FEMS Microbiology Letters 9 (1980) 121-124 © Copyright Federation of European Microbiological Societies Published by Elsevier/North-Holland Biomedical Press 121 EFFECT OF MOLYBDENUM STARVATION AND TUNGSTEN ON THE SYNTHESIS AND ACTIVITY OF NITROGENASE IN ANABAENA CYLINDRICA PATRICK C. HALLENBECK and JOHN R. BENEMANN * Sanitary EngineeringResearch Laboratory, University of California, Berkeley, CA 94 720, U.S.A. Received 23 June 1980 Accepted 16 July 1980 1. Introduction As with other nitrogen-fixing organisms, molyb- denum is required for the growth of cyanobacteria on atmospheric nitrogen [1 ] due to the association of this metal with one of the two nitrogenase proteins [2]. The results of substituting vanadium or tungsten for molybdenum in the growth media have been dif- ferent for different organisms [3-10]. In some cases there is an apparent molybdenum requirement for synthesis of nitrogenase, as in Klebsiella pneumoniae or Clostridium pasteurianum [9,10]. Replacement of molybdenum by tungsten in the growth medium ofAnabaena cylindrica permits the synthesis of active nitrogenase component II [11] ; however, nitrogenase activity itself is very low [12]. In the non-heterocystous cyanobacterium, Plecto- nema boryanum, both nitrogenase proteins appear to be synthesized when the cultures are induced under molybdenum-deficient conditions (with or without tungsten) [7]. Here we have investigated the effects of molybdenum starvation (with and without tung- sten) on in vivo and in vitro nitrogenase activity. Our results are consistent with the results obtained with Plectonema boryanum; namely that the nitrogenase proteins are synthesized in the absence of combined nitrogen under conditions of molybdenum starvation. * To whom inquiries should be addressed. Current address: Ecoenergetics, Inc., 180 Viewmont Ave., Vallejo, CA 94590, U.S.A. 2. Materials and Methods 2.1. Organism and culture methods A. cylindrica 629 was grown in modified Allen and Arnon media [2] made with distilled, deionized water and with molybdenum and tungsten omitted from the trace metal solution (nil media). These metals were added as sodium molybdate or sodium tungstate as needed. 2 M NH4C1 was added to a final concentra- tion of either 5 mM NH4C1, for maintenance of stock cultures on nil media; or 1 mM NH4C1, for growth of cultures in which depression of heterocyst differentia- tion was desired. (This amount of nitrogen supported growth to about 0.245 g dry weight/.) Nitrogen- starved cultures with active nitrogenase (grown on media containing molybdenum) were prepared as previously described [2]. Cultures were used when nitrogen starvation derepression of heterocyst differ- entiation was complete (36-48 h). The cultures were grown as either non-axenic 47-liter or axenic 2-liter batch cultures, and in vivo experiments were con- ducted in either 2-liter or 250-ml culture vessels. Lighting (2.0 • 104 erg/cm2/s), growth determina- tions, and dry weight measurements were as previ- ously described [13]. During nitrogen starvation and in vivo experiments, cultures were sparged with 99.7% argon, 0.3% carbon dioxide. In vivo [13] and in vitro [2] nitrogenase activities were determined as previously described. Determina- tion of component I protein was performed by a modification of the Laurell electroimmunodiffusion technique [14]. Downloaded from https://academic.oup.com/femsle/article-abstract/9/2/121/503718 by guest on 16 June 2020