an Abbe-3L refractometer. The density of phage. MSP8 was 1.51 g/cm3. ... buffers of conductivity 0.1 A were used (Smith,. Chromatographic and Electrophoretic ...
JOURNAL OF BACTERIOLOGY, Mar., 1966 Copyright (©) 1966 American Society for Microbiology
Vol. 91, No. 3
Printied in U.S.A.
Biochemical Characterization of an Actinophage for Streptomyces venezuelae R. A. KOLSTAD AND S. G. BRADLEY Departmenit of Microbiology, Uniiversity of Milnniesota, Minineapolis, Minniiesota Received for publication 20 July 1965
Actinophage MSP8 has been purified and concentrated by partitioning in a two-phase aqueous polymer system containing methylcellulose and sodium dextran sulfate, by dialysis, by elution chromatography with ECTEOLA-cellulose, and by gel filtration (R. A. Kolstad and S. G. Bradley, J. Bacteriol. 87:1157, 1964). Purified suspensions of the phage contained at least 101i plaque-forming units (PFU) per ml. Morphologically, purified phage MSP8 was tadpole-shaped, with a head 58 m,t wide and a tail 137 m,u long. The tail was terminated with a knob (B. G. Painter and S. G. Bradley, J. Bacteriol. 89:240, 1965). This report is concerned with the chemical composition, dry weight, density, and isoelectric point of actinophage MSP8. Phage MSP8 contained 10.4 ,ug of phosphorus by the Fiske-SubbaRow test, 34.5 ,ug of nitrogen by t1he Nessler test, and 125 jig of deoxyribonucleic acid (DNA) by the diphenylamine test per 1012 PFU. With the assumption that DNA contained 9c%, phosphorus, the calculated value for DNA was 116 ,ug/10l2 PFU. From the assumption that DNA contained 15 % nitrogen and that the nitrogen content of protein was 16%, the mean, calculated value for protein was 102 jsg/10l2 PFU. By summing the average DNA and protein values, we determined that phage MSP8 weighed 223 Aig/10l2 particles. The dry weight of the virus was measured with a purified suspension of phage which was filtered through a column of G-75 coarse Sephadex packed in deionized water. The aqueous suspension of phage was centrifuged at 41,000 X g at 4 C for 1 hr in a Spinco model SW-39 rotor. The supernatant fluid, which contained less than 5%I of the total virus, was discarded, and the pellet was dried to constant weight over anhydrous CaCI2. The dry weight of the phage was 219 ,4g/10'2 PFU. The molecular weight of phage MSP8 was estimated to be 1.1 X 108. The amino acid composition of acid-hydrolyzed phage was measured with the Spinco model 120 amino acid analyzer. The value for glycine may be high because DNA yields some glycine upon acid hydrolysis (Fraser, J. Biol. Chem.
227:711, 1957). Tryptophan, destroyed by acid hydrolysis, was not measured. Since the amide forms of the dicarboxylic amino acids were converted to the acid forms upon acid treatment, glutamic acid and glutamine were reported together, as were aspartic acid and asparagine (Table 1). The density of phage MSP8 was measured by isodensity gradient centrifugation (Polson and Levitt, Biochim. Biophys. Acta 75:88, 1963). Cesium chloride was dissolved in buffer consisting of 0.002 M KH2PO4 and 0.02 M K2HPO4, pH 7. A mixture of 3.0 ml of CsCl solution and 0.2 ml of purified phage suspension or 0.15 M NaCI was centrifuged at 40,000 X g at 4 C for 18 hr in a Spinco model SW-39 rotor. From the level of the single phage band, 0.1 ml of fluid was withdrawn with a blunted needle and syringe from the tube without phage. The refractice index of this sample was determined with TABLE 1 Aminto aicid co)m1positioni of ac tinophacge MSP8 Amino acid
MIole per cent*
Lysine .1.28 Histidine .0.66 Arginine .5.36 9.16 Aspartic acid (NH2) Threonine .7.50 4.16 Serine 9.36 Glutamic acid (NH2). 7.60 Proline 11.90 Glycine Alanine .12.56 Half cystine .2.42 Valine .9.34 Methionine .1.10 Isoleucine .5.43 7.30 Leucine .
Tyrosine. Phenylalanine .
1.82 3.74
Standard
0.06 0.10 0.27 0.85 0.88 0.09 0.26 0.50 1.05 0.66 0.31 1.42 0.12 0.37 0.08 0.20 0.15
* Average values of four determinations on acid-hydrolyzed suspensions of phage. 1372
VOL. 91, 1966
NOTES
1373
Abbe-3L refractometer. The density of phage agar in acetate-ethylenediaminetetraacetic acid MSP8 was 1.51 g/cm3. buffers of conductivity 0.1 A were used (Smith, The isoelectric point of phage MSP8 was Chromatographic and Electrophoretic Techniques, measured by two methods. In the acid-sediment- vol. 2, Interscience Publishers, Inc., New York, ation method, 2 ml of phage suspension was 1960). The isoelectric point of phage MSP8, diluted to 11 ml with an aqueous solution of HCl determined electrophoretically and corrected for to pH values between 2 and 6. Absorbancy of light electro-osmotic flow, was approximately pH 3.5. at 260 m,u was measured on the acidified suspension of virus both before and after centrifugaThis investigation was supported by Public Health tion at 6,000 X g for 20 min at 4 C. At pH 3.5 Service research grant AI-01601 from the National and lower, the phage sedimented upon centrifuga- Institute of Allergy and Infectious Diseases. The tion. The isoelectric point was also determined senior author was the recipient of a U.S. Public by agar-gel electrophoresis. Difco 0.7% Noble Health Service Predoctoral Fellowship, 1962-1965.
an
