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A cyanobacterial narB gene encodes a ferredoxin-dependent nitrate reductase
L M Rubio1, A Herrero, E Flores
1Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Spain.
Plant Molecular Biology
|February 1, 1996
Summary
The cyanobacterial narB gene was successfully expressed in E. coli, producing active nitrate reductase. This confirms narB as the structural gene for nitrate reductase in cyanobacteria.
Area of Science:
- Molecular Biology
- Biochemistry
- Cyanobacterial Research
Background:
- Nitrate reductase is crucial for nitrogen assimilation in cyanobacteria.
- The specific gene responsible for nitrate reductase activity in Synechococcus sp. PCC 7942 (narB) was previously uncharacterized at the functional level.
Purpose of the Study:
- To functionally characterize the narB gene from Synechococcus sp. PCC 7942.
- To determine if narB encodes the nitrate reductase enzyme in cyanobacteria.
Main Methods:
- Cloning the narB gene into an Escherichia coli expression vector (pTrc99A) under LacI-regulated Ptrc promoter control.
- Inducing gene expression with isopropyl-beta-D-thiogalactoside.
- Assessing nitrate reductase activity in E. coli extracts using reduced methyl viologen or reduced ferredoxin as electron donors.
Main Results:
- Expression of the narB gene in E. coli resulted in a 76 kDa polypeptide.
- The E. coli extracts exhibited nitrate reductase activity identical to that of Synechococcus nitrate reductase.
- The enzyme activity was dependent on electron donors like reduced methyl viologen or reduced ferredoxin.
Conclusions:
- The narB gene from Synechococcus sp. PCC 7942 is a structural gene encoding nitrate reductase.
- Functional expression of cyanobacterial nitrate reductase is achievable in E. coli.