Genetically engineered mutant of the cyanobacterium
            <i>Synechococcus</i>
            PCC 7942 defective in nitrate transport

Omata, Tatsuo; Ohmori, Masayuki; Arai, Nobuyuki; Ogawa, Teruo

doi:10.1073/pnas.86.17.6612

Cited by 101 publications

(69 citation statements)

References 20 publications

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“…This was in good agreement wii h the value of 30 pmol mg-' Chl h-', which was calculated from the previously published data of the short-term experiments performed under high-CO, conditions (figs. 6 and 7 in Omata et al, 1989). The average rate of nitrate uptake by the C0,-limited cells was calculated to be 22 pmol mg-' Chl h-' (1.3 pmol mL-' of nitrate taken up divided by 0.060 mg Chl h-' mL-'), which was about 65% of that calculated for the high-C0,-grown cells.…”

Section: Other Methodsmentioning

confidence: 74%

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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Section: Other Methodsmentioning

confidence: 74%

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

Suzuki¹,

Sugiyama²,

Omata³

1995

Plant Physiol.

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“…4B). nblA (whose product is responsible for phycobilisome degradation) and the nrtABCD operon (which encodes the nitrate/nitrite transporter) are known to be regulated by NtcA (32)(33)(34)(35). On the other hand, the transcripts of other NtcA-regulated genes such as glnA (which encodes glutamine synthetase type I), glnB, amt1 (which encodes the ammonium transporter), and glnN (which encodes glutamine synthetase type III) were less affected by the pamA mutation (Fig.…”

Section: Transcripts Of Nitrogen-related Genes Were Decreased By the mentioning

confidence: 99%

Identification of PamA as a PII-binding Membrane Protein Important in Nitrogen-related and Sugar-catabolic Gene Expression in Synechocystis sp. PCC 6803

Osanai

Sato

Tabata

et al. 2005

Journal of Biological Chemistry

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The PII signaling protein plays a pivotal role in the coordination of carbon and nitrogen metabolism in a wide variety of bacteria, Archaea, and plant chloroplasts. By using a yeast two-hybrid screening system, we identified a transmembrane protein, designated PamA (encoded by sll0985), as a PII-binding protein in Synechocystis sp. PCC 6803. The interaction between PII and PamA was confirmed in vitro, and the interaction was inhibited in the presence of ATP and 2-oxoglutarate, whereas the interaction was not influenced by the phosphorylation status of PII. Northern blot analyses revealed that the transcripts of a set of nitrogen-related genes, including nblA, nrtABCD, and ureG, were decreased in a pamA deletion mutant. The mRNA and protein levels of a group 2 factor SigE were also reduced by the pamA mutation, and transcripts for sugar catabolic genes, such as gap1, zwf, and gnd that are under the control of SigE, were consequently decreased in the pamA mutant. In addition, the pamA mutant was found to be unable to grow in glucose-containing media. These results indicate that PamA has a role in the transcript control of genes for nitrogen and sugar metabolism in Synechocystis sp. PCC 6803.Cyanobacteria are prokaryotes that perform oxygenic photosynthesis similar to that in higher plants and algae. These bacteria coordinately regulate various aspects of cellular metabolism in response to changes in their environment. Carbon and nitrogen are important for cell growth, and the complex metabolism of each of these elements is regulated in synchrony under all conditions. The internal balance of carbon and nitrogen in unicellular cyanobacteria is monitored by the PII sensor protein, which is highly conserved among bacteria, Archaea, and plant chloroplasts (1-3).It has been shown that PII protein regulates nitrogen metabolism in unicellular cyanobacteria (4). As in Escherichia coli and related bacteria, the PII protein of Synechococcus sp. PCC 7942 binds to ATP and 2-OG 2 in a synergistic manner (5). The intracellular 2-OG level is assumed to reflect not only the carbon status but also the nitrogen status in cyanobacteria because of the lack of canonical 2-OG dehydrogenase (6). Therefore, PII is considered to be able to integrate energy, carbon, and nitrogen signals by monitoring ATP and 2-OG levels (4). In addition to ATP and 2-OG binding, Synechococcus and Synechocystis PII proteins are phosphorylated at a serine residue under nitrogen starvation (7,8). This phosphorylation level is also affected by carbon status (9), and recent analyses revealed that PII dephosphorylation specifically responded to intracellular 2-OG concentrations (10).A PII-deficient mutant (MP2) of Synechococcus sp. PCC 7942 does not inhibit the nitrate/nitrite transporter in the presence of ammonium, which is the preferred nitrogen source for unicellular cyanobacteria; however, it inhibits the nitrate/nitrite transporter in the wild-type strain (11). High affinity bicarbonate transporters are constitutively activated regardless of the ambient carb...

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“…The major rate-limiting step of nitrate assimilation in cyanobacteria is nitrate transport into the cell (3,14), which has been shown to be inhibited by ammonium (14,15). Because inhibition of glutamine synthetase by L-methionine-DL-sulfoximine abolishes the negative effect of ammonium on nitrate transport (14), fixation of ammonium to Gln is clearly required for the regulation.…”

mentioning

confidence: 99%

“…strain PCC 7942 transports nitrite as well as nitrate (16,17) and hence is a nitrate/nitrite transporter. The product of the nrtA gene is a 45-kDa cytoplasmic membrane protein (3), which has been shown to be a nitrate/nitrite-binding lipoprotein (18). The deduced NrtB protein has structural similarities to the integral membrane components of the ABC (ATP-binding cassette) transporters, and the deduced NrtC and NrtD proteins have the sequences typical of the ATP-binding components of the ABC transporters (5), showing that the cyanobacterial nitrate/ nitrite transporter belongs to the superfamily of ABC transporters (19) or traffic ATPases (20).…”

mentioning

confidence: 99%

“…In the unicellular non-nitrogen-fixing cyanobacterium Synechococcus sp. strain PCC 7942, the genes encoding the nitrate transport system (nrtA, nrtB, nrtC, and nrtD) (3)(4)(5), NR (narB) (6,7), and NiR (nirA) (8,9) form an nirA-nrtABCD-narB operon (the nirA operon), and the transcription from the operon is inhibited by the addition of ammonium to the cyanobacterial cultures (9). Ammonium inhibits transcription through its fixation into Gln, but Gln is not the direct regulator of transcription (9,10).…”

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confidence: 99%

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Involvement of the C-terminal Domain of an ATP-binding Subunit in the Regulation of the ABC-type Nitrate/Nitrite Transporter of the Cyanobacterium Synechococcus sp. Strain PCC 7942

Kobayashi

Rodríguez

Lara

et al. 1997

Journal of Biological Chemistry

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In Synechococcus sp. strain PCC 7942, an ATP-binding cassette transporter encoded by the genes nrtA, nrtB, nrtC, and nrtD mediates active transport of nitrate and nitrite, which is inhibited by ammonium, a preferred source of nitrogen for the cyanobacterium. One of the ATP-binding subunits of the transporter, NrtC, has a distinct C-terminal domain of 380 amino acid residues. A mutant NC2, constructed by removal of this domain using genetic engineering techniques, assimilated low concentrations of nitrate and nitrite and accumulated nitrate intracellularly, showing that the domain is not essential for the transporter activities. Assimilation of low concentrations of nitrite was only partially inhibited by ammonium in NC2 but was completely inhibited in the wild-type cells. Cells of NC2 and its derivative (nitrate reductase-less strain NC4) carrying the truncated NrtC but not the cells with the wild-type NrtC accumulated nitrate intracellularly in the presence of ammonium in medium. These findings indicated that the C-terminal domain of NrtC is involved in the ammonium-promoted inhibition of the nitrate/nitrite transporter. In the presence of ammonium, NC2 could not assimilate nitrate despite its ability to accumulate nitrate intracellularly, which suggested that reduction of intracellular nitrate by nitrate reductase is also subject to inhibition by ammonium.

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Genetically engineered mutant of the cyanobacterium Synechococcus PCC 7942 defective in nitrate transport

Cited by 101 publications

References 20 publications

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

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

Identification of PamA as a PII-binding Membrane Protein Important in Nitrogen-related and Sugar-catabolic Gene Expression in Synechocystis sp. PCC 6803

Involvement of the C-terminal Domain of an ATP-binding Subunit in the Regulation of the ABC-type Nitrate/Nitrite Transporter of the Cyanobacterium Synechococcus sp. Strain PCC 7942

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