Characterization of the norB gene, encoding nitric oxide reductase, in the nondenitrifying cyanobacterium Synechocystis sp. strain PCC6803

Abstract

A norB gene encoding a putative nitric oxide reductase is present in the genome of the nondenitrifying cyanobacterium Synechocystis sp. strain PCC6803. The gene product belongs to the quinol-oxidizing single-subunit class of nitric oxide reductases, discovered recently in the denitrifier Ralstonia eutropha. Heterologous complementation of a nitric oxide reductase-negative mutant of R. eutropha with norB from Synechocystis restored nitric oxide reductase activity. With reduced menadione as the electron donor, an enzymatic activity of 101 nmol of NO per min per mg of protein was obtained with membrane fractions of Synechocystis wild-type cells. Virtually no nitric oxide reductase activity was present in a norB-negative mutant of Synechocystis. Growing cells of this mutant are more sensitive toward NO than wild-type cells, indicating that the presence of a nitric oxide reductase is beneficial for Synechocystis when the cells are exposed to NO. Transcriptional fusions with the chloramphenicol acetyltransferase reporter gene were constructed to monitor norB expression in Synechocystis. Transcription of norB was not enhanced by the addition of the NO-generating agent sodium nitroprusside.

FIG. 1.

Synechocystis norB gene region. A physical map and relevant restriction sites are shown. DNA fragments of subclones are indicated by black bars. The orientation of a kanamycin resistance cartridge (Km) is indicated by arrows. The norAB promoter of R. eutropha is depicted by a white box with an arrow.

FIG. 2.

Nitrous oxide production by R. eutropha strains. Symbols: ◊, wild-type H16; ▴, nitric oxide reductase-negative mutant HF420; •, HF420 complemented with the norB gene region from Synechocystis(pGE409); ○, HF420 complemented with Synechocystis norB under the control of the norAB promoter of R. eutropha(pGE453).

FIG. 3.

cat expression conferred by the intergenic region between dnr and norB of Synechocystis. Media were supplemented with either 18 mM nitrate or 5 mM ammonia. When the cells reached an optical density at 750 nm of 1.0, 5 mM SNP was added. Activities were measured after an additional 4 h of growth. Error bars represent standard errors from three replicates. Symbols: black bars, pSB2A; gray bars, dnr"-cat; white bars, norB"-cat.

FIG. 4.

Effect of NO on growing cells of Synechocystis. (A) Paper disks soaked with water (1) or 5 μl (2) or 15 μl (3) of a 0.5 mM SNP solution were placed on agar plates spread with a layer of Synechocystis cells. Left plate, mutant M321; right plate, wild type. Inhibition zones occurred after incubation for 36 h. (B) Suspensions of Synechocystis cells in BG-11 medium containing 0.6% agar were grown in glass tubes sealed with a rubber septum. WT, wild type; M320 and M321, norB-negative mutant strains. NO gas (10 mM) was injected before incubation (+NO).