CHAMCTERLZAHGN as BACTEREQPHAGE gm ma {fszmomms PUTiQé Them {‘09 {he Daqm 05 MI. 9.. MICHIGAN STATE UNWERSITY Lucy Fang Lee 3967 LIBRARY Tums Michigan saw ; University This is to certify that the thesis entitled Characterization of Bacteriophage GH—l for Pseudomonas putida presented by Lucy F. Lee has been accepted towards fulfillment of the requirements for Ph. D - degree mmmtry 04%. A BW / Major professor Date June 6: 1967 0-169 ABSTRACT CHARACTERIZATION OF BACTERIOPHAGE gh-l FOR PSEUDOMONAS PUTIDA by Lucy Fang Lee Bacteriophage gh—l for Pseudomonas putida A.3.12 was isolated and purified by differential centrifugation and diethylaminoethyl (DEAE) cellulose chromatography. An electron micrograph of the phage stained with uranyl ace— tate revealed a regular hexagonal outline about 50 mu across with a short, wedge—shaped tail attached at one corner of the head. The phage formed 10% as many plaques on g. putida C15 as on g, putida A.3.12, the organism used in the isolation procedure No plaques were formed on_g. fluore- scens (ATCC 9712) or g, aeruginosa. The latent period of the infectious cycle was 21 minutes and the average burst size was 103. The nucleic acid component of gh-l bacteriophage was found to be deoxyribonucleic acid (DNA) by a positive di- phenylamine reaction a negative orcinol test, and by its susceptibility to deoxyribonuclease but not ribonuclease. The double—stranded character of gh—l DNA was demonstrated by the sharpness of the rise and the extent of the hyper- chromic effect in the thermal denaturation studies. Further- more, chemical analysis of the DNA base composition showed that its mole per cent adenine (A) equaled thymine (T) and mole per cent guanine (G) equaled cytosine (C). In addition, the buoyant density of heat denatured gh-l DNA was found to Lucy Fang Lee be 0.014 g/cm3 higher than that of its native form. This difference in buoyant density is that expected between duplex DNA and its single-stranded derivative. The base composition of gh—l DNA was established to be 57% GC by direct chemical analysis of its individual bases. This value agreed with that deduced from the thermal de— naturation profile studies and with that calculated from the buoyant density measurement. The buoyant density of gh—1 phage measured by cesium chloride equilibrium centrifugation was 1.45 g/cm3, whereas that of gh-l DNA, heat—denatured gh—lDNA, and g. putida A.3.12 DNA was 1.716, 1.730, and 1.722 g/cm3 respectively. The sedimentation coefficients, S§0,w’ of gh-l and phenol-ex— tracted gh-l DNA measured by the moving boundary sedimenta— tion velocity method were 430—460 and 30.9 respectively. The molecular weight of gh—l DNA calculated from the sedi— mentation coefficient was 22.6 i .9 x 106. Studies of zone sedimentation of single-stranded poly— nucleotide chains derived from differentially labeled gh-l and T7 bacteriophage were performed in alkaline sucrose gradient. The results indicated that the molecular weight of the single—stranded polynucleotide chains of gh-1 DNA is proximate to that of single chains derived from T7 DNA, and that duplex gh-1 DNA, like T7 DNA, consists of two linear uninterrupted polynucleotide chains. Further evi— dence for gh-1 DNA being an intact linear duplex molecule Lucy Fang Lee rather than a circular duplex molecule came from the obser- vation that the buoyant density of heat denatured gh—1 DNA was 0.014 g/cm3 greater than that of native DNA. Finally, the electron micrograph of gh—l DNA clearly showed the mole— cule to be a linear DNA with two ends. CHARACTERIZATION OF BACTERIOPHAGE gh—l FOR PSEUDOMONAS PUTIDA BY Lucy Fang Lee A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1967 (.l 9,0,3, ACKNOWLEDGMENTS The author wishes to express her deepest appreciation to Dr. J. A. Boezi for his guidance, criticism, and en- couragement which made possible this study. Sincere appreciation also goes to the members of my guidance committee, Dr. R. L. Anderson, Dr. J. L. Fairley, Jr., Dr. R. G. Hansen and Dr. J. B. Kinsinger, for guiding my entire graduate program. No words of appreciation can be enough for Dr. Joseph Jen-Hwa Lee for sharing with me the joy and suffering of my graduate study and for reading my thesis. I also wish to thank Dr. Hans Ris, University of Wis- consin, for the use of his facilities and for help in ob— taining the electron micrographs, and to Dr. P. Gerhardt and Dr. R. Scherrer for the use of the Hitachi electron microscope in the Department of Microbiology of Michigan State University. Appreciation is also given to Dr. R. L. Armstrong, Kenneth Payne, and James Johnson for helpful discussion and Mrs. M. DeBacker for technical assistance. Finally, I am indebted to the financial support from the Department of Biochemistry of Michigan State University and the National Institutes of Health. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . 1 MATERIALS AND METHODS . . . . . . . . . . . . . . 6 Organism and Growth Medium . . . . . . . . . . . 6 Isolation of gh-l Bacteriophage for P, putida. 7 Assay Technique for Bacteriophage gh- -1 and P. putida . . . . . . . . . . . . . ., 7 Preparation and Assay of gh-l Antiserum . . . 8 Purification of Bacteriophage gh-l . . . . . . 9 Preparation of 32P-Labeled gh-l and 14C- Thymidine—Labeled T7 Bacteriophage . . . . . 10 One Step Growth Experiment . . . . . . . . . . 11 Preparation of gh-1 Bacteriophage and gh—l DNA for Electron Microscopy . . . . . . . . 12 CsCl Equilibrium Centrifugation of Bacterio— phage gh-l . . . . . . . . . . . . . . . . . 13 Purification of gh—l Deoxyribonucleic Acid (DNA)...................14 CsCl Equilibrium Centrifugation of DNA . . . . 15 Thermal Denaturation Profile of DNA . . . . . 16 Chemical Analysis of Base Composition . . . . 16 Moving Boundary Sedimentation Velocity Studies 17 Zone Sedimentation of DNA Through Alkaline Sucrose Gradients . . . . . . . . . . . . 18 Radioactivity Assaying Procedures . . . . . . 18 iii TABLE OF CONTENTS (Cont.) Page RESULTS . . . . . . . . . . . . . . . . . . . . . . 20 Plaque Morphology and Host Specificity . . . . 20 Kinetics of gh—l Antiserum Neutralization . . 20 One Step Growth Experiment . . . . . . . . . . 27 Bacteriophage Morphology . . . . . . . . . . . 28 CsCl Equilibrium Centrifugation of gh—l . . . 28 Characterization of gh—l Nucleic Acid as DNA . 36 Electron Microscopy of gh—l DNA . . . . . . . 36 Thermal Denaturation Profile of gh—1 DNA . . . 41 Base Composition of gh-l DNA and P, Putida A.3.12 DNA . . . . . . . . . . . . . . . . . 44 Buoyant Density of gh—l DNA and P, putida A.3.12 DNA . . . . . . . . . . . . . . . . . 46 Sedimentation Velocity Studies of gh—l Bacteriophage and gh—l DNA . . . . . . . . . 46 DNA Zone Sedimentation Studies in Alkaline Sucrose . . . . . . . . . . . . . . . . . . 56 DISCUSSION . . . . . . . . . . . . . . . . . . . . 64 REFERENCES . . . . . . . . . . . . . . . . . . . . 76 iv LIST OF TABLES TABLE Page I. Chemical analysis of DNA base compositions . 45 II. Sample calculation for sedimentation coef— ficients of gh—l DNA . . . . . . . . . . . 52 III. Sedimentation coefficients and molecular weights of gh—l and T7 DNA . . . . . . . . 57 LIST OF FIGURES Figure 1. Plaque morphology of gh-l bacteriophage 2. Ultraviolet absorbancy spectrum of purified gh-1 bacteriophage . . . . . . . . . . 3. Kinetics of neutralization of gh—l bacterio- phage by gh—l antiserum . . . . . . . . 4. One step growth experiment of bacteriophage gh—l on g. putida A.3.12 . . . . . . . . . . 5. Electron micrograph of gh—l bacteriophage . . 6. Cesium chloride equilibrium centrifugation profile of gh—l bacteriOphage . . . . . . . 7. Ultraviolet absorbancy spectrum of gh—l DNA 8. Electron micrograph of purified gh—l DNA . . 9. Thermal denaturation profile of DNA . . 10. Cesium chloride equilibrium centrifugation of gh-1 DNA, heat-denatured gh-l DNA, and P, putida A.3.12 DNA . . . . . . . . . . . . . 11. Ultracentrifuge sedimentation pattern of gh—l DNA . . . . . . . . . . . . . . . 12. Effect of gh—l DNA concentrations on sedi- mentation velocity coefficients . . . . 13. Alakline sucrose gradient profile of alkaline denatured gh-l DNA and T7 DNA . . . . . . 14. Co—sedidentation profile of alkaline-denatured gh-l DNA and T7 DNA in an alkaline sucrose gradient . . . . . . . . . . . . . . . . . vi Page 22 24 26 3O 32 35 38 4O 43 48 51 55 59 62 INTRODUCTION One area of research which has engaged the general in— terest of our laboratory has been the study of RNA metabolism in uninfected and phagadnfected bacteria. As one of the steps in the initiation of these studies in Pseudomonas putida, a number of bacteriophages were isolated. The present study describes the isolation and characterization of one of these bacteriophages, gh-l, for g. putida A.3.12. Bacteriophages of the genus Pseudomonas have not been fully characterized. Excepting a few phages of g. aeruginosa (1,2,3,4,5), no phages of other species of Pseudomonas have been the subject of a detailed analysis. Some preliminary studies have been conducted by Niblack and Gunsalus (6) con- cerning bacteriophage Pf, the host of which is P. putida C18. Another study dealing with the host range specificity of .g. fluorescens phages (7) has also been reported. Bacteriophage gh-l, as compared to other phages of P, putida A.3.12 isolated by us, forms a giant plaque, sug- gesting a small virus with a small nucleic acid molecule. It immediately received our attention. Initial cesium chloride centrifugation analysis of gh-l lysates revealed two infectious components. We chose to study the heavier component and gave it the name gh—l. We have since found that both of these infectious components were the same phage. 2 Bacteriophages are classified as either deoxyribonu- cleic acid (DNA)—containing or ribonucleic acid (RNA)-con— taining, according to their nucleic acid components. Most of the phages belong to the former class. Within this class a great variation in DNA structure and chemical composition has been observed. The DNA of T-even phages, like most DNA, is double—stranded, but it differs from others in that it contains hydroxymethylcytosine instead of cytosine (8). The DNA of SP-8 phage for B, subtilis contains hydroxymethyl— uracil in place of thymine (9); and the DNA of PBS—r1 phage for B, subtilis contains uracil in place of thymine (10). Coliphages¢X-174 and $13 contain single—stranded DNA (11, 12). Finally, coliphages MS—2 and f2 contain RNA as its genetic material rather than DNA (13,14). Studies on viral DNA have revealed a great variation in the conformation of DNA molecules. The majority of phage DNA molecules studied to date have a linear double—helical structure (15,16,17). The DNA isolated from coliphage lambda is a linear duplex molecule with unpaired, single- stranded ends. The nucleotide sequences of these single- stranded regions are complementary to each other. The linear duplex molecule can form a circular structure by in— teration of the unpaired ends (18) The single-stranded DNA molecule of QX—174 is circular (19). Rodent polyoma virus contains a circular duplex in a highly twisted con— formation (20,21): Other circular twisted duplex struc- tures have been reported for human papilloma virus (22), 3 Shope rabbit papilloma virus (23) and simian 40 virus (24). The DNA from coliphage T5 has recently been found to con— tain single—strand breaks, or gaps, in both of the duplex molecules (25). The relationship between the sedimentation coefficient and the molecular weight of DNA molecules (5 = kMa) has been studied by various groups of invemfigators. Doty, McGill, and Rice were the first to define this relationship by use of the moving boundary sedimentation method in an analytical ultracentrifuge (26). Burgi and Hershey, on the other hand, used zone sedimentation in sucrose gradient (27), and Studier used zone sedimentation in an analytical ultra- centrifuge (28). The viral DNA molecules so far studied fall within a range of molecular weights between 1.5 —130 million. T2 and T7 DNA, the molecular weights of which have been established as 130 x 106 and 25 x 106 respectively, have been used as standards for calibrating the molecular weights of unknown DNA molecules (29). T2 and T7 have further been shown to contain one DNA molecule per phage particle (30,31,32). Current researchers assume that this is generally true of all viruses. Ruben— stein and Thomas have demonstrated that the total nucleic acid content of T2 phage equaled the total mass of the isolated T2 DNA molecule. Further, it was found that both the T2 particles and their isolated T2 DNA molecules con— tained 130 x 106 daltons of nucleic acid (30). Similar results were obtained for T7. Davidson and Freifelder 4 showed that the isolated T7 DNA molecule has a molecular weight of 25 x 106 and is the entire nucleic acid content of the T7 Virus particle (32). Studies of a dozen or more other DNA-containing viruses have led to the general con— clusion that each virus particle contains a single nucleic acid molecule (29). Phage structure and phage nucleic acid material often define'de mode of phage infection and replication. Know- ledge in this regard comes mainly from the studies on T phages for E, 921;, A typical T-even phage, for example, possesses a bipyramidal hexagonal prism head wherein the DNA is located (33) and a complex tail the function of which is to serve as an adsorption and injection apparatus to the host(34L The:hUection of viral nucleic acid material into a host cell has been well documented (35). Once the viral nucleic acid gains entrance into the host cell, it performs two principal functions. First, it programs the synthesis of enzymes used in the replication of the viral nucleic acid; second, it directs the synthesis of viral structural proteins. The end result of viral infection is, therefore, the production of many new copies of both viral nucleic acids and of the structural proteins. By some mech— anism yet to be thoroughly understood, the new progeny molecules are assembled to form mature virus particles. Following the rupture of the host cell, the phage progeny are released. The yield is usually between 100 to 10,000- fold. 5 The objective of this report is to give a detailed description of the isolation and purification procedure for gh-1 bacteriophage and the characterization of some selected physical, chemical, and biological prOperties, with emphasis on the nucleic acid component. MATER IALS AND METHODS Organisms and Growth Medium Pseudomonas putida A.3.12 (kindly supplied by Dr. W. A. Wood, Michigan State University) was used as the host for bacteriophage gh—l. .E' putida A.3.12 was previously designated 2, fluorescens (36). The organism was grown at 33°C on a gyrorotary shaker, or in a micro-fermentor, in a medium containing the following, in grams per liter: yéast extract, 5; glucose, 4; NaCl, 8; (NH4)2‘HPO4, 6; KH2P04, 3; MgSO4'7H20, 1; and FeCl3, 0.005. Escherichia coli B was grown at 33°C in basal C medium (37) with 0.4% glucose to serve as host for bacteriophage T7 Coliphage T7 used in this study was obtained from Dr. D. Schoenhard, Michigan State University. In the prepara— tion of 14C-thymidine—labeled T7 bacteriophage, a thymine- requiring mutant of E, 39;; B (kindly supplied by Dr. R. Greenberg, University of Michigan) was used as host. The mutant was grown at 37°C in the basal C—glucose medium sup- plemented with 1—2 ug/ml of thymidine. In the study of host specificity, g, fluorescens (ATCC 9712), P. aeruginosa, and g. putida C18 were used. ‘g. putida, C18 was kindly supplied by Dr. I. C. Gunsalus, University of Illinois. 7 Isolation of gh—l Bacterigphage for P. putida Bacteriophage gh-l was isolated from a sample taken from the aeration tank at the Waste Water Treatment Plant in East Lansing, Michigan in the summer of 1965. The sample was centrifuged to remove debris and fil— tered through an ultrafine sintered—glass filter with a maximum pore size between 0.9—1.4 u. A sample of the fil— trate was added to an exponentially growing culture of P. putida A.3.12. After incubation overnight, the culture was centrifuged at 4,000 x g for 10 minutes. A sample of the supernatant fluid was tested for bacteriophage content by the agar layer technique. A well—isolated plaque of bacteriophage gh—l was picked and replated several times to ensure genetic homogeneity. Assay Techniques for Bacteripphage gh—l and P. putida Bacteriphage gh-l was assayed by the agar layer tech— nique of Adams (38). The technique consisted in introduc— ing a drop of host bacteria and a sample of diluted phage, containing approximately 50 to 100 plaque-forming units (PFU), into 2.5 ml of growth medium containing 0.6% agar maintained at 45°C. This mixture was then poured onto the surface of Petri plates containing a hardened layer of 2% agar and growth medium. After being incubated at 33°C for 4 hours, the plates were read for the number of plaques formed. From this count and the dilution factor, the phage titer was calculated. 8 The concentration of the host organism, g, putida A.3.12, was determined by viable cell count. A sample from the last serial dilution of an exponentially growing culture at a given turbidity was spread over the surface of agar plates with a glass rod. The plates were incubated overnight at 33°C and the number of the colonies counted. From this count, the number of viable cells per milliliter was calcu— lated. A count of 5 x 108 cells/ml gave a turbidity read— ing of one unit at 660 mu in the Beckman DU Spectrophotom- eter. For E, Eggi_B grown in basal C-glucose medium, 5 x 108 cells/ml gave a turbidity reading of 0.5 unit at 660 mu. Preparation and Assay_of gh—l Antiserum Antiserum for gh—l bacteriOphage was prepared accord- ing to the procedure of Adams (38). For this preparation, five milliliters of the purified gh-l containing 5 x 101° PFU in sterile saline solution (0.8% NaCl) were injected intraperitoneally into each of two rabbits. Two injections per week were given for three weeks, followed by a collec— tion of blood for a potency test one week after the last injection. Blood was drawn from the marginal ear vein and allowed to clot at 37°C. After storage at 5°C overnight, the blood was centrifuged to separate the serum from the red blood cells. The serum thus obtained was assayed for its ability to inactivate gh—l bacteriophage. Since the blood showed sufficient antibody activity, one rabbit was 9 then bled by cardiac puncture to obtain a large quantity of serum. The assay for gh—l antiserum was performed in the fol- lowing manner. The gh-l antiserum was prepared for testing at dilutions of 1:100 and 1:1000. The phage stock was diluted to a titer of 107 PFU/ml. A sample of 0.1 ml of this phage dilution was added to 0.9 ml of diluted serum l 2 I 15, 20, 30 minutes), 0.1 ml samples of the phage-serum mix- at 37°C. At Specified time intervals (t = 2, 5, 7 10, ture were withdrawn and diluted 12100 with growth medium. Duplicate samples of 0.05 ml of this dilution were plated out by the agar layer technique. The fraction of phage remaining after various times of antiserum inactivation was calculated. A plot of the fraction of surviving phage against time on semi-log paper gave a straight line, the slope of which equaled the specific neutralization rate constant, k. Purification of Bacteriophage gh-l A 6— to 10—liter amount of g, putida A.3.12 was grown with vigorous aeration to a density of about 5 x 108 cells/ ml. Bacteriophage at a multiplicity of about 5 were added. After 3 to 4 hours the lysed culture was centrifuged at 4,000 x g for 10 minutes to remove bacteria and cell debris. The titer of the lysates was about 2.5 x 101° PFU/ml. The virus was collected either by centrifugation at 16,000 x g for 2 hours or by precipitation with ammonium sulfate at 10 50% saturation. The preparation was then suspended in buf— fer containing 0.05 M tris(hydroxymethyl)aminomethane(Tfis) ad— justed to pH 8.0 with HCl, 0.001 M MgClz, and 0.2 M NaCl. The suspension was centrifuged at low speed, and the super- natant fraction was loaded on a diethylaminoethyl (DEAE) cellulose column (3 by 17 cm) equilibrated with 0.05 M Tris chloride buffer (pH 8.0) containing 0.2 M NaCl. At this salt concentration most contaminating material is ad— sorbed on the column, but the phage is not. The fractions containing gh—l were concentrated by centrifugation, and the resulting pellets were resuspended in the buffer des— cribed above. The yield of plaque-forming units was between 25 and 50%. Purified gh-l at a concentration of 1012 to 1013 PFU/ml was stored at 5°C. Preparation of 32P—Labeled gh-l and 14C—Thymidine—Labeled T7 .P. putida A.3.12 was grown exponentially to a cell dens— ity of 5 x 108 cells/ml in a medium containing 0.5% yeast extract and 0.5% tryptone. 32P-phosphoric acid neutralized with NaOH (carrier free, obtained from Tracer lab, Waltham, Mass.) at 5 uc/ml was added to the growth medium. Thirty minutes later, gh—l at the multiplicity of 2 was added. Lysis of bacteria occurred within four hours. The radio- active bacteriophages were purified according to the pro- cedure described previously. In view of the damage to DNA structure which could be caused by the decay of the radio- isotope, the purification procedure and analysis were 11 carried out immediately. The assay of radioactivity will be described in the later part of this section. The specific activity of gh-l bacteriophage was found to be 1.1 x 105 cpm/ A26°. The absorbancy readings were not corrected for the amount due to light scattering. E, 92;; B thy_ was grown to a density of 5 x 108 cells/ ml. The culture was~centrifuged, washed, and suspended at a concentration of 5 x 108 cells/ml in the growth medium minus thymidine. Radioactive [2—14C] thymidine (specific activity of 30 uc/mmole, obtained from Tracérlab) at a con— centration of 0.25 uc/ml was added to the culture, followed immediately by the addition of bacteriophage T7 at a multi- plicity of 0.5. The culture lysed in 4-5 hours. T7 was purified by a series of differential centrifugations at 4,000 x g for 10 minutes followed by 16,000 x g for 2 hours. The specific activity of the purified T7 was found to be 1.2 x 105 cpm/AZSO. One Step Growth Experiment The procedure described by Adams (38) was used. The host bacteria for this experiment were grown at 33°C to a concentration of about 5 x 108 cells/ml. Bacteriophages at a multiplicity of 1 to 2 were added. After allowing 5 minutes for adsorption, the culture of the infected bac— teria was diluted 1:20 into the gh—l antiserum. The con- centration of gh-l antiserum usedves sufficient to inacti— vate 95 to 99% of the free virus particles in 5 minutes. 12 After a five minute incubation period in gh-l antiserum, the infected culture was diluted 1:500 into the first growth tube. Immediately a sample from the first growth tube was further diluted 1:100 into the second growth tube. Both infected cultures were incubated at 33°C. Every 2.5 min— utes for the first 25 minutes samples were removed from the first growth tube. For the next 35 minutes, samples were taken from the second growth tube at 5—minute intervals. The samples thus obtained were assayed for plaque—forming units. Preparation of gh—l Bacteriophage and gh-l DNA for Electron Microsc0py To prepare for electron microscopy, the purified phage preparation was dialyzed against 0.1 M ammonium acetate at 4°C overnight. The dialyzed phage suspension was then mixed with an equal volume of 1% (w/v) uranyl acetate solution (39). A drop of the phage suspension was transferred to a carbon-coated supporting grid and then examined under a Siemens Elmiskop 1 electron microscope at a magnification of 40,000. These experiments were performed in Dr. Hans Ris' laboratory at the University of Wisconsin. Prelimin— ary electron microscopic studies of the virus particles were performed with the RCA EMU—2 in the Biochemistry De— partment at MSU and the Hitachi electron microscope in the Department of Microbiology. For such electron microscopic investigations, the virus was negatively stained with 2% (w/v) 13 phOSphotungstic acid solution adjusted to pH 7.0 with 1 N KOH (40). For the preparation of gh—l DNA for electron micro— scopy, phenol extracted DNA was diluted to 5 ug/ml. This diluted sample was then mixed with an equal volume of 1 M ammonium acetate solution containing 0.01% (w/v) cytochrome C (41). A Teflon Petri plate was filled with a 0.1 M ammonium acetate solution. A wet microsc0pe slide was partially immersed in the solution at an angle of about 30°. Talcum particles were sprinkled lightly over the surface of the acetate solution in the area near the slide. A sample of 0.3 ml of the DNA—cytochrome C mixture was allowed to flow gently down the glass slide. As the DNA—cytochrome C mixture entered the acetate solution, it pushed the talcum powder away and floated as a thin film on the surface of the solution. The film was compressed lightly with two Teflon rods. A Specimen for electron microscopic examina— tion was Obtained by gently touching the film with a car— bon-coated grid. The grid was then dipped in ethanol and dried on filter paper. The dry specimen was shadowed with uranium at a 6° angle while rotated at 60 rev/min. The shadowed preparation was then examined under the Siemens Elmiskop 1 electron microscope at a magnification of about 10,000. CsCl Equilibrium Centrifugation of Bacteriophage gh-l Solid cesium chloride (optical grade, obtained from 14 Gallard—Schlesinger Chemical Mfg. Corp., Carle Place, N.Y.) was added to a sample of a purified phage preparation con- taining 1.2 x 1012 PFU (absorbancy, 260 mu, of 14 units) to give an initial CsCl concentration of 1.50 g/cm3. The sus— pension was centrifuged in an SW 39 rotor at 39,000 rev/min in a Spinco Model L—2 Ultracentrifuge at 7°C for 20.5 hours. Fractions were collected from the bottom of the centrifuge tube and analyzed for absorbancy at 260 mu and for plaque— forming units. The CsCl concentration of various fractions was calculated from refractive index measurements using the equation described by Ifft, Voet, and Vinograd (42). 25°C 10 8601 25°C 13 4974 P = ’ TlD — ' ° Purification of gh-l Deoxyribonucleic Acid (DNA) A sample of gh—l containing 1.0 x 1012 PFU/ml (ab— sorbancy, 260 mu, of 14.3 units/ml) was suspended in 0.5% sodium dodecyl sulfate (pH 7.3) and then stirred with a magnetic stirring device at 5°C for 10 to 15 minutes. An equal volume of phenol was added, and the mixture was stirred for another 10 minutes. After a low-speed centri- fugation the aqueous layer was removed. Phenol extraction was repeated, and the purified nucleic acid was dialyzed overnight against 0.01 M Tris chloride (pH 7.3) with 0.1 M KCl, or against SSC (0.15 M NaCl-0.015 M trisodium citrate). For the analysis of the sedimentation velocity coef— ficient, DNA was prepared according to the procedure 15 described by Abelson and Thomas (25). The purified phage preparation was adjusted to a concentration having an ab— sorbancy of 5-15 units/ml and placed in a glass stoppered tube. An equal volume of the freshly distilled water- saturated phenol was added and the tube was rolled in a horizontal position at 60 rev/min for 30 minutes at room temperature. The mixture was centrifuged at 4°C for 5 minutes at 3,000 rev/min to separate the two phases. The phenol layer was removed with a Pasteur pipette. The pro— cedure was repeated once using a 15—minute rather than a 30—minute extraction. The final aqueous layer was removed after centrifugation and dialyzed extensively with SSC buffer. The dialysis tubing was boiled in 5% sodium bi- carbonate before use and washed excessively with distilled water. DNA preparations from E, putida A.3.12 and E, 29;; were purified as described by Armstrong and Boezi (43). DNA concentrations were calculated from the absorbancy at 260 mg by use of an extinction coefficient of 20 cmZ/mg. Diphenylamine reactions were carried out by the method described by Burton (44), while orcinol tests were performed according to the procedure of Mejbaum (45). Heat denatura— tion of gh—1 DNA was performed in 10—fold diluted SSC at 100°C for 10 minutes, followed by quick cooling. ggcl Equilibrium Centrifugation of DNA CsCl equilibrium centrifugation of gh—l DNA, heat— denatured gh—l DNA, and E, putida A.3.12 DNA was performed 16 according to the procedure described by Schildkraut, Marmur, and Doty (46). The initial concentration of CsCl was 1.710 g/cm3. E, EQEE_DNA, the buoyant density of which was taken to be 1.710 g/cm3, was used as the density marker. Centrifugation was performed at 25°C in a Spinco Model E Analytical Ultracentrifuge for 20 hours at 44,700 rev/min. The centrifuge cell was a standard cell fitted with a 1° negative wedge window. Tracings from the ultra— violet absorbancy photographs were made by use of a Joyce- Loebl double—beam recording microdensitometer. Thermal Denaturation Profile of DNA The thermal denaturation profile of DNA was determined by measuring the absorbancy at 260 mu at various tempera- tures. The buffer used in this determination was 0.01 M Tris chloride (pH 7.3) with 0.012 M KCl. Chemical Analysis of Base Composition A sample containing 2.5 x 1012 PFU of bacteriophage gh—l was suspended in 0.1 ml of 70% perchloric acid in a small, glass—stoppered test tube. The suspension, after being heated at 100°C for 1 hour with occasional agitation, was diluted to 0.5 ml with water. A black residue was removed by centrifugation. A sample of the hydrolysate was spotted on acid-washed Whatman No. 1 paper. Chroma— tography was performed by the descending method with iso— propanol—HCl-water (65:17:18) as solvent (47). The bases 17 were locatedby use of an ultraviolet lamp. After elution from the paper with 0.1 N HCl, the isolated bases were identified, and the amount of each was determined from the ultraviolet absorption spectrum. The base composition of .g. putida A.3.12 DNA was determined in a similar manner. Moving Boundary Sedimentation Velocity Studies The sedimentation studies of gh—l and its DNA and T7 DNA were carried out using the boundary sedimentation velocity method in a Spinco Model E analytical ultracentri- fuge with ultraviolet optics. The centrifugations were made at 15,220 rev/min for bacteriophage gh-1 and 42,040 rev/min for DNA. The centrifuge cell was a standard cell fitted with a Kel F centerpiece. The temperatures of the runs were set variously at 18 to 22°C. After reaching the maximal speed, ultraviolet absorbancy photographs were taken for gh—1 at 4—minute intervals and for gh-l DNA and T7 DNA at 8-minute intervals. The ultraviolet absorbancy photographs were transcribed into a density—versus-distance plot by means of a Joyce-Loebl double-beam recording micro- densitometer. The sedimentation coefficients of gh-1 and T7 DNA were measured in 1 M NaCl at a DNA concentration of 15 to 50 ug/ml and in SSC at a concentration of 25 to 75 ug/ml for gh—l bacteriophage. The sedimentation coefficient was corrected to standard conditions and is reported as 520,w- 18 -Zone Sedimentation of DNA Through Alkaline Sucrose Gradients Sedimentation of DNA in alkaline sucrose was performed according to the procedure of Abelson and Thomas (25). A gradient of 5% to 20% sucrose (w/v) in 0.9 M-NaCl and 0.1 M- NaOH was prepared at 20°C. A sample of the radioactive bacteriophage was mixed with an equal volume of 0.2 M-Na3PO4 solution and allowed to stand for 10 minutes at 20°C in order to assure the complete release of DNA from the phage head. A 0.1 ml or 0.2 m1 sample of the mixture containing approximately 1 to 2 ug/ml of DNA was layered onto the top of the 4.8 m1 sucrose gradient. A 2-m1 serological pipette was used to layer the DNA mixture gently onto the sucrose gradient. The pipette was supported mechanically and con- trolled with a screw-driven pipetting device. The mixture was then centrifuged at 20°C in a SW 39 swinging bucket rotor in a Model L-2 Spinco Ultracentrifuge for 2% to 3 hours at 35,000 rpm. At the end of the run, a hole was punched at the bottom of the centrifuge tube and seven drops per fraction were collected. The average size drop was 15.5 ul i 0.1 al. The radioactivity of each fraction was assayed according to the procedure described below. RadioactivityyAssaying,Procedure The general procedure for assaying radioactivity used throughout this investigation began with the addition of 250 ug of salmon sperm DNA to each sample followed by pre- cipitation with cold 10% trichloroacetic acid (TCA). The 19 TCA insoluble material of each sample was collected by filtering through a nitrocellulose membrane filter (Carl Schleicher & Schuell Co., Keene, N.H.). The filter was dried at 95°C for 10-20 minutes. The radioactive content was assayed in a liquid-scintillation spectrometer with a fluor containing either 0.1 g POPOP (1,4-bis—[2-(5 phenyl- oxazolyl)]—Benzene) and 4.0 g PPO (2,5—diphenyloxazole) per liter of toluene or 4.0 g BBOT (2,5—bis-[2~(5—tert— butylbenzoxazolyl)]—Thiophene) per liter of toluene. The gain and window discriminator settings for each of the isotopes were as follows: (1) 3H-gain 58% and window dis— criminator ratio 50-1000; (2) 32P, 1.3% and 100—1000; and (3) 14C, 24% and 50—800. In eXperiments where the 14C and 32P content of a sample was assayed simultaneously, an overlapping of 8% of the 32F counts was found in the 14C channel with less than 1% of the 14C counts in the 32P chan— nel. Appropriate calculations were made to account for this fact. RESULTS PlaqueyMprphology and Host Specificity BacteriOphage gh-l formed a clear, smooth plaque, 4 to 6 mm in diameter, on E, putida A.3.12. The plaque morphology is shown in Figure 1. Bacteriophage gh—l at— tacked E, putida C18, forming about 10% as many plaques as on E, putida A.3.12. No plaques were formed on E. fluorescens (ATCC 9712) or on E. aerugenosa. Klinge (7) has made an extensive study of the host specificity of Pseudomonas phages. He isolated 29 different bacteriophages for E, fluorescens and three for E, putida. None of these phages lysed E, aeruginosa. Two E, fluorescens phages lysed E. putida and no E, putida phages lysed E, fluorescens. An ultraviolet Spectrum of purified gh-l bacterio— phage is shown in Figure 2. Only preparations of gh-1 which had been purified through the DEAE-cellulose frac— tionation step exhibited such a spectrum with an absorption maximum at about 260 mu and a minimum at 240 mu. The 260— 280 mu absorbancy ratio was 1.56 and the 260-240 mu ratio was 1.36. Kinetics of gh-1 Antiserum Neutralization The kinetics of neutralization of gh-l by antiserum is illustrated in Figure 3. The rate at which a phage preparation is neutralized by antiserum obeys the following relationship 20 21 Figure 1. Plaque morphology of gh-l bacteriOphage. 23 .ommnmoaumuomfl Hlnm Umamausm mo Eduuommm wocmnuomnm umaofl>mHuHD .N musmflm 24 con .N musmflh as £52334; emu o8 EN 08 ODN OQN OQN d . 084 on. 084 . 9.: mm. oe~< O; AONVSHOSSV 25 Figure 3. Kinetics of neutralization of gh-l bacterio- phage by gh—1 antiserum. FRACTION PHASE SURVIVING |.O 0| .Ol 26 INACTIVATION 0F BACTERIOPHAGE gh-I . A IO l5 TIME (MINUTES) Figure 3. 20 27 - dP/dt = ch where - dP/dt is the rate of inactivation of phage per unit time, C is the concentration of antiserum, P is the titer of phage, and k is the specific neutralization rate constant. Integrating the above equation between PO and P, to and t, we obtain ln Po/P = kCt or log Po/P = 0.43 kCt where P0 is the initial phage titer at zero time (to), and P is the titer after t minutes of incubation with an anti- serum of concentration C. Since the dilution factor D (= 1/C) of antiserum is generally known, the equation can be rewritten as log PO/P = 0.43 kt/D The value k, the specific neutralization rate constant, can be obtained from any one measurement of inactivation by use of the above formula, or from a plot of the fraction of surviving phage against time on semi-log paper. Such a plot gives a straight line, the dope of which is k. The k determined from Figure 3 was 222. With this k value for a given antiserum it is possible to calculate the dilution of antiserum required to produce a desired amount of phage in- activation in a given time period. One Step Growth Experiment A one-step growth experiment was performed to determine the length of the latent period and the average burst size 28 of the infectious cycle. The results are given in Figure 4. The latent period, which is defined as the interval of time between adsorption of the virus to the host and lysis, was found to be about 21 minutes. The average burst size, cal- culated by dividing the number of plaque-forming units present after lysis by that present before lysis, was 103. Bacteriophage Morphology An electron micrograph of gh—l stained with 1% uranyl acetate is presented in Figure 5. The nucleocapsid is of a regular hexagonal outline of about 50 mp across. A short, wedge-shaped tail attached at one corner of the head can be seen. Two fibers attached to the wedge—shaped tail are visible on the bacteriOphage at the center of Figure 5. The morphology of gh—l is similar to that of coliphage T3 described by Bradley and Key (48), that of T7 reported by Davison and Freifelder (49), and that of bacteriophage Pf for E, putida C18 reported by Niblack and Gunsalus (6). The nucleocapsid of coliphage T3, for example, has a hex- agonal cross section of about 55-65 mu and has a short wedge—shaped tail, 14 mu long. The nucleocapsid of coli- phage T7 is about 59-65 mu across with a small tail. Bacteriophage Pf for E, putida C18 has a polyhedral head of 54 mu diameter and a 10 mp conical tail. CsCl Equilibrium Centrifugation of gh—l An analysis of a purified gh-1 preparation by equilibrium 29 Figure 4. One step growth experiment of bacteriophage gh-l on E, putida A.3.12. PFU x I0'3 30 l5- IO- ONE STEP GROWTH' EXPERIMENT LATENT PERIOD I 2| MINUTES BURST SIZE 8 I03 1 l l IO 20 30 4O 50 60 TIME, MINUTES Figure 4. 31 .ooo.ov mo :ofipmoHMHcmmE HousmESHumsH um wmoomouofle couuowam H momeEHm wfiwfiwfim.m.mwwmd cmcHmew mmB coaumnmmmnm cmcflmum one .mumumum HmdeD A>\3V RH SUH3 pmcflmum maw>flummos also mmmnm0HHmuomQ.wo.£mMHmOHUHE couuumam .m musmflm _ ‘59.. ‘te. @1334 n A .r 'a ‘ ‘ l 33 centrifugation in CsCl is presented in Figure 6. Absorb- ancy measurements at 260 mu identified three components banding at densities of 1.52, 1.50 and 1.45 g/cm3. The component banding at 1.52 g/cm3 was not infectious while the other two components were. When material from the 1.50 g/cm3 density component was collected and rebanded in CsCl, a single component of duasame density was found. When material from the 1.45 g/cm3 density component was similarly collected and rebanded, two components of densi- ties 1.50 and 1.45 g/cm3 were detected in the same ratio, as Shown in Figure 6. These results suggested that the 1.50 g/cm3 density component was an artifact produced by degradation of the 1.45 g/cm3 density component in the CsCl gradient. The 1.52 g/cm3 density component, on the other hand, was not detected upon rebanding of either of these two lighter components in the CsCl gradient. Its identity is not known. It could be, however, defective viral particles produced during the purification steps prior to CsCl equi— librium centrifugation. The 1.45 g/cm3 component was considered to be the intact viral particles. It was about three times as infectious per absorbancy unit as the 1.50 g/cm3 density component. If we assume the intact viral particle to consist of only protein and DNA, the fractional composition of each component can be calculated from its buoyant density according to the equation (50) 1/P0 = n1/P1 + n2/Pz where p0 is the measured buoyant density of the intact viral 34 .mnsu was mo mop map Eouw .om .oc GOAUUMHM “mafia wmdwflupcmo map mo Eouuon exp Eoum ddflwamE mcflmucoo H .oc coauomum .Hmnesc coauomnm waHmmm wwaOHm mum ATCHH pmnmmcv mafia: mCHEHOMstqmam cam AmGHH Uflaomv 1800a um mucmfluomnfi .Hlnm mo COHumWSMHHUGmU EDHHQHHADUT mcfluoano Edflmmo .0 mndmflm 35 (o---o) OI’O| X "3:! o 9 9 ‘0 i 2' . . . a 'T .c o u. "'0 g ’’’’’’ _-._:9w=3->(:_ _____________________ < (D D E}: o: ”I'- Z LIJ .. aqua-9 2 2 II 9.3 —=J_ D O UJ l l I o “’- O- N _ _ (W...) m» 092 'AONVBUOSBV FRACTION NUMBER Figure 6. 36 particle; p1 and p2 are the buoyant densities of DNA and protein and are taken to be 1.71 and 1.33 g/cm3; n1 and n2 are the fractional parts of DNA and protein. Accordingly, the particle banding at p = 1.45 g/cm3 was composed of 64% protein and 36% DNA. Characterization of gh—l Nucleic Acid as DNA The nucleic acid of the virus was purified by phenol extraction, and shown to be DNA by a positive diphenylamine reaction, a negative orcinol test, and by its susceptability to deoxyribonuclease but not to ribonuclease. The ultra— violet spectrum of gh—l DNA was typical of nucleic acids with a maximal absorbancy at 259 mu, as shown in Figure 7. The ratios of absorbancies at 260 mu to 280 mu and 260 to 230 mu were 1.9 and 2.2 respectively. Electron Microscopy of gh-l DNA Figure 8 is a portion of an electron micrograph of gh—l DNA prepared by the protein-monlayer technique of Klein— schmidt (41). This technique provides a direct way of ex— amining the conformation and the contour length of DNA. As seen in Figure 8, the molecule is linear with its two ends clearly visible. This structure seems to be a single un— broken DNA molecule and represents the entire DNA content of a gh-l virus particle. In areas not shown in Figure 8, neither circles nor twisted supercoils were observed, al- Q. though many multiple—looped structures or "flowers" were 37 .fiza also mo Enupommm mocmnuomnm umHOH>MHuHD .h musmflm 38 .b onsmflm 1.2 £5235; 00m 0mm 0mm Ohm CON 0mm O¢N 0mm % q n J 4 u d d 1 0.. AONVSHOSBV Figure 8. 39 Electron micrograph of purified gh—l DNA examined under the Siemens Elmiskop 1 electron microscope at an instrumental magnification of 10,000. 8 e r u g .1 F . . 1...l.4 .no not..\IAo. ‘41 present. These latter structures result when crowding.of the DNA molecules occurs on the electron microscope grid. With prOper equipment the contour length of DNA can be measured. Assuming a linear density of 196 dalton/A for DNA of the B conformation, the molecular weight of the DNA can be calculated from the length thus measured. Con— versely, with known molecular weight the length of the molecule can be estimated. Since the molecular weight of gh-1 DNA has been established by sedimentation velocity studies to be about 22.6 x 106, its molecular length should be 11.5 x 104 A or 11.5 microns. This value can be com— pared with the molecular length of 12.5 microns reported for T7 DNA. It should be mentioned here that T7 phage has a gross morphology -- size and shape -— Similar to that of gh-l phage. Furthermore, the molecular weights of the DNA of these two phages as determined by sedimentation velocity studies were found to be close. For these reasons and for the purpose of making comparison, many of the studies in this investigation were performed on both gh—1 and T7. Thermal Denaturation Profile of gh-l DNA The thermal denaturation profile for gh-l DNA is given in Figure 9. The absorbancy of gh-l DNA remained constant until about 77°C. It rose sharply thereafter and reached a maximum by 80°C. 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