R. Premakumar

Nitrogen Fixation by Azotobacter vinelandii Strains Having Deletions in Structural Genes for Nitrogenase.

Phenotypic reversal of Nif- mutant strains to Nif+ under molybdenum-deficient conditions has been cited as evidence that Azotobacter vinelandii possesses two nitrogen fixation systems: the conventional molybdenum-enzyme system and an alternative nitrogen-fixation system. Since explanations other than the existence of an alternative system were possible, deletion strains of A. vinelandii lacking the structural genes for conventional nitrogenase (nifHDK) were constructed. These strains were found to grow in molybdenum-deficient nitrogen-free media, reduce acetylene (at low rates), and incorporate molecular nitrogen labeled with nitrogen-15. Thus it can be concluded that the phenotypic reversal phenomenon cannot be due to altered phenotypic expression of nif mutations under molybdenum-deficient conditions, but is due to the existence of an alternative nitrogen-fixation system in A. vinelandii as originally proposed.

Evidence for Two Dinitrogenase Reductases under Regulatory Control by Molybdenum in Azotobacter Vinelandii

Abstract Evidence for an alternative nitrogen fixation system which is expressed under conditions of molybdenum deficiency has been reported in Azotobacter vinelandii (Bishop, P.E., Jarlenski, D.M.L. and Hetherington, D.R., Proc. Natl. Acad. Sci. U.S.A. (1980) 77, 7342–7346). In the present report we describe the existence of activity for a dinitrogenase reductase-like enzyme (alternative reductase) in Mo-deficient cell-free extracts of Nif− mutant strains of A. vinelandii which lack either conventional dinitrogenase reductase (strains UW1 and UW3) or contain a defective enzyme (strain UW91) under conditions of Mo-sufficiency. Nitrogenase activities were determined by the acetylene reduction method in a complementation assay where extracts of strain UW91 served as a source of dinitrogenase and extracts of strains UW1, UW3 or UW91 served as a source of alternative reductase. Strains that lack dinitrogenase reductase activity in the presence of Mo, were shown to have alternative reductase activity under Mo-deficient conditions. Two-dimensional gel electrophoretic analysis showed these extracts to contain a protein of similar mobility as the conventional dinitrogenase reductase. Molybdenum and tungsten repressed the formation of the alternative reductase whereas vanadium mimicked Mo deprivation. In conclusion, the results with the Nif− strains provide evidence for the presence of two reductase activities, one of which is expressed in the presence of Mo (dinitrogenase reductase) and the other in the absence of Mo (alternative reductase).