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Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

First Advisor

Carl A. Westby


Azospirillum brasilense is a bacterium that exists in a close association with the roots of many grain crops and forage grasses, (26, 64, 66, 83, 95, 97) and is capable of carrying out all the major reactions of the nitrogen cycle except nitrification, (8, 27, 74). These include nitrogen fixation, assimilatory and dissimilatory nitrate reduction, assimilatory and dissimilatory nitrite reduction, ammonia assimilation, and denitrification. For these reasons A. brasilense has gained attention as an agronomically important bacterium as it shows potential as a possible biofertilizer (2, 7, 26, 75, 81, 102). However, relatively little work has been focused on nitrate (N03-) and nitrite (N02-) reduction or ammonia (NH4+) assimilation in this bacterium, (100). The first step in both assimilatory and dissimilatory nitrate reduction is the conversion of N03- to N02-. A. brasilense accomplishes this conversion by a common assimilatory/dissimilatory nitrate reductase or separate assimilatory (ANR) and dissimilatory (DNR) reductases, (8, 83). The N02- produced from this conversion can subsequently proceed through one of two pathways. The assimilatory pathway, which converts NO2- to NH4+, is catalyzed by an assimilatory nitrite reductase (ANIR), (83). The NH4+ formed here is then available for biosynthesis and is taken up through two NH4+ assimilation pathways, the glutamine synthetase/glutamate synthase (GS/GOGAT) dehydrogenase (GDH), route, (97). The route or the glutamate assimilatory NO2- to NH4+ conversion is repressed by NH4+, (79). In the dissimilatory pathway, N03- or N02- is used as a terminal electron acceptor in a dissimilatory pathway at low p02, producing gaseous nitrogenous products, (102). This step is catalyzed by a separate nitrite reductase (DNIR) and an nitrous oxide reductase (N20R). The pathway of inorganic nitrogen utilization by Escherichia coli is well known, (13, 17, 23, 28, 54, 55, 86, 90, 91). Although E. coli is incapable of true denitrification, it does convert NO2- to NH4+ under anaerobic conditions, (18, 53). The first step here, N03- conversion to NO2- , is catalyzed by a single DNR. This reductase consists of alpha, beta and gamma subunits that are coded for by the nar C, H and I genes, respectively, (13, 14, 15, 23, 91) of the nar operon. E. coli then converts NO2- to NH4+ using a single DNIR, (18, 19, 76, 92). The NH4+ formed here is then available for biosynthesis. This DNIR activity is unaffected by NH4+, (92). The research described in this thesis would not have been possible without the work previously conducted in this laboratory. This research began in 1987 when B. Buttaro produced a gene library for A. brasilense and screened for the nitrate reductase (NR) gene, (11). The library consisted of 25 kb fragments of A. brasilense genomic DNA expressed in E. coli NR- mutants RK5267 and RK529S. These experiments led to the isolation of BW1 and BW2. Secondly, in 1988, studies examining N03- , N02- , and purine nitrogen assimilation were conducted by K. Y. Choi, (16). Here, various A. brasilense mutants defective in N03- and N02- assimilation were isolated and characterized, and revertants were obtained and studied. In addition, N03- and N02- assimilation studies were also conducted in BW1 and BW2. Finally, in 1989, a number of A. brasilense mutants lacking N03- and N02- assimilatory or dissimilatory genes were obtained using chemical and transposon mutagenesis techniques by T. Gors, (37). In addition, these mutants were phenotypically characterized by aerobic and anaerobic growth and N02- utilization and excretion studies. Also, NR complementation studies were conducted in which Sp79TC1 asnd [sic] Sp79TC2 were obtained. Here we describe work on nitrate and nitrite reductase and the enzymes of ammonia assimilation in A. brasilense Sp7. The activities of these enzymes were studied in recombinant E. coli strains, A. brasilense Sp7 mutant, revertant, transconjugant strains, and control strains in order to understand how genes controlling the expression of these enzymes are regulated. Southern blotting techniques were implemented to further analyze the genetic material of the E. coli recombinants and the A. brasilense transconjugants.

Library of Congress Subject Headings

Escherichia coli
Denitrifying bacteria



Number of Pages



South Dakota State University