UBC Theses and Dissertations
Ammonium and methylammonium uptake by the nitrogen-fixing bacterium Azotobacter vinelandii Moore, Richard Atwood
Azotobacter vinelandii, grown with ammonium as a nitrogen source, was shown to possess an active transport system which could concentrate ammonium 44 to 58 fold. Ammonium uptake was inhibited by the glutamate analog methionine sulfone. The properties of the ammonium uptake system (transport and metabolism) were investigated using the ammonium analog methylammonium. The uptake of methylammonium was inhibited by arsenate indicating that phosphate bond energy was required. Methylammonium uptake was also inhibited by the electron transport inhibitor, cyanide, and the uncoupler, carbonyl cyanide- m-chlorophenyl hydrazone. However, it was shown that these agents served to deplete ATP pools in A. vinelandii. Uptake of methylammonium was sensitive to a Tris-Mg⁺⁺ shock treatment suggesting the possible involvement of a periplasmic binding protein, however, methylammonium-binding activity was not found in periplasmic extracts. A. vinelandii was shown to exhibit a positive chemotactic response toward ammonium as well as acetate, glucose and sucrose. Comparison of outer membrane proteins from nitrogen-fixing cells and ammonium-grown cells revealed the production of a 44,000 dalton protein in membranes from nitrogen-fixing cells. Inner membranes from nitrogen-fixing cells contained a 41,000 dalton protein which was present in low amounts in the membranes of ammonium-grown cells. It was shown that the outer membranes of ammonium-grown cells contained a major protein which was "heat modifiable" in that its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined by the temperature of solubilization prior to electrophoresis. Methylammonium was shown to be metabolized to N-methylglutamine. Strain JK301, an L-methionine-D,L-sulfoximine-resistant mutant of A. vinelandii, was unable to catalyse N-methylglutamine synthesis in vivo or in cell-free extracts and lacked detectable methylammonium uptake activity. Glutamine synthetase in cell-free extracts of JK301 had a Km for glutamate approximately three-fold higher and a Vmax approximately fourfold lower than enzyme from the wild type strain. It was concluded that methylammonium uptake reflects, in part, metabolism to N-methylglutamine by glutamine synthetase.
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