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Journal of Bacteriology, October 2003, p. 5838-5846, Vol. 185, No. 19
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.19.5838-5846.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Monomeric NarB Is a Dual-Affinity Nitrate Reductase, and Its Activity Is Regulated Differently from That of Nitrate Uptake in the Unicellular Diazotrophic Cyanobacterium Synechococcus sp. Strain RF-1

Tung-Hei Wang,1 Hongyong Fu,2 and Yuh-Jang Shieh1,2*

Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan 114,1 Institute of Botany, Academia Sinica, Taipei, Taiwan 11529, Republic of China2

Received 1 November 2002/ Accepted 17 July 2003

Synechococcus sp. strain RF-1 is a unicellular freshwater cyanobacterium that fixes N2 aerobically and exhibits a circadian rhythm for nitrogenase activity under a light-dark regimen. Synechococcus sp. strain RF-1 also utilizes nitrate, nitrite, or ammonium for growth. Under the diazotrophic growth, the nitrate uptake in Synechococcus sp. strain RF-1 was induced by nitrate or nitrite but repressed by ammonium. In contrast, a prominent nitrate reductase (NR) activity was detected in diazotrophically grown cells using the reduced methyl viologen assay. The NR activity was not inhibited by ammonium and only slightly enhanced by nitrate. The different expression patterns of nitrate uptake and NR in Synechococcus sp. strain RF-1 were reflected in general at the transcript level determined by reverse transcriptase PCR. Under both nitrate-induced and uninduced conditions, the in situ NR activity exhibited similar biphasic kinetics for nitrate. The recombinant NR encoded by the narB gene of Synechococcus sp. strain RF-1, expressed in E. coli, also showed the biphasic kinetics with similar pH and temperature profiles. By in-gel NR activity assay, the recombinant NarB was found to exist as a single form. Both the high- and low-affinity NR activities of the recombinant NarB showed the same thermostability. When modified at the N terminus by a polyhistidine tag, the recombinant NR activity was shifted from biphasic to hyperbolic kinetics and showed only a single Km for nitrate, indicating the functional importance of the NarB N-terminal structure in NR kinetics.

* Corresponding author. Mailing address: Institute of Botany, Academia Sinica, Nankang, Taipei, Taiwan 11529, Republic of China. Phone: 886-2-27899590, ext. 322. Fax: 886-2-27827954. E-mail: yjshieh{at}gate.sinica.edu.tw.

Journal of Bacteriology, October 2003, p. 5838-5846, Vol. 185, No. 19
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.19.5838-5846.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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