Comparison Of Oxygen Sensitivities Of Nitrous Oxide Reductase Of Pseudomonas aeruginosa PAO1 and Pseudomonas stutzeri ZoBell

Abstract

Nitrous oxide, a potent greenhouse gas and a key agent in stratospheric ozone depletion, can be produced as an end or intermediate product of denitrification or as a byproduct of nitrification. Some denitrifiers can reduce exogenous N2O to N2 by using nitrous oxide reductase (N2OR) encoded by nosZ gene. The activity of N2OR is greatly decreased in the presence of oxygen (O2) gas, although several strains were reported capable of reducing N2O under aerobic conditions. Based on our studies, we hypothesize that O2 is an un-competitive inhibitor for N2OR. To better understand this mechanism, Clark-type microsensors and H-shaped chambers were used to calculate N2O and O2 reduction rates of various bacterial strains. From microsensor studies, we identified Pseudomonas stutzeri strain Zobell and Pseudomonas aeruginosa strain PAO1 having O2 tolerant and O2-sensitive N2OR, respectively. Gene knock out of PAO1 and ZoBell nosZ genes ensured null N2OR activity by microsensor assay. The gene nosZ originated from the same strain or different strain was re-introduced via plasmid to the knockout mutants to clarify the role of NosZ on the N2OR activities. The phenotypes of the wildtype and mutants were analyzed by monitoring N2O and O2 consumption in real time using microsensors. PAO1ΔnosZ_p.Z.nosZ and ZoBellΔnosZ_p.P.nosZ were both unexpectedly found to be N2OR - oxygen sensitive which rejects our hypothesis that the difference in nosZ genes is responsible for the difference in oxygen sensitivities of N2OR. However, nosZ genes were re-introduced into original positions and switched on the genome to further understand ZoBell N2OR’s oxygen tolerance mechanism. PAO1ΔnosZ_g.P.nosZ and ZoBellΔnosZ_g.Z.nosZ showed the N2O and O2 consumption behaviors exactly as the wildtype as expected: i.e., their N2OR were oxygen-sensitive and -tolerant, respectively. nosZ switch on the genome, between PAO1 and ZoBell unexpectedly did not show any N2OR activity based on microsensor studies. This implies the need for further exploration and optimization of genetic manipulation of all nos genes in the cluster to fully understand the oxygen tolerance mechanism of ZoBell.