Transposon (Tn5)
mutagenesis
of Pseudomonas perfectomarina with the plasmid pSUP2021
[(pBR325-Mob(RP4))::Tn5]
and the chromosomally integrated RP4 plasmid in Escherichia coli
as the donor, produced three distinct groups of mutants that were
defective
in nitrous oxide respiration. One group of mutants lacked the
structural
protein of N2O reductase, the second synthesized
a
copper-free apoprotein; and a third group expressed a low level of
intact
enzyme. The mutants provided evidence for N2O
being
the immediate precursor of dinitrogen in denitrification and documented
the essentiality of the copper enzyme. Synthesis of N2O
reductase depended strongly on the growth conditions, with N2O-grown
cells expressing the lowest level of enzyme. Regulatory responses of
mutants
elicited by nitrate or oxygen were unaltered when compared with
wild-type
behavior.