Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification

Abstract

The structural gene, nirS, for the respiratory nitrite reductase (cytochrome cd1) from Pseudomonas stutzeri was identified by (i) sequencing of the N-terminus of the purified protein and partial sequencing of the cloned gene, (ii) immunoscreening of clones from a lambda gt11 expression library, (iii) mapping of the transposon Tn5 insertion site in the nirS mutant strain MK202, and (iv) complementation of strain MK202 with a plasmid carrying the insert from an immunopositive lambda clone. A mutation causing overproduction of cytochrome c552 mapped on the same 8.6 kb EcoRI fragment within 1.7 kb of the mutation affecting nirS. Two mutations affecting nirD, which cause the synthesis of an inactive cytochrome cd1 lacking heme d1, mapped 1.1 kb apart within a 10.5 kb EcoRI fragment contiguous with the fragment carrying nirS. Nir- mutants of another type that had low level synthesis of cytochrome cd1, had Tn5 insertions within an 11 kb EcoRI fragment unlinked to the nirS+ and nirD+ fragments. Cosmid mapping provided evidence that nirS and nirD, and the previously identified gene cluster for nitrous oxide respiration are closely linked. The nirS gene and the structural gene for nitrous oxide reductase, nosZ, are transcribed in the same direction and are separated by approximately 14 kb. Several genes for copper processing are located within the intervening region.