FEMS Microbiology Letters 6 (1979) 45-46 © Copyright Federation of European Microbiological Societies Published by Elsevier/North-Holland Biomedical Press 45 ADENYLATE ENERGY CHARGE IN CHEMOORGANOTROPHIC THIOBACILLUS FERROOXIDANS KG-4 PAIVI M. SORMUNEN * and OLLI H. TUOVINEN ** Department of General Microbiology, University of Helsinki, SF-O01 O0 Helsinki 1O, and Department of Microbiology, University of Helsinki, SF-00710 Helsinki 71, Finland Received 19 March 1979 1. Introduction The adenylate energy charge is a measure of the cellular adenine nucleotide pool indicating the ener- getic state and the overall metabolic regulation of energy generation and expenditure [1-3] : ATP + 0.5 ADP ATP + ADP + AMP In active cells not subjected to metabolic stress or deprivation, the energy charge has been shown to stabilize within the range of 0.8-0.9 [ 1,4]. In the present study the adenylate energy charge was mo- nitored during the growth of a heterotrophic strain of Thiobacillus ferrooxidans. The results show that the energy charge values are comparable to those reported for other microorganisms. Furthermore, the energy charge is shown to indicate metabolic changes, possibly such as diauxie, which may not be apparent by the conventional measures of growth. 2. Materials and Methods T. ferrooxidans strain KG-4 was originally supplied by Dr. F. Shafia (California State Polytechnic College, Pomona, CA 91768) and maintained on glucose [5, 6]. Growth experiments were carried out using 50 ml * Present addresses: Lumatek Ltd., P.O. Box 9, SF-02211 Espoo 21, Finland; ** Department of Microbiology, The Ohio State University, Columbus, Ohio 43210, U.S.A. cultures in 250 ml shake flasks at 180 rev./min and 28°C. The growth on glucose was followed by mea- surement of absorbance at 440 nm. 1-ml samples of cultures were taken at intervals for determination of adenine nucleotides. A survey of the literature indicated that a varying proportion of the intra-cellu- lar nucleotides may escape to the medium if the bacteria are first filtered or centrifuged [7,8] : the size of this fraction is greatly dependent on the physiological condition of the bacteria. In the present study, therefore, only the total adenine nucleotide pool in the samples was determined. (Normally the extracellular level of nucleotides is almost negligible in comparison to their intracellular pool). Samples were extracted with perchloric acid followed by neutralization and the enzymic conversion of AMP and ADP by the myokinase and pyruvate kinase reac- tion as described in detail by Ball and Atkinson [9]. ATP was assayed by the luciferine-luciferase bio- luminescence reaction using a Lumac Celltester (Lu- mac Systems AG, Basel) to monitor the light emis- sion. All the reagents including the stocks of AMP, ADP and ATP were checked for purity with respect to contaminating adenine phosphonucleotides. The myokinase and pyruvate kinase enzymes were pur- chased from Sigma Chemical Co. and Boehringer GmbH, respectively, and checked for purity. The luciferine-luciferase enzyme of Pholinus pyralis was obtained from Lumac Systems AG (Lumit NS). The recovery of known amounts of nucleotides using the entire procedure of extraction, enzymic conver- sion and measurement varied between 92-102%. The recovery was routinely determined when measuring different batches of samples. Downloaded from https://academic.oup.com/femsle/article-abstract/6/1/45/474736 by guest on 10 June 2020