ISOLATION, SEQUENCING AND MUTATIONAL ANALYSIS OF A GENE-CLUSTER INVOLVED IN NITRITE REDUCTION IN PARACOCCUS-DENITRIFICANS

By using the gene encoding the C-terminal part of the cd(1)-type nitrite reductase of Pseudomonas stutzeri JM300 as a heterologous probe, the corresponding gene from Paracoccus denitrificans was isolated. This gene, nirS, codes for a mature protein of 63144 Da having high homology with cd(1-)type nitrite reductases from other bacteria. Directly downstream from nirS, three other nir genes were found in the order nirECF. The organization of the nir gene cluster in Pa. denitrificans is different from the organization of nir clusters in some Pseudomonads. nirE has high homology with a S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase (uro(')gen III methylase). This methylase is most likely involved in the heme d(1) biosynthesis in Pa. denitrificans. The third gene, nirC, codes for a small cytochrome c of 9.3 kDa having high homology with cytochrome c(55X) of Ps. stutzeri ZoBell. The 4th gene, nirF, has no homology with other genes in the sequence databases and has no relevant motifs. Inactivation of either of these 4 genes resulted in the loss of nitrite and nitric oxide reductase activities but not of nitrous oxide reductase activity. nirS mutants lack the cd(1)-type nitrite reductase while nirE, nirC and nirF mutants produce a small amount of cd(1)-type nitrite reductase, inactive due to the absence of heme d(1). Upstream from the nirS gene the start of a gene was identified which has limited homology with nosR, a putative regulatory gene involved in nitrous oxide reduction. A potential FNR box was identified between this gene and nirS.