The nos genes of Pseudomonas
stutzeri are required for the anaerobic respiration of nitrous
oxide,
which is part of the overall denitrification process. A nos-coding
region of ca. 8 kilobases was cloned by plasmid integration and
excision.
It comprised nosZ, the structural gene for the
copper-containing
enzyme nitrous oxide reductase, genes for copper chromophore
biosynthesis,
and a supposed regulatory region. The location of the nosZ gene
and its transcriptional direction were identified by using a series of
constructs to transform Escherichia coli and express nitrous
oxide
reductase in the heterologous background. Plasmid pAV5021 led to a
nearly
12-fold overexpression of the NosZ protein compared with that in the P.
stutzeri wild type. The complete sequence of the nosZ gene,
comprising 1,914 nucleotides, together with 282 nucleotides of
5'-flanking
sequences and 238 nucleotides of 3'-flanking sequences was determined.
An open reading frame coded for a protein of 638 residues (Mr,
70,822) including a presumed signal sequence of 35 residues for protein
export. The presequence is in conformity with the periplasmic location
of the enzyme. Another open reading frame of 2,097 nucleotides, in the
opposite transcriptional direction to that of nosZ, was
excluded
by several criteria from representing the coding region for nitrous
oxide
reductase. Codon usage for nosZ of P. stutzeri showed a
high
G + C content in the degenerate codon position (83.9% versus an average
of 60.2%) and relaxed codon usage for the Glu codon, characteristic
features
of Pseudomonas genes from other species. E. coli
nitrous
oxide reductase was purified to homogeneity. It had the Mr
of the P. stutzeri enzyme but lacked the copper chromophore.