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UBC Theses and Dissertations

Regulation of sucrose metabolism and constitutive nitrogen fixation in Azotobacter vinelandii Jacobson, Marty


Azotobacter vinelandii was found to exhibit diauxie growth in sucrose containing Burk's medium supplemented with 29 mM ammonium acetate as nitrogen source. The diauxie growth curve was determined to be a result of preferential utilization of acetate as carbon source during the first part of the growth curve. No detectable disappearance of [U-¹⁴C] sucrose was noted in samples taken before the lag separating the two portions of the diauxie curve. Repression of both sucrose transport and metabolizing activity was observed in pre-lag, acetate-utilizing cells when the medium contained a high (58 mM) sucrose concentration. When a lower initial sucrose concentration (7 mM) was used, both sucrose transport and glucose-6-phosphate dehydrogenase activity were induced but acetate was still used preferentially and diauxie growth observed. Mutant strains which fix N₂ in the presence of NH₄⁺ exhibit diauxie growth curves as well and are severely reduced in their ability to reduce C₂H₂ (fix N₂) while utilizing acetate in the pre-lag growth phase. In vivo nitrogenase activity in the presence of NH₄⁺ increases dramatically after the lag. The low nitrogenase activity in pre-lag cultures is not due to a reduced supply of energy or reductant because measurement of nitrogenase in cell free extracts provided with ATP and dithionite yields the same results. The amount of nitrogenase protein detectable as antigenically cross reactive materials varies with the density of the culture. We conclude that the presence or utilization of acetate in some way interferes with the synthesis or stability of nitrogenase. A derepressed mutant Azotobacter vinelandii (UW59) was isolated as a revertant of a Nif⁻ mutant (UW2. Proc. Natl. Acad. Sci., USA 69:3501-3503). A derivative of UW59, UW590, expresses approximately twice the activity of UW 59 when grown with either N₂ or NH₄⁺ as nitrogen source. Biochemical and genetic characterization was undertaken to determine the nature of the mutation(s) resulting in the constitutive phenotype. Strains UW59 and UW590 were used as donors in genetic transformation of UW2. Results indicate that the nif⁵⁹ mutation .is responsible for the constitutive (Nif[sup c] ) phenotype whereas the nif⁵⁹⁰ mutation permits good growth on N₂ but not constitutivity. The rate of NH₄⁺ uptake by UW590 was lower than that of the wild type at pH 5.9. The decreased NH₄⁺ uptake ability was correlated with an increased constitutive synthesis of nitrogenase at low pH. A defect in CH₃NH₃⁺ uptake was detected in UW59 and UW590 at pH 7.3 and pH 5.9. A kinetic analysis of the derepressed mutant UW59 revealed that at high CH₃NH₃⁺ concentrations the mutant is significantly different from the wild type. The results suggest that the constitutive nitrogenase synthesis might be due to an alteration in NH₄⁺ (CH₃NH₃⁺) assimilation.

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