Kuhlemeier et al, 1984;Andriesse et al, 1990; X74597 in the GenBank/ EMBL/DDBJ data bases) is supposed to be the structural gene of NR since (a) it is required for expression of NR activity and assimilation of nitrate and (b) it encodes a polypeptide homologous to assimilatory NR of Klebsiella pneumoniae (Lin et al, 1993) and periplasmic NR of Alcaligenes eutrophus (Siddiqui et al, 1993). Mutant strains of narB have no appreciable activity of NR, suggesting that the cyanobacterium has only one NR activity (Kuhlemeier et al, 1984). To examine whether this is true also in the C0,-limited cells, we constructed a targeted mutant of narB (AnarB::km') and measured the NR activity of the mutant cells grown under the C0,-limited conditions.…”

Section: Effects Of Co Limitation On Growth Nitrate Uptake and Nitmentioning

confidence: 99%

Regulation of Nitrite Reductase Activity under CO2 Limitation in the Cyanobacterium Synechococcus sp. PCC7942

Suzuki¹,

Sugiyama²,

Omata³

1995

Plant Physiol.

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During photoautotrophic growth under C0,-limited conditions, cells of Synechococcus sp. PCC7942 excreted into the medium about 30% of the nitrite produced by reduction of nitrate. No nitrite was excreted under C0,-sufficient conditions. After transfer of high-C0,-grown cells to C0,-limited conditions, nitrite reductase activity started to decline within 0.5 h and decreased to 50% of the initial leve1 in 3 h, whereas nitrate reductase activity was virtually unchanged. Nitrite started to accumulate i n the medium about 3 h after the transfer of the cells to COJimited conditions and reached a concentration of >0.4 mM at 17 h. These findings suggested that the nitrite excretion was due to an imbalance of the activities of nitrite reductase and nitrate reductase. Since ammonium, the product of nitrite reduction, was not detected in the medium, it was concluded that the step of nitrite reduction limits the rate of nitrate assimilation under C0,-limited conditions. The extent of decrease i n nitrite reductase activity under C0,-limited conditions was much larger than that caused by rifampicin (an inhibitor of RNA synthesis) treatment under high-CO, conditions. Addition of CO, , i n the form of sodium bicarbonate, to the C0,-limited culture increased the nitrite reductase activity, but rifampicin inhibited this increase. These findings suggested the presence of a mechanism that irreversibly inactivates nitrite reductase under C0,-limited conditions.

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“…Among cyanobacteria, the NR-related genes are best characterized in Synechococcus elongatus strain PCC7942, where three genetic loci, narA, narB, and narC, have been identified and characterized as essential for the nitrate reduction (17,18). The narB locus has been identified as the NR structural gene (narB) (4), which is clustered with the NiR gene (nirA) (23,40) and the nitrate and nitrite transporter genes (nrtABCD) (30)(31)(32) to form the nirA-nrtABCD-narB operon (40).…”

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A Novel Gene ( narM ) Required for Expression of Nitrate Reductase Activity in the Cyanobacterium Synechococcus elongatus Strain PCC7942

Maeda

Omata

2004

J Bacteriol

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A new class of mutants deficient in nitrate assimilation was obtained from the cyanobacterium Synechococcus elongatus strain PCC7942 by means of random insertional mutagenesis. A 0.5-kb genomic region had been replaced by a kanamycin resistance gene cassette in the mutant, resulting in inactivation of two genes, one of which was homologous to the recently characterized cnaT gene of Anabaena sp. strain PCC7120 (J. E. Frías, A. Herrero, and E. Flores, J. Bacteriol. 185:5037-5044, 2003). While insertional mutation of the cnaT homolog did not affect expression of the nitrate assimilation operon or the activity of the nitrate assimilation enzymes in S. elongatus, inactivation of the other gene, designated narM, resulted in specific loss of the cellular nitrate reductase activity. The deduced NarM protein is a hydrophilic protein consisting of 161 amino acids. narM was expressed constitutively at a low level. The narM gene has its homolog only in the cyanobacterial strains that are capable of nitrate assimilation. In most of the cyanobacterial strains, narM is located downstream of narB, the structural gene of the cyanobacterial nitrate reductase, suggesting the functional link between the two genes. NarM is clearly not the structural component of the cyanobacterial nitrate reductase. The narM insertional mutant normally expressed narB, indicating that narM is not the transcriptional regulator of the structural gene of nitrate reductase. These results suggested that narM is required for either synthesis of the prosthetic group of nitrate reductase or assembly of the prosthetic groups to the NarB polypeptide to form functional nitrate reductase in cyanobacteria.

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Cloning of a third nitrate reductase gene from the cyanobacterium Anacystis nidulans R2 using a shuttle cosmid library

Cited by 33 publications

References 15 publications

A novel nitrite reductase gene from the cyanobacterium Plectonema boryanum

A novel nitrite reductase gene from the cyanobacterium Plectonema boryanum

Regulation of Nitrite Reductase Activity under CO2 Limitation in the Cyanobacterium Synechococcus sp. PCC7942

A Novel Gene ( narM ) Required for Expression of Nitrate Reductase Activity in the Cyanobacterium Synechococcus elongatus Strain PCC7942

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