World Journal of Microbiology & Biotechnology 10, 600 Short Note: Role of membrane potential in ammonium inhibition of nitrogenase activity in the cultured cyanobiont Nostoc ANTH S. Singh, B.B. Singh and P.S. Bisen* Ammonium at 5mM completely inhibited nitrogenase activity of Nostoc ANTH but only slightly inhibited the membrane potential, indicating that these two events are independent and that nitrogenase activity is not regulated by the latter. Key words: Ammonium, cyanobiont, nitrogenase, Nostoc ANTH, membrane potential. A .1= .1¢ O =~ e => o _E == Z 6 4 0 t 2 3 ~ $ Time (h) o lOO go E ¢u == 8o == e 7O Figure 1. Relationship between NH~ inhibition of nitrogenase activity (0) and membrane potential (C)) in Nostoc ANTH. Cells were used in their exponential growth phase. NH4GI (5 mM) was added at zero time (0) and nitrogenase activity and membrane potential then measured. The data are means of two independ- ent experiments with four replicates each. The maximum varia- tion from the mean value was <5%. For sustained nitrogenase activity in heterocystous cyano- bacteria an energized membrane is required but NH + and quaternary ammonium compounds can collapse this mem- brane potential (A M) (Haaker et el. 1980). Thus, nitrogenase activity is usually highly dependent on A M. Nostoc ANTH, a free-living isolate, was axenically grown in BG-110 medium as described by Singh & Bisen (1994). NH4C1 when added was at 3 mM. The pH of the medium was adjusted to 7.0 with 10 mM Hepes/NaOH. Nitro- B.B. Singh and P.S. Bisen are with the Department of Microbiology, Barkatullah University, Bhopal-462026, India; fax: 755 510621. S. Singh was with the Department of Microbiology, Barkatullah University, Bhopal, India; he is now with the Department of Microbiology, North Maharastra University, Jalgaom-425001, India. *Corresponding author. (~ 1994 Rapid Communications of Oxford Ltd 600 World ]ournal of Microbiology & Biotechnology, Vol IO, 1994 genase activity was assayed by the acetylene reduction technique. Membrane potential (A~,) was measured by following accumulation of the lipophilic cation triphenyl- [SH]methyl phosphonium (TPMP +) as described by Hawkesford et el. (1982). The observed relationship between NH + inhibition of nitrogenase activity and A M is shown in Figure I. NH +, which inhibited nitrogenase activity by 45% within I h, did not inhibit A ~ significantly. Further, nitrogenase activity was completely inhibited when cells were incubated with 5 mM NH + for 5 h. NH + may inhibit the generation of reducing equivalents to nitrogenase by lowering the A M (Haaker et al. 1980) in which case electron transport to nitrogenase will be extremely sensitive to change in A M and would indicate that nitrogenase and A~, are inter-related. The obligatory A M for nitrogenase activity has been reported to be - 100 mV (inside negative) for Anabaena variabilis and Plectonema boryanurn. Nostoc had a Au/of - 100 mV (inside negative), even in the presence of NH + sufficient to support nitrogenase activity, but no detectable nitrogenase activity was observed under such conditions (Figure 1). Therefore, it is concluded that, unlike A. variabilis and P. boryanum, the NH + inhibition of nitrogenase activity in the cultured cyanobiont Nostoc ANTH appears not to be regulated by A M. References Haaker, H., Laane, C. & Veeger, C. 1980 Dinitrogen fixation and proton motive force. In Nitrogen Fixation, eds Stewart, W.D.P. & Gallon, J.R. pp. 113-136. London: Academic Press. Hawkesford, M.J., Reed, R.H., Rowell, P. & Stewart, W.D.P. 1982 Nitrogenase activity and membrane electrogenesis in the cyano- bacterium Plectonema boryanum. EuropeanJournal of Biochemistry 127, 63-66. Singh, S. & Bisen, P.S. 1994 Evidence for the assimilation of ethylenediamine as nitrogen source by the cyanobiont Nostoc ANTH. World Journal of Microbiology and Biofechnology 10, in press. (Received in revised form 3 May 1994; accepted 4 May 1994)