Transposon (TnS) mutagenesis of Pseudomonas perfectomarina with the plasmid pSUP2021 [(pBR325-Mob(RP4))::TnS] and the chromosomaily 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 N20 reductase, the second synthesized a copper-free apoprotein; and a third group expressed a low level of intact enzyme. The mutants provided evidence for N20 being the immediate precursor
Mutants with defective respiratory nitrite utilization (Nir- phenotype) were obtained by transposon Tn5 insertion into genomic DNA of the ZoBell strain of Pseudomonas stutzeri. Three representative mutants were characterized with respect to their activities of nitrite and nitric oxide reduction, cytochrome cd1 content, and pattern of soluble c-type cytochromes. Mutant strain MK201 overproduced cytochrome c552 about fourfold by comparison with the wild type, but possessed an in vitro functional cytochrome cd1. Mutant strain MK202 lacked cytochrome cd1 and, simultaneously, had low amounts of cytochrome c552 and the split alpha-peak c-type cytochrome. Strain MK203 synthesized nitrite reductase defective in the heme d1 prosthetic group. Irrespective of these biochemically distinct Nir- phenotypes, all mutants preserved the nitric oxide-reducing capability of the wild type. The mutant characteristics demonstrate that cytochrome cd1 is essential for nitrite respiration of P. stutzeri and establish the presence of a nitric oxide-reducing system distinct from cytochrome cd1. They also indicate the functional or regulatory interdependence of c-type cytochromes.
The novel multicopper enzyme nitrous oxide reductase from Pseudomonas perfectomarina was purified to homogeneity to study its properties and distribution in various pseudomonads and other selected denitrifying genera by immunochemical techniques. Quantitation of immunochemical crossreactivity by micro-complement fixation within the denitrifying pseudomonads of Palleroni's ribosomal ribonucleic acid group I corresponded to the taxonomic positions established by nucleic acid hybridization. The assignment of P. perfectomarina to the stutzerigroup (as strain ZoBell) was consolidated by immunochemical crossreactivity based on nitrous oxide reductase. Crossreactivity of nitrite reductase (cytochrome cd1) with a respective P. perfectomarina rabbit antiserum was limited to strain DSM 50227 of P. stutzeri; although it could not contribute information towards broader relationships within rRNA group I, it lent further prove to the unity of these two species.
We propose the transfer of Pseudomonas perfectomarina to the species Pseudomonas stutzeri as strain ZoBell.The proposal is based on 60% deoxyribonucleic acid (DNA)-DNA homology with P . stutzeri ATCC 1758ST, a similar guanine-plus-cytosine content, and the conformity of more than 100 nutritional and general characteristics.
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