Isolation and analysis of mutants of Pseudomonas aeruginosa unable to assimilate nitrate

Abstract

Pseudomonas aeruginosa can reduce nitrate to nitrite and eventually to nitrogen gas by the denitrification pathway, thereby providing the organism with a mode of respiration and ATP generation in the absence of oxygen. P. aeruginosa can also reduce nitrate to nitrite through an assimilatory pathway that provides the cell with reduced nitrogen for biosyntheses. In order to establish whether this organism synthesizes a single nitrate reductase protein that functions in both pathways, or produces one for each pathway, we isolated mutants blocked in the assimilation of nitrate. These mutants are unaffected in the reduction of nitrate be the denitrification pathway, although they produce low or undetectable levels of assimilatory nitrate reductase. On the basis of transductional analysis, the mutations were found to be distributed among four genes designated nasA, nasB, nasC, and nasD. Shifting a nasA mutant from anaerobic to aerobic growth eliminated the culture's ability to reduce nitrate, i.e. the anaerobic nitrate reductase cannot function in the presence of oxygen. Thus P. aeruginosa can synthesize two distinct proteins which reduce nitrate to nitrite: an assimilatory nitrate reductase and a dissimilatory nitrate reductase. If conditions of growth are fully aerobic, the latter is not synthesized and does not function. The former, synthesized under the control of at least four genes, is repressed by readily available nitrogen sources.