The structural gene,
nirS,
for the respiratory nitrite reductase (cytochrome cd1) from Pseudomonasstutzeri
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.