Isolation and Characterization of Nitrate and Nitrite Reduction and Ammonia Assimilation in Azospirillum Brasilense SP7; Mutants and Expression of the Genes in Recombinant Escherichia Coli Strains

Not much is known about nitrate or nitrite assimilation in A.brasilense. Nitrate and nitrite dissimilation (denitrification) is also poorly understood. A thorough understanding of the overall metabolism of nitrate in A. brasilense is needed before the microbe could be properly exploited for agricultural purposes. An understanding of the interrelationship between denitrification and nitrogen fixation in this microbe could play an important role in agriculture, i.e. A. brasilense use as a biofertilizer. The research described herein would not have been possible without the work that was previously done in this laboratory by B. Buttaro starting in 1987 and by K. Y. Choi that was started in 1988. These studies resulted in the isolation and use in this study of recombinant Escherichia coli strains carrying A. brasilense NR and NIR genes (BW-1 and BW-2 and of various A. brasilense mutants defective in nitrate and nitrite assimilation enzymes. Here we describe the isolation of two mutants (NIRrp and a spontaneous partial revertant) of A. brasilense Sp7 generated by the chemical mutagen ethyl methane sulfonate (EMS) and 6 additional mutants deficient in uptake of nitrogen compounds or deficient in glutamate synthase, GOGAT, (glutamine 2-oxoglutarate amidotransferase, EC 1.4.1.13) generated by Tn5 transposition. All mutants were phenotypically characterized by: 1) aerobic and anaerobic growth tests, 2) NO2 excretion/utilization analysis, 3) enzyme assays, 4) resting cell tests, 5) physiological testing and 6) cell morphology descriptions. The overall goal was: 1) to gain a better understanding of how the genes controlling nitrate/nitrite assimilation and dissimilation regulate these processes and 2) to determine if a single set of enzymes was used for both nitrate/nitrite assimilation and dissimilation or if separate enzyme sets were used for each process. Finally, a 27 kb fragment of Sp7 DNA carrying nitrate/nitrite reduction genes was placed in an NR- mutant of A. brasilense to further study the genes controlling Sp7 NR and NIR. It also would determine if the passage of the ANR/ANIR genes through E.coli would destroy the genes subsequent expression in A. brasilense. The results of all the phenotypic characteristic testing lead to the development of proposed genetic models for A. brasilense Sp7, A. brasilense EMS mutants, Sp72 and Sp721 and transconjugants of A. brasilense, Sp79TC1 and Sp79TC2. These models propose regulatory mechanisms of NO3- and NO2- reduction and NH4+ assimilation. The aerobic/anaerobic growth testing of the various mutants indicates a strong possibility that NO3-/NO2- assimilation/dissimilation in A. brasilense exists as two distinct enzyme sets