StiUwater, OK 74074 and b MSU-DOE Plant Research Laboratory, Michigan State ... and Bio-Rad AG 50W-X8 ion~exchange resin (Bio-Rad Laboratories).
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Biochimica et Biophysica Acta, 588 (1979) 193--200 © Elsevier/North-Holland Biomedical Press
BBA 29109
EFFECT OF NITROGEN STARVATION ON THE LEVEL OF ADENOSINE 3',5'-MONOPHOSPHATE IN A N A B A E N A VARIABILIS
ELIZABETH E. HOOD a, SUSAN ARMOUR a, JAMES D. OWNBY a, AVTAR K. HANDA b and RAY A. BRESSAN b
a Department of Cell, Molecular, and Developmental Biology, Oklahoma State University, StiUwater, OK 74074 and b MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, M148824 (U.S.A.) (Received April 17th, 1979)
Key words: Cyclic AMP level; Nitrogen starvation; (Anabaena variabilis)
Summary Low levels of adenosine 3',5'-monophosphate (cyclic AMP) were detected in the cyanobacterium Anabaena variabilis using a protein binding assay and two radioisotopic labelling methods. The basal concentration of intracellular cyclic AMP ranged from 0.27 pmol/mg protein in A. variabilis Kutz grown under heterotrophic conditions to 1.0--2.7 pmol/mg protein in A. variabilis strain 377 grown autotrophically. Extracellular cyclic AMP was found to comprise as much as 90% of the total cyclic AMP in rapidly growing cultures. When A. variabilis strain 377 was starved of nitrogen, a 3--4-fold increase in intracellular cyclic AMP was observed during the 24 h period coincident with early heterocyst development. Introduction Cyclic AMP has been shown to be an important regulator of metabolic and developmental processes in such diverse organisms as bacteria, cellular slime molds, and higher animals. Among algae, factors having the chemical characteristics of cyclic AMP have been identified in Chlamydomonas [1], Euglena [2], Chlorella [3], Ochromonas [4], Cylindrotheca [5], and Anacystis (Rickenberg, H.V., unpublished data). In none of these cases, however, is the precise function of cyclic AMP known. We know of no work showing that cyclic AMP plays an essential metabolic role in organisms whose primary mode of nutrition is autotrophic. In eubacteria, cyclic AMP is an essential component of the apparatus by
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which many enzymes of carbohydrate metabolism are regulated, although some species may not form this nucleotide [6]. Carbohydrate metabolism in bluegreen algae (cyanobacteria) apparently is not subject to the same regulatory controls found in eubacteria and higher animals [7], b u t the close ultrastructural and physiological similarities of these organisms to eubacteria [8] suggest that they may likewise utilize cyclic AMP in some forms of metabolic control. In the work reported here, we have begun to examine the possible role of cyclic AMP in cyanobacteria by measuring levels of the nucleotide in Anabaena variabilis by a protein binding method and t w o isotopic labelling methods. Of particular interest was the possibility that cyclic AMP might have a regulatory function in the response of the organism to nitrogen starvation. Materials and Methods Growth conditions. A. variabilis strain 377 was originally obtained from the University of Texas Culture Collection of algae. Growth conditions were as described previously [9]. Briefly, the cyanobacteria were cultured to the exponential growth phase at 27°C in the medium of Allen and Arnon [ 10] supplemented with 2 mM NH3C1 and 1 mM NaNO3. Nitrogen starvation was then initiated by one o f t w o methods, either addition of L-methionine-DL-sulfoximine to a final concentration of 0.75 pM, or transfer to Allen and Arnon's medium lacking combined nitrogen. The possibility that cyclic AMP measured in cyanobacterial cultures might arise from eubacterial contamination was a major concern in this work. A culture of A. variabilis strain 377 which had been freed of bacterial contaminants by ultraviolet irradiation [11] was used in all experiments with this organism described below. Stock and experimental cultures were routinely assayed for sterility by plating aliquots onto Difco nutrient agar, brain heart infusion agar, and agar containing 1% sucrose and 0.5% peptone. A bacterial contaminant occasionally encountered in this study was a non-motile, Gram-negative rod identified as Flavobacterium sp. In preliminary work we observed that cultures of Anabaena heavily contaminated with Flavobacterium contained approximately the same levels of cyclic AMP as did sterile cultures as measured b y the protein binding assay. Chromatographic separation systems. Solvent system used in the separation of labelled nucleotides were: methanol/ethyl acetate/NH4OH/1-butanol (3:4:4:7, v/v), solvent system A; methanol/1 M ammonium acetate (7:3, v/v), solvent system B; isopropanol/NH4OH/H20 (7:1:2, v/v), solvent system C. Materials used in the chromatographic purification steps included PEI-cellulose and MN300 cellulose TLC plates (Brinkmann Instruments, Inc.), Dowex 50X8-200 ion-exchange resin (Sigma Chemical Co.), neutral alumina (J.T. Baker), and Bio-Rad AG 50W-X8 ion~exchange resin (Bio-Rad Laboratories). Partial purification and assay o f cyclic AMP by the protein binding assay. Cyanobacterial filaments were allowed to settle out of the growth medium for 10 min and excess medium was decanted. The latter was also retained in some experiments and assayed for cyclic AMP. The concentrated cells were then treated with perchloric acid at a final concentration of 0.4 N and cyclic [3HIAMP (50 000 cpm) was added as an internal standard.
195 The concentrated cells were sonicated with a Branson Model 185 cell disrupter, centrifuged at 8000 × g for 10 min, and the acid-soluble extract was neutralized with KOH. After removal of KC104, nucleotides were extracted from the neutralized supernatant b y adsorption onto washed Norit A~activated charcoal as described b y Hardman et al. [12]. The eluate from the charcoal was then dried b y a stream of air at r o o m temperature and the nucleotides redissolved in 0.6 ml distilled water. The samples were divided at this time and half of each was treated with commercial (Sigma Chemical Co.) beef heart cyclic nucleotide phosphodiesterase, as described b y Bressan et al. [13]. Both enzyme-treated and control halves of each sample were then applied to columns of Bio-Rad AG50 ion-exchange resin (100--200 mesh) in the H ÷ form and nucleotides were removed by eluting with water. The first 2 ml of eluate were discarded and the next 5 ml were collected, air
