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Isolation, and characterization of a bacteriophage active against pseudomonas acidovorans Kropinski, Andrew Maitland Boleslaw 1969

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ISOLATION, AND CHARACTERIZATION OF A BACTERIOPHAGE ACTIVE AGAINST PSEUDOMONAS ACIDOVORANS by ANDREW MAITLAND BOLESLAW KROPINSKI B.Sc. (Bacteriology and Biochemistry) University of British Columbia, 1965 A THESIS SUBMITTED IN.PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF MICROBIOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April , 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and S t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s thes, is f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t . m y w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Date 7^7 ABSTRACT A phage was i s o l a t e d from sewage which was ac t ive against the non-f luorescent Pseudomonad, P_. acidovorans. I t was given the des ignat ion 0W-14. Th i s i s the f i r s t phage reported to be ac t ive aga ins t P_. acidovorans. 0W-14 was l a r g e r than prev ious ly reported Pseudomonas phages. I t had an i cosahedra l head some 90 my i n diameter and a c o n t r a c t i l e t a i l 140 my long and 20 my i n diameter. The phage tended to form large aggregates. 0W-14 formed haloed plaques on three s t r a i n s of the host organism, -4 but mutated at a high frequency (4 - 9 x 10 ) to a c l e a r plaque type when grown on P_. .acidovorans #14. The haloed plaque form was considered to be the w i l d type and was given the des ignat ion 0W-14a+. The c l e a r plaque mutant appeared s table and was given the des ignat ion 0W-14a. Adsorpt ion of 0W-14a+ was b i p h a s i c , with 35% of the phage populat ion having a decreased adsorpt ion capac i ty . The K value was —9 1.9 x 10 ml /min. More normal k i n e t i c s were observed with 0W-14a and with 0W-14a+ i n low-sa l t b r o t h . The K value for 0W-14a was 4.2 x I O - 9 . The burst s i z e for 0W-14a+ obtained from one-step growth e x p e r i -ments was 300, with a l a t e n t per iod of 67 minutes. 0W-14a had a 50% higher burst s i z e but the same la tent p e r i o d . The average burst s i z e was markedly a f fec ted by the p h y s i o l o g i c a l age of the b a c t e r i a l c u l t u r e , and by the m u l t i p l i c i t y of i n f e c t i o n . S e n s i t i v i t y of the phage to heat , pH, son ica t ion and u l t r a -v i o l e t l i g h t was i n v e s t i g a t e d . At l ea s t 70% of the l e t h a l e f fec t s o f UV i r r a d i a t i o n could be reversed by p h o t o r e a c t i v a t i o n . Thermal i n a c t i v a t i o n k i n e t i c s of 0W-14a+ i n broth were b iphas i c at 55 C and 60 C, the AH* being 75,700 c a l o r i e s / m o l e . 0W-14a appeared to be more t h e r m o - l a b i l e . N u c l e i c a c i d i s o l a t e d from 0W-14a+ was double-stranded DNA. There was a s i g n i f i c a n t discrepancy between the moles % GC ca l cu la ted 3 from buoyant densi ty determinations (1.666 g/cc -6% GC) and Tm determina-t ions (98.4 C - 71.9% GC). The DNA was unusual a l so i n that i t y i e l d e d f i v e bases upon h y d r o l y s i s . The f i f t h base was not one of those commonly found i n DNA. I t has not been i d e n t i f i e d . The base composit ion of the DNA as determined by chromatographic separat ion and q u a n t i t a t i o n of the bases was: Adenine, 21.8 moles %; Guanine, 28.2 moles %; Thymine, 11.1 moles %; Cytos ine , 26.6 moles %; Unknown, 12.3 moles ?<>; based upon the r e l a t i o n s h i p : [ A + G ] = 1 [ T + C + Unknown ] i i i TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS . . . 2 I. Organisms 7 II. Media 7 III. Isolation of phage and general properties . . . . 10 1. Isolation of phage 10 2. Plaque morphology 11 3. Host range 11 4. Relative efficiency of plating . . . . . . 11 5. Production of a high t i t r e lysate 11 6. Phage purification 12 7. Plaque morphology mutants 13 8. Electron microscopy 13 IV. Kinetics of adsorption 14 V. Intracellular phage development 15 1. One-step growth experiment 15 2. Effect of culture age on burst size . . . . . 16 VI. Lysis inhibition, 16 1. Turbidimetric method 16 2. One-step growth method 17 VII. Thermal inactivation 17 i v Table of Contents (Continued) Page V I I I . Sonic S e n s i t i v i t y of phages . . . . . . . . . . 1 9 IX. pH i n a c t i v a t i o n . . . . . . . . 19 X. S e n s i t i v i t y to u l t r a v i o l e t l i g h t and photoreac t iva t ion . 20 1. UV i n a c t i v a t i o n . . . 20 2. Photoreac t iva t ion . . 21 X I . Phage r e s i s t a n t mutants and the c a r r i e r s tate . . . . . 22 1. I s o l a t i o n of r e s i s t a n t s t r a i n s . . . • . . . . 2 2 2. Microscopy . . . . . . 2 3 a. M o t i l i t y 23 b. Phase contrast microscopy , •. . . 2 3 3. Confirmation of the c a r r i e r s tate . . . . . . 2 3 X I I . Recombination . 24 X I I I . P h y s i c a l and chemical proper t i e s of 0W-14 DNA . . . . 2 5 1. I s o l a t i o n of DNA . . . 25 a. Perch lorate method 25 b . Tryps in-phenol method . . . . . . . . . . 2 5 c. I s o l a t i o n of P_. acidovorans DNA 26 2. General proper t i e s of the phage n u c l e i c a c i d . . . 27 a. UV spectrum 27 b. C a l c u l a t i o n of DNA concentrat ion 27 c. C o l o r i m e t r i c assays 27 d. Enzyme s e n s i t i v i t y 28 V Table of Contents (Continued) Page 3. M e l t i n g temperatures 28 4. Buoyant densi ty 29 a. General method . . . . . . 29 b. Preparat ion of denatured DNA 30 i . A l k a l i n e denaturat ion 30 i i . Thermal denaturat ion 31 5. Chemical composition of phage DNA . 31 a. Hydro lys i s . . . . 3 1 i . Hydro lys i s to free bases 31 i i . Hydro lys i s to free saccharides . . . . . 3 2 i i i . Attachment s i t e of saccharide i n 0W-14 DNA . 32 b . Paper chromatography . . . 3 3 i . Separation of bases . . . . . . . . 33 i i . Separation of saccharides . . . . . . . 3 3 c. Quant i ta t ive estimate of base composition . . . 33 RESULTS AND DISCUSSION . . . . • . . 35 Sect ion I . I s o l a t i o n and general proper t i e s of 0W-14 . . . 35 1. I s o l a t i o n . . 3 5 2. Plaque morphology 37 3. Host range . 3 8 4. R e l a t i v e e f f i c i e n c y of p l a t i n g 38 v i Table of Contents (Continued) Page 5. Phage p u r i f i c a t i o n . . . . . . 41 6. Plaque morphology mutants . . . . . . . . 4 6 7. E l e c t r o n microscopy 48 Sect ion I I . K i n e t i c s of adsorpt ion 55 Sect ion I I I . One-step growth experiment 60 Sect ion IV. L y s i s i n h i b i t i o n . . . . 65 Sect ion V . Thermal i n a c t i v a t i o n . 67 Sect ion V I . Sonic s e n s i t i v i t y of phages 71 Sect ion V I I . pH i n a c t i v a t i o n 73 Sect ion V I I I . S e n s i t i v i t y to U l t r a v i o l e t l i g h t and photoreac t iva t ion . . 75 Sect ion IX. Phage r e s i s t a n t mutants and the c a r r i e r s tate 78 Sect ion X. Recombination . 85 Sect ion X I . Phage 0W-14 DNA 85 1. General proper t i e s of the n u c l e i c a c i d . . . . 85 2. Me l t ing temperatures . . . . . . . . . . 86 3. Buoyant densi ty ana lys i s . . . . 88 4. Chemical composition 90 GENERAL DISCUSSION . . . . 98 BIBLIOGRAPHY 100 v i i LIST OF FIGURES Page F i g . 1. Plaques of 0W-14 on P. acidovorans #29 37 F i g . 2. Mutant d i s t r i b u t i o n 47 F i g . 3. E l e c t r o n micrograph of 0W-14 50 F i g . 4. 0W-14 t a i l d e t a i l and empty heads 51 F i g . 5. 0W-14 showing contracted t a i l sheath 51 F i g . 6. 0W-14 showing contracted t a i l sheath 52 F i g . 7. E f f e c t of 0.1 M NaClO^ on phage i n t e g r i t y 52 F i g . 8. 0W-14 aggregate 53 F i g . 9. K i n e t i c s of adsorpt ion of phage to P. acidovorans 57 F i g . 10. One-step growth curve 61 F i g . 11- E f f e c t of c e l l age on average burst s i z e 63 F i g . 12. T u r b i d i m e t r i c demonstration of l y s i s i n h i b i t i o n 66 F i g . 13. K i n e t i c s of thermal i n a c t i v a t i o n 69. F i g . 14. Arrhenius p l o t of thermal i n a c t i v a t i o n of 0W-14 70 F i g . 15. Sonic s e n s i t i v i t y of three d i f f e r e n t phages 72 F i g . 16. pH i n a c t i v a t i o n of 0W-14a+ and 0W-14a 74 F i g . 17. UV i n a c t i v a t i o n and photoreac t iva t ion of T and 0W-14a+ 76,77 F i g . 18a. P. acidovorans #29 grown at 30 C. 81 18b. P. acidovorans #29 grown at 18 C. 81 v i i i L i s t of Figures (Continued) Page F i g . 19a. P. acidovorans #29-20 grown at 30 C 82 19b. P. acidovorans #29-20 grown at 18 C 82 F i g . 20. M e l t i n g p r o f i l e s of phage and host DNAs 87 F i g . 21. Microdensitometer scans of CsCl dens i ty gradients 89 F i g . 22. Spectra of a novel base found i n 0W-14 DNA 94 i x LIST OF TABLES Page Table I . Physiology of ,growth of various Pseudomonas phages 2,3 Tab le I I . Pseudomonas phage n u c l e i c ac ids 5,6 Table I I I . Sources of Organisms 8,9 Table IV. L i s t of i s o l a t e d Pseudomonas phages 36 Table V . Host range of 0W-14 39 Table V I . R e l a t i v e e f f i c i e n c y of p l a t i n g 40 Table V I I . Aggregation and disaggregat ion of 0W-14 preparat ions 43 Tab le V I I I . Incidence of plaque morphology mutants 45 Table IX. C h a r a c t e r i s t i c s of some phage r e s i s t a n t • mutants 80 Table X a . Chromatographic separat ion of known n u c l e i c a c i d bases 91 Table Xb. Chromatographic separat ion of 0W-14 DNA b'ses 91 Table X I . S p e c t r a l proper t i e s of bases i s o l a t e d from 0W-14 DNA 92 Table X I I . S p e c t r a l proper t i e s of a novel base found i n 0W-14 DNA 93 Table X I I I . Phys ico-chemical proper t i e s of 0W-14 DNA 96 ACKNOWLEDGEMENT To Professor R . A . J . Warren for h i s pat ience , h e l p f u l d i scuss ions and d e t a i l e d proof reading and correc t ions that he made to t h i s t h e s i s . To D r . M. Weintraub for opening to us the e l ec tron microscope f a c i l i t i e s of the Federa l A g r i c u l t u r e Research S t a t i o n , and to Miss E l s i e Jang for preparing the exce l l ent e l e c t r o n micrographs. To D r . J . Tremaine for h i s d i scuss ions on the thermodynamics of thermal i n a c t i v a t i o n . To Professor D . J . C l a r k for donating h i s time to i n s t r u c t me on phase contrast photography. To Professor M. Smith for h i s i n t e r e s t and good advice on studying the proper t i e s of the phage n u c l e i c a c i d . To D r . R. Stace-Smith for prov id ing the use of h i s Joyce-Loebl microdens i -tometer on numerous occas ions . To Mrs. Joan H i l l for t r y i n g to exp la in the i n t r i c a c i e s of the G i l f o r d spectrophotometer and i n a i d i n g i n obta in ing the Tm data . To Mrs . Janet O'Dor and Barry Walsh for t h e i r continuous harassment and unsuccessful attempt at keeping me on one bench. To my parents and parent - in - law without whose encouragement and f i n a n c i a l ass i s tance t h i s would not have been p o s s i b l e . And not the l e a s t to Miss Jeanette Bellamy for her exce l l ent typing and pat ience . Dedicated to: SHARON AND MARY CATHERINE 1 INTRODUCTION The phages of the phytopathogenic Pseudomonads have rece ived a great amount of a t t e n t i o n due to the considerable economic importance of t h e i r host organisms. Phages have been i s o l a t e d which are ac t ive against: P_. p h a s e o l i c o l a (Katznelson and Sutton, 1951); P_. morsprunorum, P_. v i r i d i l i v i d a (Crosse and G a r r e t t , 1963); P_. a t r o f a c i e n s , P_. p i s i , P_. coronafaciens (Sutton and Katznelson, 1953); P_. t a b a c i , P_. angulata (Fu l ton , 1950); P_. syringae (Baigent, de Vay, and S t a r r , 1963); P_. solanacearum (Matsui , 1952). With the exception of the l a t t e r , a l l the other reports were p r i m a r i l y concerned with the i s o l a t i o n and host-range proper t i e s of the phages as a means of c l a s s i f y i n g t h i s confusing group of organisms. Matsui has concerned himsel f with the physiology and genetics of a P_. solanacearum phage, SP^ (Matsui, 1952; Matsu i , 1953 a, b ) . The phages of P_. aeruginosa (P_. pyocyanea) have been s tudied i n great d e t a i l , but again, with some exceptions, the purpose has been taxonomic, s ince t h i s organism i s assoc iated with a number of c l i n i c a l mani fes tat ions . Some of the best s tudied P_, aeruginosa phages have been: F l , F2, and F3 ( A l f o l d i , 1956; Lovas, Egyessy and A l f o l d i , 1957); p8 and P2 (Jacob, 1952); phage 2 (Grogan and Johnson, 1964 a, b , ) ; a group of phages from a lysogenic s t r a i n (Feary, F i s h e r and F i s h e r , 1964). Phages against another human pathogenic Pseudomonad - P_. pseudomallei -were i s o l a t e d from stagnant waters i n Vietnam by Lederc and Sureau (1956). The most thoroughly Invest igated group of Pseudomonas phages -Table 1. Proper t i e s of var ious Pseudomonas phages - Physiology of Growth Phage Host bacterium Temp. Latent per iod Rise per iod Burst s i z e Ref er< PX4 P. aeruginosa 2 25 C- 52 min 103 1 PX2 P. aeruginosa 1 37 85 162 1 PX3 P. aeruginosa 2 37 35 88 1 PX7 P. aeruginosa R629 . 37 50 : 55 1 CB3 P. aeruginosa l c 37 45 75 1 7v P. aeruginosa Ps-7 37 23 9 min 230 2 1 P. aeruginosa 37 45-50 100 3 2 aeruginosa 37 37 15 111 4 K P. aeruginosa (Kitasato s t r a i n ) 35 50 130 5 Seven P. aeruginosa 35-40 40-100 6 p8 P. pyocyanea 13 37 55-65 40-60* 7 P 2 P. pyocyanea 13 37 25-30 50-70* 7 F l P. pyocyanea 3R 37 30 48 150-250 8 F2 P. pyocyanea 3R 37 15 21 30-60 8 F3 P. pyocyanea 3R 37 20 ' 22 120-180 8 PX4 P. f luorescens .14 25 60 23 1 PX10 P. f luorescens 22 25 35 80 N 1 3 Phage Host bacterium Temp. Latent Rise Burst Reference per iod per iod s i z e PX12 P. f luorescens 35 25 40 50 1 PX1 P. put ida A.3 .12 25 35 106 1 gh-1 P. put ida A.3 .12 33 21 34 103 9 PX14 P. gen icu la te 4 25 30 15 120 10 Pg8 P. a tro fac iens 55-60 15 2.1 11 SP-L P_. solanacearum S 95 15 130 12 SPjh-]** P. solanacearum B19 120. 25 70 12 * determined using minimal-glucose'medium * * a host-range mutant of SP-^  1. Olsen, Metca l f and Todd (1968). 2. Feary, F i s h e r and F i s h e r (1964). 3. van den Ende, Don, E l f o r d , C h a l l i c e , Dawson, and Hotchin (1952). 4. Grogan and Johnson (1964 b ) . 5. Takeya, M o r i , Ueda, and Toda (1959). 6. O'Cal laghan and Grogan (1967). 7. Jacob (1952). 8. A l f o l d i (1956) . 9. Lee and Boezi (1966). 10. Olsen (1967). 4 11. Sutton (1966). 12. Matsui (1953 b ) . i s o l a t e d against P_. aeruginosa, P_. f luorescens , P_. put ida and P_. gen icu la ta - i s that reported by Olsen , Metcal f and Todd (1968). In th i s study, the physiology of growth, s e n s i t i v i t y to c i t r a t e , pH and osmotic shock, as w e l l as the phage morphology and the GC content of the DNA were s tud ied . Phages ac t ive against P_. f luorescens and P_. put ida had been i s o l a t e d prev ious ly by Kl inge (1959) and Lee and Boezi (1966). In t h i s l a t t e r paper, the propert ie s of phage gh-1 were s tudied i n depth. U n t i l the present research only two phages had been i s o l a t e d against nonf luorescent , nonphytopathogenic, Pseudomonads - P_. f r a g i (Roberts and Doetsch, 1966) and I?, s t u t z e r i (Espejo and Canelo, 1968). The l a t t e r phage i s qui te unique i n that the phage coat contains an apprec iable amount of l i p o i d a l m a t e r i a l . The o r i g i n a l i n t e n t i o n of t h i s research was to i s o l a t e a phage against a nonf luorescent P.seudomonad.- P_. acidovorans. In p a r t i c u l a r a. temperate transducing phage was des ired s ince the genetics of t h i s group of organisms has rece ived l i t t l e a t t e n t i o n . Although severa l phages a c t i v e against P_. acidovorans were i s o l a t e d , a l l were l y t i c phages. This thes i s describes the proper t i e s of one of them - 0W-14. Tables I and II summarize the proper t i e s of var ious Pseudomonas phages, and i s based upon a search of the l i t e r a t u r e up to the end of 1968. 5 Table I I . Proper t i e s of var ious Pseudomonas phages - Nuc le i c Acids Phage Host Bacterium Nuc le i c Ac id %GC Method Refere 2 P. aeruginosa ds DNA* 54.7 b , c** 1 SD1 P. aeruginosa ds DNA 53.8 a,c 2 PX2 P. aeruginosa ds DNA 68.2 a 3 PX3 P. aeruginosa ds DNA 45.0 a 3 PX7 P. aeruginosa ds' DNA 54.6 a 3 CB3 P. aeruginosa ds DNA 60.4 a 3 B3 P. aeruginosa ds DNA 4 D3 P. aeruginosa ds DNA 4 E79 P. aeruginosa ds DNA 4 F116 P. aeruginosa ds DNA 4 Pf P_. aeruginosa ss DNA 5 PP7 P. aeruginosa ss RNA 6 7s P. aeruginosa ss RNA 50.4*** c 7 PX4 P. f luorescens ds DNA 44.4 a 3 PX10 P. f luorescens ds DNA 53.0 . a 3 PX12 P. f luorescens ds DNA 55.8 a 3 Pf P. put ida ds DNA 62.0 a,b 8 PX1 . P. put ida ds DNA 52.5 a 3 gh-1 P. put ida ds DNA 57.0 a , b , c 9 PM2 P. s t u t z e r i ds DNA 43.0 a,b 10 PX14 P_. gen icu la ta ds DNA 53.8 a 3 6 * ds (double-stranded); ss ( s ing le - s tranded) ; RNA ( r i b o n u c l e i c a c i d ) ; DNA (deoxyribonucle ic acid) * * a (determined from Tm); b(determined from buoyant dens i ty ) ; c (determined by q u a n t i t a t i v e chromatography a f ter a c i d hydro lys i s ) * * * Adenine (23.8 moles%); Guanine (24.6 moles%); Cytosine (25.8 moles%); U r a c i l (25.8 moles%) 1. Grogan and Johnson (1964 a ) . 2. Shargool and Townsend (1966). 3. Olsen, Metca l f and Todd (1968) . 4. Davidson, F r e i f e l d e r and Holloway (1964). 5. Takeya and Amako (1966). 6. Bradley (1966). 7. Feary, F i s h e r and F i s h e r (1963). 8. Nib lack and Gunsalus (1965). 9. Lee and Boezi (1966). 10. Espejo and Canelo (1968). 7 MATERIALS AND METHODS I . Organisms The sources of the organisms used are l i s t e d i n Table I I I . The organisms were maintained i n standard minimal base stabs with 0.5% yeast extract (S tan ier , P a l l e r o n i and Doudoroff, 1966) at 4 C, and were t rans ferred every two months. I I . Media Modif ied L u r i a broth was used throughout as the medium for b a c t -e r i a l and phage growth, and as a d i l u e n t . The composition was, i n g per l i t r e : tryptone ( D i f c o ) , 10; yeast extract ( D i f c o ) , 5; N a C l , 5; manni to l , 1. The medium was made-up i n d i s t i l l e d water, adjusted to pH 6.5, and s t e r i l i z e d by autoc lav ing . B a c t e r i o l o g i c a l agar (Difco) was added to a f i n a l concentrat ion of 1% (w/v) for p l a t i n g medium, whi le 0.6% agar was added to the overlays used i n t i t r i n g phage. L u r i a broth without added NaCl was used for the adsorpt ion studies -L u r i a broth (-NaCl) - and had the fo l lowing composit ion, i n g per l i t r e : tryptone, 1; yeast e x t r a c t , 0.5; manni to l , 2. The minimal medium contained, i n g per l i t r e : Na^HPO^, 5.3; lOL^PO^, 2.3; NH^Cl, 1.0; MgS0 4 (added a f t er autoc lav ing ) , 0.3; mannito l , 4 .0 . The pH was adjusted to pH 7.0 p r i o r to autoc lav ing . Table I I I : Sources of organisms Organism S t r a i n Source P. acidovorans #14; #29; . #114; #146 D r . R, S t a n i e r , Dept. of Ba c t e r io lo g y , U n i v e r s i t y of C a l i f o r n i a , Berkeley . AK-11 Norleucine enrichment 15666 Dr. E . F . L e s s e l , 15667 Curator of B a c t e r i a , 15668 American Type Culture C o l l e c t s p. t e s t o s t e r o n i #78; #138 11996 D r . R . D r . E . Stanier , F . L e s s e l p. mucidolens ATCC 4687 Culture C o l l e c t i o n , Dept. of Microb io logy , U n i v e r s i t y of B r i t i s h Vancouver. Columbia p. putrefac iens Hammer Dept. of Microbio logy ( U . B . C . ) p. o v a l i s ATCC 950 Dept. of Microbio logy ( U . B . C . ) p. tae tro lens ATCC 4683 Dept. of Microbio logy ( U . B . C . ) p. synxantha ATCC 796 Dept. of Microbio logy ( U . B . C . ) p. convexa ATCC 795 Dept. of Microbio logy ( U . B . C . ) p. aeruginosa ATCC 9027 Dept. of Microbio logy ( U . B . C . ) p. aeruginosa ATCC 9721 Dept. of Microbio logy ( U . B . C . ) p. f r a g i ATCC 4975 Dept. of Microbio logy ( U . B . C . ) E . c o l i K12 3000 D r . R Dept. . A . J . Warren, of Microbio logyj U n i v e r s i t y of B r i t i s h Columbia Vancouver. 9 Strain Organism Source E. coli Dr. R.A.J. Warren Appendix 1. Characteristics of Organisms of the P_. acidovorans Group "The acidovorans group consists of non-pigmented, nutri-tionally versatile aerobic pseudomonads which share a distinctive nutritional spectrum and certain unique metabolic properties. A l l strains are multitrichous." (Stanier, Palleroni and Doudoroff, 1966). Table 41. Acidovorans group. The group characters of greatest differential value in the recognition of the acidovorans group, based on the analysis of 26 strains Characters Number of positive strains Ideal phenotype 1. Poly-^-hydroxybutyrate as cellular 20 + reserve material Utilization of 2. L-arabinose 0 — 8. D-Glucose 0 — 4. D-Galactose 0 — 5. 2-Ketogluconate ' 0 — 0. Saccharate 25 + 7. Pelargonate 0 _ 8. Adipate, pimelate, suberate, azelate, 20 + sebacate 9. Glycollate 24 + 10. Laevulinate 26 + 11. Itaeonate 26 + 12. m-Hydroxybenzoate 25 18. .Norleucine 25 + 14.' Putrescine 0 — Table 42. Acidovorans group. Number of strains of different species or groups of species of aerobic pseudomonads conforming to the selected fourteen characters {Table 41) which define the ideal phenotype of the acidovorans group No. of characters of 'ideal' phenotype 14 13 12 11 10 8 Acidovorans group Fluorescent group Pseudomallei group P. mullivorans P. stutzeri P. mallophilia Alcaligcncs group P. lemoignei 24 18 8 24 81 44 48 27 1 23 10 7 6 8 23 . . . 2 1 . 10 III. Isolation of Phage and General Properties 1. Isolation of phage Activated sludge or raw sewage from the Greater Vancouver Sewage Processing Plant (Iona Island) was " c l a r i f i e d " by centri-fugation at 20,000 x j» for 5 min. A 10 ml sample of the supernatant was added to 10 ml of double-strength Luria broth, and the mixture inoculated with about two drops of an overnight culture of P_. acidovorans #14. The flask was incubated under static conditions at 30 C for 48 hr. A few drops of chloroform were added to the flask, which was then shaken vigorously. The suspension was centrifuged at 6000 x £ for 10 min. to remove bacterial cells and debris, and the supernatant was carefully decanted and stored over several drops of chloroform. The enrichment was diluted and plated with P_. acidovorans #14 by the overlay method (Adams, 1959). A single plaque was picked from one of the plates with a sterile wire and suspended in Luria broth. The phage was purified by repeated single plaque picks. Subsequently, a great number of phages were isolated from raw sewage collected from the same source using various other Pseudomonas species as host organisms. 2. Plaque morphology The typical plaque morphology was determined using the overlay method with thick (40 ml medium) Luria agar plates which had been dried previously at 37 C for several hours (Hershey, and Rotman, 1949) . 3. Host range The host ranges of the phages were determined by spot testing a •I o high t i t r e lysate (ca. 1 x 10 pfu/ml) on overlays containing the various organisms, and incubating the plates overnight at 30 C or 37 C. 4. Relative efficiency of plating A pure line of phage was diluted appropriately and eight equal volumes were plated with the various host organisms. After an over-night incubation period the plaque counts were tabulated, and the means and standard deviations calculated. 5. Production of a high t i t r e lysate A number of 500 ml erlenmyer flasks each containing 150-200 ml of Luria broth were inoculated with 10 ml of an overnight culture of P_. acidovorans #29. The f l a s k s were incubated at 30 C and 250 rpm i n a Metabolyte model G77 shaker water-bath (New Brunswick S c i e n t i f i c Co., New Brunswick, N.J.) u n t i l the cultures reached an o p t i c a l density at 650 my of 1.5-2.0. This was equivalent to g 10-13 x 10 c e l l s / m l . Phage 0W-14 was added to give a m u l t i p l i c i t y of approximately 1, and incubation was continued f o r a further 6 hr. Using t h i s method i t was possible to r o u t i n e l y obtain lysates with t i t r e s of 1-3 x 1 0 1 1 pfu/ml. 6. Phage p u r i f i c a t i o n Phage lysates were freed of whole c e l l s and debris by c e n t r i -fugation at 20,000 x £ for 5 min. The turbid supernatant f l u i d was c a r e f u l l y decanted and centrifuged at 14,000 rpm f o r 4 hr. using the A-28 rotor (approximate capacity 2000 ml) i n an International Model B-60 preparative u l t r a c e n t r i f u g e (International Equipment Co., Needham Heights, Mass.). The clear supernatant, containing l e s s than 2% of the t o t a l plaque forming u n i t s , was removed by suction and discarded. The phage p e l l e t was allowed to resuspend i n 0.05M T r i s (hydroxymethyl)aminomethane-HCl- 0.01 M c i t r i c a c i d -0.005 M NaCl buffer pH 8.1 at 4 C for several days. The highly turbid suspension was digested with DN'ase I (Worthington Biochemical Corp., Freehold, N.J.) and RN'ase A (Schwartz Bioresearch Inc., Orangeburg, N.Y.), both at 10 Ug/ml, f o r 1 hr at 37 C. The suspension 13 was chilled to 4 C and ice-cold 95% ethanol added dropwise to a final concentration of 16%. After 2 hr. the suspension was centrifuged at 6000 x j» for 10 min., and the supernatant removed and stored. 7. Plaque morphology mutants A total of twenty discrete a+-type plaques (turbid plaque type) were picked from overlay plates into small tubes containing 1 ml of sterile Luria broth. The suspensions were mixed vigorously and stored at 4 C for 1-2 days. The tube contents were then diluted and plated using P_. acidovorans #14 as the host. On the plates con--1 -2 taining the lowest dilutions (10 and 10 ) of the phage the a-f—type plaques formed a confluent lawn on which i t was possible to distinguish clear areas (a-type plaques). A l l distinct, large, clear plaques were totalled to give the minimum value of the mutational frequency, while the total of a l l possible clear plaques gave the maximum value. 8. Electron microscopy Partially dried preparations of 0W-14 were stained with 2% phosphotungstic acid, pH 7.2. The grid was allowed to air dry before being examined with a Phillips EM-200 electron microscope at an operating voltage of 60 KV. Magnifications, before printing, ranged from 15,000 to 27,800 X. IV. Kinetics of Adsorption An exponentially growing culture of P_. acidovorans #29 was diluted Q with fresh, ice-cold, Luria broth to a density of 1 x 10 cells/ml. Ten m i l l i l i t r e s of this c e l l suspension were added to a 125 ml erlenmyer flask which was then placed in a shaker water-bath. In a l l experiments, unless stated otherwise, the temperature was maintained at 30 C, and the speed at 250 rpm. After a five minute period for temperature equilibration, 0.1 ml of phage suspension, suitably diluted, was added to the flask to give an i n i t i a l multiplicity of infection (moi) of 0.01. At one minute intervals thereafter, 0.1 ml samples were removed from the adsorption flask to 9.9 ml of ice-cold Luria broth contained in large broth tubes (2.2 x 20 cm) stored i n an ice bucket. These were mixed vigorously using a Vortex Junior Mixer (Scientific Industries Inc., Queens Village, N.Y.). At the termination of the experiment, 5 ml samples from each broth tube were centrifuged in the cold at 20,000 x j> for 5 min to sediment bacterial c e l l s . The supernatants were then appropriately diluted and plated with P_. acidovorans #29 as the host organism. In the case of experiments run to show the effect of sodium chloride on adsorption, a culture of P_. acidovorans grown i n normal Luria broth was diluted into Luria broth (-NaCl). The phage lysate was similarly diluted. The adsorpt ion rate 'constants were ca l cu la ted using the equat ion" derived by Schles inger (1932): K = 2 . 3 x l o g 1 0 Po/Pt N x T where K i s the v e l o c i t y constant for c e l l attachment; Fo i s the number of plaque forming un i t s at zero time; F t i s the number unadsorbed at time T_; and, |J i s the concentrat ion of b a c t e r i a i n c e l l s / m l . V . I n t r a c e l l u l a r Phage Development 1. One-step growth experiment A 0.3 ml volume of an overnight cu l ture of P_. acidovorans #29 was added to 10 ml of f re sh L u r i a broth i n a 125 ml erlenmyer f l a s k . This c e l l suspension was incubated with aerat ion at 30 C 9 for 3 h r . 'The log phase c e l l s (approximate densi ty 1 x 10 c e l l s / m l ) g were d i l u t e d to a densi ty of 1 x 10 c e l l s / m l wi th fresh L u r i a b r o t h . A 10 ml sample of t h i s suspension was t rans ferred to a fresh 125 ml erlenmyer f l a s k which was incubated i n a shaker bath . A f t e r 5 m i n . , 0.1 ml of an appropr ia te ly d i l u t e d phage suspension was added to give a moi of 0.01. A f t e r 7 min the contents of the adsorpt ion f l a s k were d i l u t e d in to tubes of L u r i a broth i n a s t a t i c water-bath as fo l lows: 1/100 (tube 1) , 1/1,000 (tube A) and 1/10,000 (tube B) . 0.1 ml samples were p la ted at regular i n t e r v a l s from tubes A and B (the burst tube) . The number of unadsorbed phage was determined a f t er 8 min by cen tr i fug ing a 3 ml sample from tube A at 4 C for 5 min. at 20,000 x £ and p l a t i n g t r i p l i c a t e 0.2 ml samples of the supernatant. 2. E f f e c t of c u l t u r e age on the burst s i z e Eight 125 ml erlenmyer f la sks each conta in ing 10 ml of L u r i a broth were inocu la ted with an overnight cu l ture of P_. acidovorans 8 #29 to give an i n i t i a l c e l l densi ty of 1.2 x 10 c e l l s / m l . The f l a sks were incubated i n a shaker water-bath. At hourly i n t e r v a l s , g the contents of the f l a sks were d i l u t e d to give 1 x 10 c e l l s / m l and a modif ied one step growth experiment was performed using the d i l u t e d c u l t u r e . Quadrupl icate samples were p la ted from tube A at 10 m i n . , and from tube B at 150 min. A l s o , four samples were p la ted to determine the number of free phage p a r t i c l e s a f t er the adsorpt ion p e r i o d . The means of the plaque counts from these three sources were used i n the determination of the average burst s i z e . L y s i s I n h i b i t i o n 1. T u r b i d i m e t r i c method An overnight c u l t u r e of P . acidovorans #29 was used to inocu la te three 125 ml sidearm f lasks each conta in ing 10 ml of medium. A f t e r approximately one hour's incubat ion , 0W-14a+ was added to one f l a s k at a moi of 5, the second f l a s k was inoculated with 0W-14a at the same moi, whi le the t h i r d was maintained as an uninfected c o n t r o l . Every t h i r t y minutes t u r b i d i t y readings on the three f l a sks were made using a K l e t t Summerson P h o t o e l e c t r i c co lor imeter ( K l e t t Mfg. C o . , N . Y . ) equipped with a 540 f i l t e r . 2. One-step growth method o To 10 ml of log phase c e l l s (1 x 10 c e l l s / m l ) phage was added at a moi of 5. A f t e r a 10 min per iod for adsorpt ion , more phage was added, again at a moi of 5. A f t e r a fur ther 10 min, the f l a s k contents were d i l u t e d and p la ted i n the manner out l ined for the one-step growth experiment. V I I . Thermal I n a c t i v a t i o n Large broth tubes (2.2 x 20 cm) conta in ing 10 ml of L u r i a broth were placed i n deep water-baths at 50, 55, 60 and 65 C. A f t e r a 10 min. per iod for temperature e q u i l i b r a t i o n , a small volume of phage suspension, genera l ly l e s s than 0.2 ml , was added to each tube to give 3 1.5-3.5 x 10 pfu /ml (except the tube at 65 C, i n which the i n i t i a l t i t r e was 2 x 10 p f u / m l ) . The tubes were mixed v igorous ly and r e -placed immediately i n t h e i r appropriate water-baths. At regular i n t e r v a l s , 0.5 ml samples were removed to small tes t tubes c h i l l e d i n an ice^bucket. At the end of the experiment samples were p l a t e d . The rate constants for the thermal i n a c t i v a t i o n of 0W-14 were ca l cu la ted using the fo l lowing equation ( P o l l a r d , 1953): - k A = l o g e Nt/No T where k^ i s the rate constant; No the number of plaque forming u n i t s at zero time; and Nt the number of plaque forming u n i t s at any time T_. The ra te constants are expressed i n r e c i p r o c a l minutes (min ^) . In the c a l c u l a t i o n of the a c t i v a t i o n energies , the fo l lowing equation was employed (Neilands and Stumpf, 1958): E = l o g 1 0 (2.3 x R x T ^ ) k A 1 T - T l2 1 where E i s the Arrhenius energy of a c t i v a t i o n ; k . l and k „ 2 are the — - J A A rate constants for thermal i n a c t i v a t i o n at temperatures T^ and T^, r e s p e c t i v e l y ; and R i s the u n i v e r s a l gas constant . Because the heat of a c t i v a t i o n (AH*) d i f f e r s by about 600 c a l o r i e s from the Arrhenius energy of a c t i v a t i o n , the fo l lowing c o r r e c t i v e equat ion was appl ied (Dixon and Webb, 1964): AH* = E - RT V I I I . Sonic S e n s i t i v i t y of Phages Quadrupl icate 2.8 ml samples of phages S13, T l and 0W-14a+, each containing 1 x 10 p f u / m l , were subjected to t h i r t y second bursts of acoust ic energy from a Biosonic II (Bronwi l l S c i e n t i f i c , Rochester, N . Y . ) equipped with a needle probe. The frequency of u l t r a s o n i c output of t h i s instrument i s 20 Kcps - 400 cps. A s e t t i n g of 70 was used throughout, being equivalent to 87.5 Watts. The samples were c h i l l e d i n ice-water p r i o r to and during s o n i c a t i o n to reduce heat denaturat ion . Then they were d i l u t e d and 0.1 ml samples were p la ted on the appropriate hosts: E. c o l i B for S13 and T l , and P_. acidovorans #29 for 0W-14a+. IX. pH I n a c t i v a t i o n L u r i a broth was adjusted to pH 2-12 by the a d d i t i o n of IM HC1 or IM NaOH, and 4.5 ml amounts were added to test tubes i n an i c e -water bath . A f t e r 15 m i n . , 0.5 ml samples of phage preparat ions , s u i t a b l y d i l u t e d with p h y s i o l o g i c a l s a l i n e to 3 x 10^ p f u / m l , were added to each tube and the tubes shaken v i g o r o u s l y . A f t e r 30 min. incubat ion , 0.1 ml samples were p la ted from each tube. S e n s i t i v i t y to U l t r a v i o l e t L i g h t and Photoreac t iva t ion 1. UV i n a c t i v a t i o n A mixture of col iphage T l and 0W-14a+, each at a t i t r e of 1 x 10 p fu /ml , was prepared using L u r i a broth as the d i l u e n t . One m i l l i l i t r e samples of the phage suspension were placed i n d i s -posable p l a s t i c P e t r i dishes ( M i l l i p o r e F i l t e r C o r p . , Bedford, Mass.) of 2.5 cm diameter and i r r a d i a t e d at a dis tance of 50 cm with a General E l e c t r i c 15 Watt germic ida l lamp ( p r i n c i p l e wave-length at 2575 A ) . The samples were s t i r r e d continuously during the i r r a d i a t i o n per iod with a magnetic s t i r - b a r and s t i r r e r . The i r r a d i a t e d samples were then d i l u t e d and p la ted under yel low, nonphotoreact ivat ing , l i g h t . P_. acidovorans #29 was used as the host for 0W-14a+ and E. c o l i B was used for T l . The p lates were incubated at 30 C and 37 C, for 0W-14a+ and T l r e s p e c t i v e l y , i n l i g h t - p r o o f boxes. The ra te constants were c a l c u l a t e d from the l i n e a r region of the graphed r e s u l t s using the fo l lowing equation ( P o l l a r d , 1953) - k u v = l o g e Nt/No T where k i s the ra te constant for u l t r a v i o l e t l i g h t i n a c t i v a t i o n ; uv No i s the number of pfu/ml at the "beginning" of the l i n e a r reg ion of i n a c t i v a t i o n ; and, Nt i s the number of pfu/ml remaining a f t e r seconds i r r a d i a t i o n . 2. Photoreac t iva t ion In order to obta in photoreac t iva t ion , the U V - i r r a d i a t e d phage was p la ted under white l i g h t , and the uninverted p la tes were incubated 12 inches from a lamp f i t t e d with twin Westinghouse 15 Watt Cool White or General E l e c t r i c 15 Watt Dayl ight f luorescent bulbs ( p r i n c i p l e wavelength approximately 5700 A i n both cases) . I r r a d i a t i o n was continued overnight at the appropriate incubat ion temperatures. The photoreact ivable sector (Dulbecco, 1950) was c a l c u l a t e d us ing the fo l lowing equation: P = 1 - k s _p_ k uv where P i s the proport ion of the phage photoreact ivated; k i s s P_ the ra te of U V - i n a c t i v a t i o n of the phage as measured by incubat ion i n the l i g h t ; and, k i s the ra te constant for U V - i n a c t i v a t i o n ° uv as measured by incubat ion i n the dark. 22 X I . Phage Res i s tant Mutants and the C a r r i e r State 1. I s o l a t i o n of r e s i s t a n t s t r a i n s S u f f i c i e n t 0W-14a was added to 10 ml of a log phase cu l ture of P_. acidovorans #29 (1 x 10 c e l l s / m l ) to give a moi of 5. The mixture was incubated for 10 min. at 30 C, fo l lowing which 0.1 ml samples were spread on L u r i a agar p l a t e s . The co lonies which appeared on the p la tes a f t er incubat ion at 30 C for 48 hr were picked with s t e r i l e tooth p icks to overlay Q plates conta in ing a high concentrat ion (5 x 10 pfu /over lay) of 0W-14a. These p la tes were incubated overnight at 30 C . Those co lonies which grew best were t rans ferred to tubes of tryptone broth and regrown with aerat ion at 30 C . These broth cu l tures were s e r i a l l y t r a n s f e r r e d four times to d i l u t e out any r e s i d u a l 0W-14a. The cu l tures were then tested f o r ^ t h e i r res i s tance to 0W-14a+ and 0W-14a. The presence of an i n t r a c e l l u l a r form of the phage was checked for by spot t e s t i n g the cu l tures on overlays conta in ing P_. acidovorans #29. The r e s i s t a n t , non-phage c a r r y i n g cu l tures were p u r i f i e d by severa l cyc les of spreading and s i n g l e colony s e l e c t i o n . Because the continuence of the c a r r i e r s tate depends upon phage r e i n f e c t i o n of s e n s i t i v e c e l l s , those cu l tures confirmed as being phage in fec ted were p u r i f i e d by s treaking-out on overlays containing 0W-14a. The co lonies which arose were inoculated in to tubes of L u r i a broth and t rans ferred s e r i a l l y as described above. 2. Microscopy a. M o t i l i t y The m o t i l i t y of the cu l tures was determined by the hanging drop method using log phase c e l l s and a magni f i cat ion of 400 x. b . Phase contrast microscopy The samples were prepared e s s e n t i a l l y as described by Shaw (1968), and photographed with a Zeiss microscope using Adox KB14 f i l m (Adox Fotowerkej F r a n k f u r t / M a i n ) . 3. Confirmation of the c a r r i e r s tate " C a r r i e r " cu l tures can be d i f f e r e n t i a t e d from lysogenic cu l tures by the fac t that i n the former case only a p o r t i o n of the c e l l s a c t u a l l y carry the phage genome, whi le i n the l a t t e r case, a l l the c e l l s are " infected". To show which s tate ex i s ted i n the phage in fec ted c e l l s of P_. acidovorans, l og phase c e l l s of a " c a r r i e r " cu l ture were harvested by c e n t r i f u g a t i o n at 4 C, and the c e l l 24 p e l l e t washed three times with an equal volume of i c e - c o l d L u r i a b r o t h . The washed and resuspended c e l l suspension was d i l u t e d to 10" , and three 1.0 ml samples were p la ted by the Standard P la te Count Method (Standard Methods, 1960). Three 0.1 ml samples were p la ted from each of the 10" , 10 and 10~ d i l u t i o n s by the overlay method with P_. acidovorans #29. A f t e r appropriate incubat ion periods the t o t a l numbers of plaques and co lonies were tabulated . X I I . Recombination An overnight c u l t u r e of P_. acidovorans #29 was inoculated in to fresh L u r i a broth and regrown to mid-long phase. This cu l ture was d i l u t e d to give 8 a densi ty of 1 x 10 c e l l s / m l , and 5 ml of t h i s suspension was t rans ferred to a s t e r i l e , screw-capped tube. Phages 0W-14a+ and 0W-14h were added at a moi of 5 for each. A f t e r 10 min incubat ion on a tube r o l l e r , the c e l l s were harvested by c e n t r i f u g a t i o n and the supernatant d i s -carded. The c e l l p e l l e t was resuspended i n 5 ml of f resh L u r i a broth and incubat ion was continued for a fur ther two hours . A few drops of chloroform were then added to the tube, which was shaken v igorous ly for a minute. The tube contents were centr i fuged to remove b a c t e r i a l c e l l s and d e b r i s , and the supernatant was d i l u t e d and p la ted by the overlay method on P_. acidovorans #29 and on a mixed i n d i c a t o r composed of the l a t t e r organism and P_. acidovorans #29-5. A f t e r overnight incubat ion at 30 C the recombinants were i d e n t i f i e d and tabulated . 25 X I I I . P h y s i c a l and Chemical Propert i e s of 0W-14 DNA 1. I s o l a t i o n of DNA a. Perchlorate method ( F r e i f e l d e r , 1966) A 7.5 M s o l u t i o n of sodium perchlorate i n 0.001 M EDTA was adjusted to pH 7-9 by the dropwise a d d i t i o n of IN NaOH, and 50 ml of i t was added to 113 ml of a p u r i f i e d phage suspension 12 (10 pfu/ml) with s t i r r i n g . The f i n a l concentrat ion of perch lorate was approximately 2.3 M. The s o l u t i o n was s t i r r e d slowly at room temperature for 30 m i n . , fo l lowing which the p r e c i p i t a t e d m a t e r i a l was removed by c e n t r i f u g a t i o n . The n u c l e i c a c i d was p r e c i p i t a t e d from the supernatant by the a d d i t i o n of two volumes of 95% ethanol , and the f ibrous m a t e r i a l c o l l e c t on a magnetic s t i r - b a r . This m a t e r i a l was washed once with 66% ethanol and then d i s so lved i n 0.1 x SSC (0.015 M NaCl - 0.0015 M N a ^ i t r a t e buf fer pH 7 .0) . A f t e r s evera l days s t i r r i n g at 4 C, the i n s o l u b l e m a t e r i a l was removed by c e n t r i f u g a t i o n and the n u c l e i c a c i d p u r i f i e d fur ther by the i sopropanol p r e c i p i t a t i o n method of Marmur (1961). b. Tryps in-phenol method A p a r t i a l l y p u r i f i e d preparat ion of 0W-14 (10 pfu/ml) was digested with 0.1 mg/ml t r y p s i n 1-300 ( N u t r i t i o n a l B i o --3 chemicals C o r p . , C leve land, Ohio) i n the presence of 10 M CaCl^ for 8 hr at 37 C . Then the mixture was kept at 4 C for severa l days. The h igh ly viscous s o l u t i o n , containing less than 1% of the t o t a l pfu input , was deprote in ized by shaking with b u f f e r - s a t u r a t e d phenol . The DNA was p r e c i p i t a t e d by the a d d i t i o n of two volumes of 95% ethanol , r ed i s so lved i n SCC (0.15 M NaCl - 0.015 M Na^ci trate buf fer pH 7 .0) , and p u r i f i e d fur ther by the i sopropanol p r e c i p i t a t i o n method (Marmur, 1961). c. I s o l a t i o n of P_. acidovorans DNA P_. acidovorans DNA was p u r i f i e d by a mod i f i ca t ion of the sodium dodecyl su l fa te -pheno l method (Mandel, 1966) from 17.8 g, wet weight, of c e l l s . A f t e r the i n i t i a l d e p r o t e i n i z a t i o n with phenol , the aqueous phase was deprote in ized s i x consecutive times by shaking s lowly at 4 C with chloroform - isoamyl a l c o h o l (24; 1, v / v ) . The DNA was p r e c i p i t a t e d from the aqueous l ayer with two volumes of absolute ethanol and d i s so lved i n SSC b u f f e r . A f t e r removing r e s i d u a l phenol by e x t r a c t i n g severa l times with 0.1 volume of e ther , the s o l u t i o n was treated with r ibonuclease (DN'ase f r e e ) , 50 yg /ml , f or 30 min. at 37 C, a f t e r which the depro te in i z ing step was repeated once. The DNA was p u r i f i e d fur ther by p r e c i p i t a t i n g twice with i s o -propanol (Marmur, 1961). General proper t i e s of the phage .nucle ic a c i d a. UV spectrum The UV spectrum of 0W-14 DNA d i s so lved i n 0.1 x SSC was obtained using a Unicam SP.800B Spectrophotometer (Unicam Instruments L t d . , Cambridge, England) . b . C a l c u l a t i o n of DNA concentrat ion DNA concentrat ions were estimated using an e x t i n c t i o n co-2 e f f i c i e n t of 20 cm /mg based upon the o p t i c a l densi ty at 260 mu (Lee and Boez i , 1966). c. C o l o r i m e t r i c assays The diphenylamine test (Ashwell , 1957) was used to determine the presence of DNA, whi le the o r c i n o l assay (Ashwell , 1957) was used for RNA determinations; d. Enzyme s e n s i t i v i t y Phage DNA was adjusted to a concentrat ion of, 50 ]ig/ml i n SSC. Three tubes were set-up each conta in ing 0.84 ml of DNA and 0.05 ml of SSC-0.018 M MgCl buf fer pH 7.0. To the f i r s t tube was added 0.01 ml of DN'ase; to the second 0.01 ml of RN'ase; and, to the t h i r d 0.01 ml of SSC. In each case the f i n a l enzyme concentrat ion was 10 ]ig/ml. The tubes were i n -cubated at 37 C for 30 m i n . , then c h i l l e d on i c e . To each tube was added 0.1 ml of 30% HCIO^ to p r e c i p i t a t e undegraded n u c l e i c a c i d . A f t e r a fur ther 15 min. the tubes were c e n t r i -fuged and the OD 260 of the supernatants determined. 3. M e l t i n g temperatures A Beckman Model DUR recording quartz spectrophotometer (Beckman Industr ies I n c . , F u l l e r t o n , C a l i f . ) equipped with a G i l f o r d 2000 m u l t i p l e sample adsorbance recorder ( G i l f o r d Instrument Laborator ies I n c . , O b e r l i n , Ohio) was the bas i c u n i t used i n the determination of the mel t ing temperatures (Tm) . A thermos ta t i ca l ly c o n t r o l l e d waterbath with pump (Haake constant temperature c i r c u l a t o r , Model F) was used to heat and c i r c u l a t e the ethylene g l y c o l to the inner thermospacers f l a k i n g the cuvette chamber. The rate of heat ing of the f l u i d was automat ica l ly c o n t r o l l e d by a v a r i a b l e speed motor and c o n t r o l l e r (Gerald K. H e l l e r C o . , Las Vegas, Nevada). P r i o r to the Tm determination the phage DNA was d ia lyzed over-night at 4 C against 300 volumes of 0.1 x SSC. The d ia lyzed DNA so lu t ions were d i l u t e d i n 0.1 x SSC to obta in samples with 0.4 -0.6 OD 260 un i t s per m i l l i l i t r e . Three m i l l i l i t r e samples of the so lut ions were t rans ferred to g lass-s toppered quartz cuvettes of 1 cm l i g h t path . These were then placed i n the cuvette chamber along with a c o n t r o l cuvette containing 0.1 x SSC b u f f e r . The temperature was r a i s e d r a p i d l y to approximately 50 C, then the regu la tor was adjusted to give a rate of increase of 1 C every 4 min. 4. Buoyant densi ty a. General method The method used was e s s e n t i a l l y that of Mandel, Sch i ldkraut and Marmur (1968), us ing a Beckman Model E a n a l y t i c a l u l t r a -centr i fuge (Beckman Instruments I n c . , Palo A l t o , G a l i f . ) at 44,000 rpm for 22 hr at 20 C. _E. c o l i DNA (Worthington) was used as the densi ty marker and i t was assumed that i t s densi ty was 1.710 g / c c 3 (Mandel et a l . , 1968). Kodak Commercial F i l m (Eastman Kodak C o . , Rochester, N . Y . ) was used for the UV photography, and the negatives were developed using D - l l developer. The f i lms were scanned using a Joyce-Loeb l double-beam recording microdensitometer. The buoyant densi ty of the phage DNA was determined using the fo l lowing equation (Mandel et a l . , 1968): p = 1.710 - 0.0089 ( r 2 - r 2 ) g / c c 3 o where r and r are the peak distances from the centre of o r o t a t i o n of the phage DNA and the _E. c o l i marker, r e s p e c t i v e l y . The GC content of the phage DNA was c a l c u l a t e d us ing the formula of S c h i l d k r a u t , Marmur and Doty (1962): GC = p - 1.660 0.098 b . Preparat ion of denatured phage DNA for buoyant densi ty a n a l y s i s . i . A l k a l i n e denaturat ion . A small sample of 0W-14 DNA i n DNA i n SSC was adjusted to pH 12.5 by the dropwise a d d i t i o n of IN NaOH. A f t e r standing at 22 C for 30 m i n . , the s o l u t i o n was d i l u t e d by the a d d i t i o n of 0.02 M T r i s - H C l buf fer pH 8.5, and adjusted to pH 8.5 by the c a r e f u l a d d i t i o n of 0.2 M HC1. This s o l u t i o n was d i l u t e d fur ther with buf fer to a concentrat ion of 50 ug /ml . i i . Thermal denaturat ion . Phage DNA i n SSC was d i l u t e d 1/10 in to d i s t i l l e d water, and a small sample heated at 100 C for 10 min. The so lu t ion , was r a p i d l y c h i l l e d on i c e , fo l lowing which i t was d i l u t e d to 50 ug/ml with 0.02 M T r i s - H C l buf fer pH 8.5. 5. Chemical composition of the phage DNA. a. Hydro lys i s i . Hydro lys i s to free bases. Phage DNA i n SSC was p r e -c i p i t a t e d from s o l u t i o n by the add i t i on of three volumes of 95% ethanol . The p r e c i p i t a t e was washed with anhydrous acetone and d r i e d . A 1 mg sample was placed i n a quartz -g lass h y d r o l y s i s tube (9 mm externa l diameter; 6 mm i n t e r n a l diameter) , sealed at one end, and 0.71 ml of 88% formic a c i d was added. A f t e r f reez ing the contents of the tube i n a mixture of d r y - i c e and acetone, the tube was sealed under reduced pressure . Hydro lys i s was c a r r i e d out for 45 min at 175 C, with the h y d r o l y s i s tube i n s i d e a c a s t - i r o n guard tube i n case of explos ion . A f t e r coo l ing to room temperature, the contents of the tube were again frozen i n a mixture of d r y - i c e and acetone. Gentle heat ing of the end of the tube was used to permit the c o n t r o l l e d re lease of the i n t e r n a l pressure . The tube contents were evaporated to dryness and the res idue was d i s so lved i n 50 u l of 20% isopropanol - 0.1 N HC1. 32 i i . Hydro lys i s to free saccharides . The DNA was prepared for h y d r o l y s i s as descr ibed above. A 1 mg sample of the DNA was placed i n a h y d r o l y s i s v i a l together with 1 ml of 1 N HC1. The v i a l was sealed under reduced pressure , and h y d r o l y s i s was c a r r i e d out at 100 C for 90 min. A f t e r c o o l i n g , the contents of the v i a l were t r a n s -f e r r e d to a centr i fuge tube. To t h i s was added an equal volume of a s l u r r y of acid-washed charcoal (Nori t A ) . The mixture was swir led gently for 5 min. at room temperature, then centr i fuged at low speed. The supernatant was removed c a r e f u l l y wi th a Pasteur p ipe t t e and evaporated to dryness. The res idue was d i s so lved i n 100 y l of 10% i sopropano l . i i i . Nature of the attachment s i t e of the saccharide compo- nent of 0W-14 DNA. The method of A l e g r i a and Kahan (1968) was used to determine whether or not the saccharide component of 0W-14 DNA was cova lent ly bonded to the DNA. The phage DNA (214 yg) i n SSC was p r e -c i p i t a t e d by the a d d i t i o n of an equal volume of i c e - c o l d 10% t r i c h l o r o -a c e t i c a c i d (TCA). A f t e r c h i l l i n g the a c i d i f i e d s o l u t i o n at 0 C for 10 m i n . , the p r e c i p i t a t e d mater ia l was c o l l e c t e d by c e n t r i f u g a t i o n . The supernatant was c a r e f u l l y decanted and extracted with e t h y l ether to remove TCA. The p e l l e t was resuspended i n a volume of water equal to that of the supernatant, and the presence of carbohydrate i n the two f r a c t i o n s was assayed for by the Anthrone method (Sp iro , 1966). b. Paper chromatography i . Separat ion of bases. The descending method of paper chromatography on Whatman #1 paper was used to separate the free bases. Three solvent systems were employed: (a) _t - butanol - 88% formic a c i d -water (16:1:4, v /v) (Roberts, 1961); (b) n - butanol - methanol - water -concentrated ammonium hydroxide (60:20:20:1, v /v) (Randerath, 1965); (c) i so -propano l - concentrated h y d r o c h l o r i c a c i d - water (65:17:18, v /v) (Bendich, 1957). A f t e r i r r i g a t i o n of the paper the chromatogram was d r i e d and the bases were located by t h e i r f luorescence i n shortwavelength (ca . 254 my ) U V - l i g h t . i i . Separation of sacchar ides . Two solvent systems were used to separate sugars on Whatman #1 paper: (a) e t h y l acetate - p y r i d i n e -water (120:50:40, v / v ) ; (b) i so -propano l - water (160:40, v /v) (Smith, 1960). The sugars were located by developing the chromatogram with e i t h e r s i l v e r n i t r a t e reagent or ani l ine-d iphenylamine reagent (Smith, 1960). c. Quant i ta t ive es t imat ion of 0W-14 DNA base composition Phage DNA (500 yg) was hydrolyzed as described above ( 5 . a . i . ) . The yel low res idue remaining a f t er evaporation of the formic a c i d was red i s so lved i n 100 y l of 10% i so -propanol - 0.1 N HC1. The t o t a l hydrolysate was appl ied to a sheet of Whatman #1 chromato-graphy paper. Separat ion of the bases was accomplished by using solvent system C. A f t e r time s u f f i c i e n t to ensure the best poss ib le separat ion of the bases, the chromatogram was removed from the tank and d r i e d . The areas showing f luorescence were cut out as w e l l as corresponding areas from an unspotted area of the chromato gram. The l a t t e r areas served as the c o n t r o l s . The f luorescent areas and the contro l s were e luted with 0.1 N HC1, with the exception of thymine, i n which case 0.01 N HC1 was used. E l u t i o n was c a r r i e d out i n a humidi f ied container using the second method described by Heppel (1968). The eluates were adjust ed to a uniform volume and spectra were obtained for the bases i n a c i d i c and a l k a l i n e s o l u t i o n s . The concentrat ions of the i n d i v i d u a l bases were c a l c u l a t e d from t h e i r molar e x t i n c t i o n c o e f f i c i e n t s i n a c i d i c s o l u t i o n : 3 3 Adenine 13.1 x 10 at 265.5 my; Cytosine 10.0 x 10 at 276 my; Guanine 11.4 x 10 3 at 248.5 my; Thymine 7.89 x 10 3 at 264.5 my (Besch and Goldwyn, 1966). RESULTS AND DISCUSSION S e c t i o n I . I s o l a t i o n and General Propert i e s of the Phage 1. I s o l a t i o n Various Pseudomonas phages were i s o l a t e d by enrichment c u l t u r e from raw sewage or ac t iva ted sludge (Table I V ) . At present , only the phage ac t ive against P_. acidovorans #14 has been inves t iga ted i n any depth. The o r i g i n a l enrichment contained only one plaque type: a s m a l l , d i s c r e t e , c l e a r plaque. However, when the o r i g i n a l enrich=-ment was r e t i t r e d a f t er approximately one month at 4 C , i t was noted that the number of c l e a r plaque formers had decreased markedly, and the presence of a few haloed plaques was observed. The l a t t e r were chosen for fur ther study and given the name 0W-14. Since i t l a t e r t r a n s p i r e d that the two phages were r e l a t e d the c h a r a c t e r i s t i c s of both were i n v e s t i g a t e d . L y t i c agents were a lso i d e n t i f i e d upon Mitomycin C induc t ion (0.05 ug/ml Mitomycin C i n L u r i a broth) of P_. acidovorans and P_. t e s t o s t e r o n i c u l t u r e s . Also some examples of c r o s s - r e a c t i v i t y between these two groups of organisms was obtained wi th the induced c u l t u r e s . The nature of these agents, whether phage or b a c t e r i o c i n , was not fur ther i n v e s t i g a t e d . 36 Table IV. Prev ious ly unreported phages i s o l a t e d from raw Vancouver sewage. Organism Phage i s o l a t e d P_. acidovorans #29 + —" acidovorans #14 + P_. acidovorans 15666 + P_. acidovorans 15667 + P_. t e s t o s t e r o n i #78 + P. t e s t o s t e r o n i #138 + P. t e s t o s t e r o n i 11996 + P_. mucidolens 4687 + P_. putrefac iens (Hammer) + P_. convexa 795 + P_. synxantha 796 + P. taetro lens 4683 + P. o v a l i s 950 + 2. Plaque morphology F ive d i s t i n c t types of plaques were observed: (a) a - type: small plaque, approximately 2 mm i n diameter, with a very small c l ear centre and a f a i r l y t u r b i d ha lo; the halo appeared f a i n t l y r inged , (b) a-type: plaque approximately 1.5 mm i n diameter with a wide c e n t r a l c l e a r zone and a very small i n d i s t i n c t ha lo , (c) h-type: a spontaneously a r i s i n g host range mutant of a_ which produced the same type of plaque, (d) t - type: the same s i z e as af*", with a very small c l e a r c e n t r a l zone bordered by a s i n g l e d i s t i n c t c l e a r r i n g and a t u r b i d ha lo , (e) sc - type: approximately the same s i z e as a_+, but with a l a r g e r c l e a r zone, surrounded by a f a i r l y t u r b i d ha lo ; the halo sometimes appeared as f a i n t , concentr i c , r i n g s . F i g . 1 shows the d i f ference between the a + and a- type plaques. Host-range of 0W-14a+ and d e r i v a t i v e s . Organism P_. acidovorans #29 I?, acidovorans #14 P_. acidovorans #114 P_. acidovorans #146 P_. acidovorans AK-11 P. acidovorans 15666 P_. acidovorans 15667 P_. acidovorans 15668 P_. t e s t o s t e r o n i #78 P_. t e s t o s t e r o n i #138 P_. t e s t o s t e r o n i 11996 P_. aeruginosa 9027 P_. aeruginosa 9721 P. o v a l i s 950 P_. f r a g i 4975 P_. convexa 795 E . c o l i k l2 E_. c o l i B * nd - not tested L y s i s by: 0W-14a+ 0W-14a 0W-14h + + + + + -+ + -+ + nd* nd + nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd 38 3. Host range 0W-14 showed a l i m i t e d host range, l y s i n g only three of the seven d i f f e r e n t s t r a i n s of P_. acidovorans tested and none of the r e l a t e d organism, P. t e s t o s t e r o n i . The c l e a r plaque mutant, 0W-14a, had an i d e n t i c a l host range, and the plaques on the three hosts were always haloed for a+ and c l e a r for a_. The host range mutant, 0W-14h, d id not l y s e P_. acidovorans #14 nor P_. acidovorans AK-11 -hosts for the parent s t r a i n - but d id ly se P_. acidovorans #29 and _P. t e s t o s t e r o n i #78 (Table V ) . I t i s i n t e r e s t i n g to note that 0W-14h d id not l y se P_. t e s t o s t e r o n i 11996, s ince P_. t e s t o s t e r o n i 78 and the former are supposedly i d e n t i c a l organisms (S tan ier , P a l l e r o n i and Doudoroff, 1966). 4. R e l a t i v e e f f i c i e n c y of p l a t i n g The determination of the r e l a t i v e e f f i c i e n c y of p l a t i n g (REP) on var ious host s t r a i n s appeared to be dependent to some extent , upon the s t r a i n of P_. acidovorans used to prepare the phage stock (Table V I ) . For a given phage stock the highest t i t r e s were always obtained when the phages were t i t r e d on P_. acidovorans #29. The plaques formed on s t r a i n AK-11 were small and i n d i s t i n c t , so that the REP could not be determined accurate ly with t h i s s t r a i n . Table V I : Re la t ive e f f i c i e n c y of p l a t i n g of 0W-l4a+ and 0W-14a Phage Growing host P. acidovorans #14 P. acidovorans #29 0W-14a+ P. acidovorans #14 3.3 x 10 5.0 x 10 -1 P l a t i n g hosts P. acidovorans #29 0W-14a P l a t i n g hosts P_. acidovorans #14 5.0 x 1 0 - 1 3.3 x 1 0 _ 1 P. acidovorans #29 5. Phage p u r i f i c a t i o n Various methods were used i n an attempt to p u r i f y 0W-14. These inc luded ammonium s u l f a t e p r e c i p i t a t i o n , DEAE-ce l lu lose column chromatography, adsorpt ion chromatography on magnesium pyrophosphate ge l (Sch i to , 1966), and d i f f e r e n t i a l c e n t r i f u g a t i o n . The f i r s t three of these methods gave very poor recover ies of p f u . D i f f e r e n t i a l c e n t r i f u g a t i o n proved most use fu l because of i t s s i m p l i c i t y and the good recover ies obtained, but i t was not without i t s p i t f a l l s s ince the phage p a r t i c l e s suspended i n buf fer were almost q u a n t i t a t i v e l y sedimented i n 10 min. at 6000 x j». Lee and Boezi (1966) found i t necessary to centr i fuge at 16,000 x for 2 h r . to sediment P_. put ida phage gh-1. This phenomenon was inves t iga ted fur ther i n an attempt to obta in d isperse phage suspensions. The r e s u l t s of a number of experiments are presented i n Table V I I . I t was.shown subsequently, that the pH of the b u f f e r , i n the range of 6.5 to 8 .1 , had no e f fec t on the sedimentation proper t i e s of the phage p a r t i c l e s . The a d d i t i o n of ethanol to a f i n a l concentrat ion of 16% r e s u l t e d i n the greatest increase i n nonsedimentable plaque forming u n i t s . Such preparat ions s t i l l exh ib i ted a high degree of l i g h t s c a t t e r i n g . Under the e l e c t r o n microscope, a great number of phage aggregates were observed, but very l i t t l e b a c t e r i a l debris was present . The i n a c t i v a t i o n of phage i n lysates i s caused usua l ly by the adsorpt ion of the p a r t i c l e s to fragments of c e l l w a l l s . However, p a r t i c l e s may be i n a c t i v a t e d by i n t e r a c t i o n with so luble components of the host c e l l . Burnet and Freeman (1937) observed that crude b a c t e r i a l p o l y -saccharide preparat ions would i n h i b i t homologous phage. However, i n a c t i v a t i o n d i d not depend n e c e s s a r i l y upon a s t e r e o s p e c i f i c r e l a t i o n s h i p between the two e n t i t i e s s ince Ashenburg et a l , (1940) found that s a l i n e suspensions of Aerobacter capsular po lysacchar ide , as w e l l as s t a r c h , glycogen and gum a r a b i c , were as e f f e c t i v e as the homologous capsular polysaccharide i n i n h i b i t i n g plaque formation by a K l e b s i e l l a pneumonia phage. A r e l a t i o n s h i p between these substances and the i n a c t i v a t i o n by aggregation of 0W-14 does not appear l i k e l y s ince P_. acidovorans does not s tore i n t r a c e l l u l a r polysaccharide (Stan ier , P a l l e r o n i and Dourdoroff , 1966). The existence of e x t r a c e l l u l a r sl ime or capsular polysaccharide i n th i s organism has not been examined. C e l l w a l l l ipopo lysacchar ides can i n a c t i v a t e col iphages T3 , T4 and T7, and the F e l i x 0-1 phage of Salmonella minnesota. I n a c t i v a t i o n i n the l a t t e r case appeared to be due to adsorpt ion fol lowed by t a i l contrac t ion and phage DNA e j e c t i o n (Lindberg , 1967). L ipopolysacchar ide i s o l a t e d from P_. acidovorans by the phenol-water method (Osborn, 1966) appeared not to i n a c t i v a t e . 0 W - 1 4 . Proteinaceous m a t e r i a l s , other than antiphage and normal sera , .43 f a b l e V I I : Aggregation phenomena of p a r t i a l l y p u r i f i e d suspensions of 0W-14. Addi t ions % pfu nonsedimentable None 11 D i s t i l l e d water 2:10 (v/v) 13 D i s t i l l e d water 9:1 (v/v) 68 95% ethanol 1:10 (v/v) 55 95% ethanol 2:10 (v/v) 82 95% ethanol 3:10 (v/v) 72 10" 2M EDTA pH 7 1:10 (v/v) 11 0.05M T r i s pH 7. 4 9:1 (v/v) 26 0.05M KP0 4 pH 7. 4 9:1 (v/v) 27 Phage 0W-14, suspended i n 0.05M Tri s (HCI) -O.OIM C i t r i c a c i d -0.005M NaCl buf fer pH 8 .1 , at a t i t r e of 1.1 x 1 0 1 2 pfu/ml was treated at 4 C as o u t l i n e d above. A f t e r 1 hr suspensions were centr i fuged at 6000 x j> for 10 m i n . , and the pfu remaining i n the supernatant were t i t r e d . can have antiphage a c t i v i t y . B r i n t o n , B u z z e l l and Lauf fer (1954) found that p a r t i a l l y p u r i f i e d preparat ions of EL c o l i B/6 f i laments ( p i l i ) markedly i n h i b i t e d the plaque forming a b i l i t y of col iphage T2, T4, T5 and T6. This p o s s i b i l i t y i s ru l ed out i n the present case s ince no fore ign p a r t i c u l a t e matter appeared to be respons ib le for the aggregation of 0W-14 ( F i g . 8 ) Das and M a r s h a l l (1967) showed that case in i n h i b i t e d the a b i l i t y of a S_. aureus phage to form plaques . Though case in was not present , some s i m i l a r i t i e s between t h e i r observations and the present r e s u l t s are apparent. With both S^ . aureus phage and 0W-14a, an increase i n the t i t r e of the preparat ions was observed when the pH was adjusted to 4-5, suggesting that the adsorpt ive agent had an i s o e l e c t r i c point i n th i s r e g i o n , and that i t s n e u t r a l i z a t i o n re leased the bound phage. However, t h i s does not exp la in the r e s u l t s with 0W-14a+ (see Sect ion I X ) . The fac t that ethanol d i s soc ia t e s the l a r g e r aggregates a lso suggests an i o n i c i n t e r a c t i o n , s ince i t i s known that t h i s solvent lowers the d i e l e c t r i c constant of s o l u t i o n s . Examples of unexplained spontaneous aggregation are to be found i n the l i t e r a t u r e . Putnam (1951) observed that p a r t i c l e s of T6 tended to aggregate and s e t t l e out i f t h e i r concentrat ion was 12 greater than about 4 x 10 p f u / m l , whi le more d i l u t e suspensions were d i sperse . I t was observed wi th 0W-14 that d i l u t i o n of the phage 1/10 in to buf fer had l i t t l e e f f ec t on the number of plaque forming un i t s which remained nonsedimentable. Lanni (1958) observed that Table VIII: Quantitative d i s t r i b u t i o n of plaque morphology mutants i n d i v i d u a l 0W-14a+ plaques. Clone # T o t a l PFU ( x 10" 6) ( x Min. 1 130 57 2 37 17 3 267 220 4 399 44 5 243 42 6 94 102 7 50 10 8 318 71 9 448 145 10 221 62 11 308 115 12 39 15 13 64 10 14 78 23 15 20 1 16 119 73 17 48 .74 18 55 24 19 29 3 20 80 41 Mutation frequency r 3) ( x io 4 ) Max. Min. Max. 270 4.38 20.77 55 4.59 14.86 560 8.24 20.97 117 1.10 2.93 103 1.73 4.24 155 10.85 16.49 34 2.00 6.80 135 2.23 4.25 590 3.24 13.17 160 2.81 7.96 159 3.73 5.16 33 3.85 8.46 38 1.56 5.94 55 2.95 7.05 11 0.50 5.50 147 6.15 12.35 104 15.42 21.67 38 4.36 6.91 16 1.03 5.52 87 5.13 10.88 Means: 152.4 57.5 143.4 3.77 9.41 when p u r i f i e d suspensions of T5 i n buf fer were d i l u t e d in to n u t r i e n t broth and stored at 4 C the plaque count decreased 80-90% i n severa l weeks. This t i t r e was res tored to the s t a r t i n g value by-heat ing the suspension at 44 C for 10 min. 6. Plaque morphology mutants When grown on P_. acidovorans #14, 0W-14a+ mutated at a high frequency to the c l e a r plaque forming type (Table V I I I ; F i g . 2) . I f the c l e a r areas were picked and p la ted out, c l e a r and haloed plaques were obtained i n approximately equal numbers. The . -4 minimal estimate of the mutation frequency was 3.8 x 10 ; the -4 maximum estimate was 9.4 x 10 . In the case of two c lones , -3 frequencies of 2.2 x 10 were observed. When P_. acidovorans #29 was used as the host the mutation was not observed. Because of t h i s mutation and the higher burst s i z e of the a-type phage (see Sect ion I I I ) , great d i f f i c u l t y was encountered i n prepar ing a high t i t r e l y s a t e of predominantly the a+-type phage when P_. acidovorans #14 was used as the host . The spontaneous mutation of a t u r b i d to a c l e a r plaque type i s a qui te common occurrence, having been seen with a number of phages from d i f f e r e n t sources. Phage 7 m of P_. aeruginosa mutates to a c l e a r plaque type at a frequency of 10 - ^ to 10 (Feary, F i s h e r and F i s h e r , 1964). For col iphage T2H, the observed 100 20 0 300 400 5 . T O T A L P F U (x IO" 6 ) F i g . 2. Graph ica l representat ion of the occurrence of c l e a r plaque. mutants as a funct ion of the t o t a l number of plaque forming un i t s i n i n d i v i d u a l plaques. 0 - i n d i v i d u a l experimental r e s u l t s . x - x - p l o t of the mean. frequency was approximately 10 (Hershey, 1946). Hydrogenomonas  f a c i l i s phage 0HF shows th i s type of mutation (Pootjes, 1964), and Murphy (1954) reported a high frequency of mutation of the t u r b i d w i l d type phage plaque type of B a c i l l u s megatherium (s ic ) to a c l e a r plaque type. Thus the values obtained for 0W-14 f a l l w i t h i n the general range reported for t h i s type of mutation. The exactness of these r e s u l t s i s i n doubt s ince d i f f i c u l t y was encountered i n d i s t i n g u i s h i n g the c l e a r plaques on lawns of the haloed plaque type. In a d d i t i o n , the d i f ferences i n adsorpt ion k i n e t i c s (see Sect ion II) and burst s i zes (see Sect ion III) between the two phage types would inf luence the r e s u l t s . The only accurate , but h i g h l y l a b o r i o u s , means of determing the true mutation frequency i s by the s i n g l e burst method ( L u r i a , 1951). The a-type of the phage appeared to be more s tab le than the a-t—type s ince revers ion was r a r e l y , i f ever, observed i n t h i s type. 7. E l e c t r o n microscopy Phage 0W-14 had a regu lar i cosahedra l head 87 my i n diameter. The p a r t i c l e had a t a i l which i s about 20 my i n diameter and 140 my long . Subunits could be seen i n the t a i l s of some of the p a r t i c l e s ( F i g . 3 ) . In some cases, the t a i l appeared to be c o n s t r i c t e d where i t j o ined the head. The baseplate was approximately .50 my i n diameter, and appeared to carry t a i l pins ( F i g . 4) about F i g . 3. P_. acidovorans phage 0W-14, PTA, x 245,200, sca le 100 Fig. 3. F i g . 4. 0W-14 t a i l d e t a i l , and empty heads, PTA, x 367,500, sca le 100 my. 0W-14 showing contracted sheath, and exposed core , PTA. x 254,800 sca le 100 my 52 53 Fig. 8. 0W-14 aggregate, PTA, x 133, 100, scale 100 mu. 4 x 8 my. A number of empty heads were observed which had re ta ined to a cons iderable extent the angular nature of the complete head ( F i g . 4 . ) . A few complete phage p a r t i c l e s showing empty heads were a l so observed i n the prepara t ion . A great many free t a i l s 130 my i n length were seen i n the e l e c t r o n micrographs In order to demonstrate the presence of a c o n t r a c t i l e t a i l sheath sodium p e r c h l o r a t e , buffered to pH 7-9, was added to a concentra-t i o n of 0.025 M ( F r e i f e l d e r , 1966). At th i s low concentrat ion , a number of phage p a r t i c l e s with contracted sheaths were observed ( F i g . 5; F i g . 6 ) . Some of these contracted p a r t i c l e s seemed to have a c o l l a r at the juncture of the head and t a i l . The con-trac ted sheath (25 x 40 my) exposed the t a i l core - a s lender p r o j e c t i o n 7 x 130 my. A s k i r t of p r o j e c t i o n s , o r i g i n a t i n g i n the reg ion of the former base p l a t e , fanned out around the exposed core . At higher concentrat ions of sodium perch lorate (>0.1 M) the phage head ruptured ( F i g . 7) and the v i s c o s i t y of the medium increased . Phage 0W-14 showed a marked tendency to aggregate ( F i g . 8 ) . No b a c t e r i a l debr is appeared to be present to account for t h i s aggregat ion. Phage 0W-14 f a l l s in to Brad ley ' s morphological c l a s s i f i c a t i o n group A, which contains a l l phages having c o n t r a c t i l e t a i l s (Bradley, 1968). Morpho log ica l ly i t resembles other Pseudomonas phages, but i t i s cons iderably l a r g e r than those prev ious ly reported , the heads of which range from 50 - 60 my i n diameter (Lee and Boez i , 1966; Bradley , 1967; Olsen et a l . , 1968). Aggregation of phage p a r t i c l e s has been reported p r e v i o u s l y , but i n most cases i t appears to involve adsorpt ion to pieces of c e l l u l a r d e b r i s . The considerable number of aggregates and the number of phage p a r t i c l e s involved per aggregate could exp la in the unusual ly high l i g h t s c a t t e r i n g seen with p u r i f i e d prepara-t ions of 0W-14. . Sec t ion I I . K i n e t i c s of Adsorpt ion The r e s u l t s of the adsorpt ion experiments are presented i n F i g . 9. The concentrat ion of unadsorbed phage decreased exponent ia l ly with f i r s t order k i n e t i c s . Adsorpt ion was b iphas i c i n each case. In the case of 0W-14a+, the time required for adsorpt ion of 50% of the phage to the host c e l l s was approximately 3.9 min. This c o r r e s --9 ponded to an adsorpt ion constant (K value) of 1.9 x 10 ml /min. The ra te of adsorpt ion decreased markedly (K of 3 x 10 ml/min. ) or appeared to stop a f t e r only 60% (range 49-72%) of the phage had adsorbed. At 0 C , the rates of adsorpt ion were almost the same as at 30 C, and again adsorpt ion was b i p h a s i c . In attempt to f i n d the a f f ec t of NaCl concentrat ion on the adsorpt ion r a t e , without r e s o r t i n g to the use of buf fers - as a l l previous experiments had used L u r i a broth - a complex medium c o n t a i n -ing a low concentrat ion of NaCl was prepared. The concentrat ion of -3 NaCl was c a l c u l a t e d to be approximately 2 x 10 M. Using th i s medium the ra te of adsorpt ion increased 1.6 f o l d , 50% of the phage b inding to -9 the c e l l s i n 2.2 min. The adsorpt ion rate constant was 3.0 x 10 ml /min. The k i n e t i c s of adsorpt ion appeared to be "normal", with a decrease i n the rate occurr ing a f t e r 93% of the free phage had been i r r e v e r s i b l y bound. In the case of the spontaneously occurr ing mutant, 0W-14a, the rate of adsorpt ion was 2.2 f o l d f a s t e r than 0W-14a+, with 50% of the phage being adsorbed i n 1.7 m i n . , g i v i n g a K value of 4.2 x -9 10 ml /min . As was the case with the wi ld - type phage i n l ow-sa l t bro th , the change i n the rate of adsorpt ion occurred a f t er a p p r o x i -mately 94% of the free phage had bound to the Pseudomonas c e l l s . I t appeared that the adsorpt ion of 0W-14a?was unaffected by the NaCl concentrat ion of the medium, or at l e a s t not to the same extent as was the case with 0W-14a+. Because the adsorpt ion stage of the one-step growth experiment i s u s u a l l y c a r r i e d out with the phage -ce l l mixture under s t a t i c cond i t i ons , the rate of adsorpt ion was examined under these condit ions I f , a f t e r 1 min. incubat ion of the phage and c e l l s at 250 rpm, the speed of r o t a t i o n was reduced to 50 rpm, the subsequent rate of adsorpt ion of 0W-14a+ was markedly depressed. The time required for 50% adsorpt ion of the phage was increased to 0.2 m i n . , equivalent to a 57 F i g . 9. Adsorpt ion of 0W-14a+ and 0W-14a to P_. acidovorans #29 Log phase c e l l s at 1 x 10 per m i l l i l i t r e were . in fec ted with phage at a moi of 0.01. 0 - 0 , adsorpt ion of 0W-14a+; A - A, adsorpt ion of 0W-14a; X - X, adsorpt ion of 0W-14a+ i n low-sa l t L u r i a b r o t h . -10 v e l o c i t y constant for c e l l attachment of 7.3 x 10 ml /min. As a r e s u l t of t h i s , the c l a s s i c a l one-step growth experiment ( E l l i s and Delbruch, 1939) was modif ied as ou t l ined i n M a t e r i a l s and Methods (V) . Two poss ib l e explanations of the unusual adsorpt ion k i n e t i c s of 0W-14a+ are: a. Sagik i n h i b i t i o n phenomenon. Sagik (1954) noted that col iphage T2 was present i n f r e s h l y prepared lysa tes of E. c o l i B i n an i n h i b i t e d s t a t e . Standing for a per iod of days i n the co ld or d i l u t i o n in to d i s t i l l e d water brought about a great increase i n the t i t r e of the l y s a t e . Concomitant with t h i s r i s e were the appearance of regular plaque morphology and normal iza t ion of the heat i n c a t i v a t i o n and adsorpt ion k i n e t i c s . In fresh lysates which contained both i n h i b i t e d and normal phage p a r t i c l e s , the adsorpt ion rates were very slow; even a f t e r an extended per iod for adsorpt ion , 43% of the t o t a l phage remained unadsorbed. I t i s u n l i k e l y that my r e s u l t s are due to p a r t i c l e aggregation because (a) no r i s e i n t i t r e was observed when 0W-14a+ was d i l u t e d in to or d i a l y z e d against 0.005 M N a C l , and (b) no a c t i v a t i o n , or delay, was observed i n the heat i n a c t i v a t i o n studies with t h i s phage. S i m i l a r l y , i f i n h i b i t i o n was to account for the p e c u l i a r r e s u l t s the nature of the i n h i b i t o r y substance must be such that i t binds to the phage preventing i t s easy access to the c e l l u l a r attachment s i t e s , but does not b ind to the adsorpt ive organel les of the phage. I t i s pos s ib l e with t h i s theory to exp la in the s a l t e f f ec t s by t h e e l u t i o n of the i n h i b i t o r o f f the phage p a r t i c l e s . b . Phenotypic and genotypic mutants. Schles inger (1932), Delbruck (1940 a) and Garen (1954) a l l demonstrated a degree of heterogenei ty , with respect to adsorpt ive p r o p e r t i e s , i n phage populat ions . The percentage of phage p a r - , t i d e s i n a l y sa te with decreased adsorpt ive capaci ty i s very low, u s u a l l y being l e s s than 1% of the t o t a l . However, there are some notable exceptions . In lysa tes of P_. aeruginosa phage 7v some 20% of the p a r t i c l e s had a decreased capaci ty to absorb (Feary, F i s h e r and F i s h e r , 1964), and ex trapo la t ion of the data of Schade and Adler (1967) with 0 x ind ica tes that a large f r a c t i o n (25%) adsorbed to the host at a slow r a t e . Three explanations are pos s ib l e to exp la in .our r e s u l t s with respect to the mutation theory: (a) a propor t ion of the phage, for an unknown reason, adsorbs at a lower r a t e , (b) for a proport ion of the phage the s a l t concentrat ion i s too high for optimal adsorpt ion , and (c) the increased NaCl concentrat ion i s such that i t causes the r e v e r s i b l e b inding of a proport ion of the phage to the c e l l s . These p o s s i b i l i t i e s could be inves t iga ted fur ther fo l lowing an enrichment for the mutant us ing the method of Delbruck (1940). The f i n d i n g that the a+ form of the phage adsorbed at a slower rate than the a. form has a number of precedents i n the l i t e r a t u r e . Minamishima et a l . (1968) presented evidence that the r+ form of t h e i r f ibrous P. aeruginosa phage Pf2 was adsorbed at a 1.5 f o l d slower ra te than the r_ mutant. Cohen and Arbogast (1950) showed that col iphage T4r+ adsorbed very . s l owly to I S . c o l i B i n mineral medium supplemented with l i m i t i n g (0.2 ug/ml) tryptophan, whi le T4r adsorbed r a p i d l y . At high concentrat ions of the adsorpt ion co fac tor , the adsorpt ion of the two phages became almost i d e n t i c a l . As an optimal concentrat ion of NaCl i s required for T l adsorpt ion (Puck, Garen and C l i n e , 1951) i t i s p o s s i b l e that the mutant and w i l d - t y p e phages of P_. acidovorans d i f f e r i n , the optimal s a l t concentrat ions required for adsorpt ion . Sect ion I I I . One-step Growth Experiment In order to determine the l a t e n t per iod and the average burst s i z e of phage 0W-14, one-step growth experiments were c a r r i e d out w i th , i n most cases, P_. acidovorans #29. The l a t e n t per iod ( F i g . 10) was between 61 and 66 min. This was fol lowed by a gradual r i s e ( r i s e period) i n the phage t i t r e for a fur ther 37-45 min. The burst s i z e computed by d i v i d i n g the t o t a l number of phage p a r t i c l e s re leased by the number of in fec ted c e l l s was found to be, on the bas is of s i x d i f f e r e n t experiments, 300. The range of burs t s i z e s , using mid- log phase c e l l s , was 214-470. F i g . 10. One-step growth curve of 0W-14a+ and P. acidovorans #29. The burst s i z e was markedly dependent upon the age of the cu l ture ( F i g . 11). The burs t s i zes were found to range from 30 for s ta t ionary phase c e l l s to about 600 for l a t e log phase c e l l s . As the c e l l s entered the log phase of growth, the average burst s i z e increased r a p i d l y to a maximum of l a t e log phase and then decreased as the c e l l s entered s ta t ionary phase. The same type of dependence curve was a l so observed when P_. acidovorans #14 was used as the host . The adsorpt ion rate constant for P_. acidovorans #14 was very low, so that est imations of the burst s i z e could be obtained only by passing the in f ec t ed c u l t u r e through a 0.45 u m i l l i p o r e membrane ( M i l l i p o r e F i l t e r C o r p . , Bedford, Mass.) and washing the c e l l s free of unadsorbed phage p r i o r to running the one-step growth experiments. In the case of the mutant, 0W-14a, the l a t en t per iod and the r i s e per iod were the same as those of the w i l d type, but the burst s i z e was about 50% greater . Attempts to l y s e in fec ted c e l l s prematurely with chloroform or lysozyme were unsuccess fu l . 0W-14 i s d i s t i n g u i s h e d from the other Pseudomonas phages by i t s long l a t e n t and r i s e p e r i o d s , and the high burst s i z e (Table I ) . The burs t y i e l d as a func t ion of the c u l t u r e age of the host c e l l s has been inves t iga ted p r e v i o u s l y . Delbruck (1940 b) working with E_. c o l i and i t s phage compared r a p i d l y d i v i d i n g c e l l s with a 24 h r . aerated c u l t u r e . The one-step growth experiment r e s u l t s i n d i c a t e d an increase i n the length of the l a t en t and r i s e per iods , and a decrease i n the burs t s i z e for s ta t ionary phase c e l l s . Heden (1951) us ing 0 ! 2 3 4 5 6 7 8 HOURS F i g . 11. E f f e c t of c e l l age on phage development as measured by the one-step growth method. 0 - 0 , growth of P_. acidovorans #29 at 30 C measured i n OD 650 u n i t s / m l . © - ©, average burst s i z e from c e l l samples taken at s p e c i f i e d times and in fec ted with 0W-14a+. ! phage T2 and E. c o l i B, compared phage re lease from in fec ted c e l l s i n the l ag and log phases of growth. Though he noted considerable and reproduc ib le v a r i a t i o n i n the average burst s i z e s , the l a t e n t per iod was of constant l ength . His r e s u l t s unexpectedly showed the highest phage y i e l d at the t r a n s i t i o n from lag to log phase growth, which was fol lowed by a r a p i d decrease during logar i thmic growth. These r e s u l t s are d i f f i c u l t to compare with those of the other workers s ince the method used for c u l t i v a t i o n of the host was r a d i c a l l y d i f f e r e n t . Baer and Krueger (1952), us ing B a c i l l u s mycoides N phage, found l i t t l e d i f f erence i n phage product ion by l a g or l og phase c e l l s . From a search of the l i t e r a t u r e i t appears that the present p u b l i c a t i o n i s the only i n v e s t i g a t i o n of the burst from l a g through log to s ta t ionary phase c e l l s . This work was considerably s i m p l i f i e d by the longer d i v i s i o n cyc le of the organism (approximately 60 min. i n L u r i a broth at 30 C with adequate aerat ion) and the considerably modif ied one-step growth experiment method. The higher burs t s i z e of the mutant 0W-14a appears to have few p a r e l l e l s i n the l i t e r a t u r e , and may be due to the more complete l y s i s of the in fec ted c e l l s rather than any ac tua l d i f f erence i n the growth cyc le of the phage (see Sect ion IV ) . Cohen and Arbogast (1950) observed that T4r+ had a higher burs t s i z e than T 4 r . Sect ion IV. L y s i s I n h i b i t i o n T h i r t y minutes a f t e r i n f e c t i o n of a c u l t u r e of P_. acidovorans #29 wi th 0W-14a, the t u r b i d i t y of the cu l ture decreased markedly and then remained constant for the remainder of the experiment ( F i g . 12). Fol lowing i n f e c t i o n of the same s t r a i n with 0W-14a+, there was a s l i g h t decrease i n the t u r b i d i t y of the c u l t u r e , but then there was a gradual increase i n t u r b i d i t y during the next 3 h r . This increase i n t u r b i d i t y appeared to be caused by growth of the organism rather than to disaggregat ion ( F i g . 12). Thus a c u l t u r e in fec ted with 0W-14a+ appears to be l y s i s i n h i b i t e d . L y s i s i n h i b i t i o n i s charac ter i zed by an increase i n the length of the l a t en t per iod and an increase i n the burst s i z e fo l lowing super in fec t ion by an r+-type phage of a cu l ture p r i m a r i l y in fec ted with an r+ phage. Th i s has been shown with the T even coliphages by Doermann (1948), Rutberg and Rutberg (1965) and Bode (1967). Attempts at demonstrating l y s i s i n h i b i t i o n by phage super in fec t ion of P_. acidovorans #29 were unsuccess fu l , though the burst s i z e was usua l ly cons iderably depressed over that of the c o n t r o l . In the case of these experiments the c o n t r o l was run simultaneously with a m u l t i p l i c i t y of i n f e c t i o n of 0.01. This depression of the average burst s i z e by the use of a high moi a f fec ted the w i ld - type and i t s mutant, 0W-14a, s i m i l a r l y . The i n a b i l i t y to demonstrate l y s i s i n h i b i t i o n with 0W-14a+ may have been due to the fac t that the aH T-+• a mutation i s not i » — a — " r ™ " — ™ — i 0 1 2 3 ' 4 H O U R S F i g . 12. Growth,of P_. acidovorans #29 in fec ted at zero time with 0W-14a+ (X - X) or 0W-14 a (© - © ) ; Uninfected c o n t r o l (Q analagous to the r+ >• r mutation seen i n the T even co l iphages , or to the condi t ions used not being conducive to the product ion of th i s s t a t e . The unusual decrease i n the average burst s i z e warrants fur ther d i s c u s s i o n . I t i s be l i eved genera l ly that the burst s i z e i s independent of the m u l t i p l i c i t y of i n f e c t i o n s . However, a high m u l t i p l i c i t y of i n f e c t i o n has been shown e i t h e r to increase the burst s i z e or to decrease i t . Delbruck and L u r i a (1942) showed that the m u l t i p l e i n f e c t i o n of _E. c o l i B with col iphages a tand 3 r e s u l t e d i n a 45-100% increase i n the burs t s i z e . The p o s s i b i l i t y of l y s i s i n h i b i t i o n was ru led-out s ince the l a t e n t per iod was unaffected. P r i c e (1950) showed that the burst s i z e of a phage i n Staphylococcus muscae cu l tures v a r i e d d i r e c t l y with the increase i n the m u l t i p l i c i t y of i n f e c t i o n . C e l l s of S h i g e l l a sonnei gave smaller bursts fo l lowing i n f e c t i o n with a large excess of Coliphages T4 or T7 (Barry and Goebel , 1951). These r e s u l t s and those obtained with P_. acidovorans and 0W-14 can not be explained at present . Sect ion V . Thermal I n a c t i v a t i o n 0W-14a+ was e s s e n t i a l l y s table at 50 C i n L u r i a b r o t h , but for every 5 r i s e i n temperature between 55 C and 65 C , there was an approximate s i x f o l d increase i n the rate of i n a c t i v a t i o n . The ra te -1 -1 -1 constants were 0.033 min. , 0.18 min. , and 1.06 min. at 55 C , 60 C, and 65 C r e s p e c t i v e l y . These values corresponded to h a l f - l i v e s ranging from 21 to 0.65 min. At 55 C and 60 C i n a c t i v a t i o n was b iphas i c ( F i g . 13). A p p r o x i -mately 35% of the phage p a r t i c l e s appeared to have some degree of r e s i s t a n c e . This f r a c t i o n appeared to be s tab le at 55 C but slowly i n a c t i v a t e d at 60 C, the rate constant being 0.032 min. The c l e a r plaque mutant, 0W-14a, showed b iphas i c i n a c t i v a t i o n at 55 C but not at 60 C. Using the Arrhenius p l o t of the thermal i n a c t i v a t i o n of 0W-14a+ ( F i g . 14) the heat of a c t i v a t i o n (AH*) was c a l c u l a t e d to be 75,700 c a l o r i e s / m o l e . 0W-14a was found to be s l i g h t l y more thermo- lab i l e the AH*'being approximately 62,500 c a l o r i e s / m o l e . B iphas ic thermal i n a c t i v a t i o n k i n e t i c s , though qui te common i n the animal v i r u s e s , have not been observed often i n bacteriophages . Except ions , wi th the percentage of the populat ion having d i f f e r i n g thermal s t a b i l i t y i n parentheses, are some of the N e i s s e r i a phages (2-10%; Phelps , 1967); B a c i l l u s cereus phages a, B, and y (4.9%); M e y n e l l , 1962); P. aeruginosa phage 7m (29%; Feary, F i s h e r and F i s h e r , 1964); and, 0W-14a+ (35%; present research) . Adams (1953), working w i t h col iphage T5, found a f r a c t i o n of the phage preparat ion was s tab le a f t e r heat ing for s evera l hours i n buffered s a l i n e at 50 C. The major i ty of the phage i n th i s f r a c t i o n were phenotypica l ly r e s i s t a n t , r a t h e r than genotyp ica l ly r e s i s t a n t , s ince t h e i r progeny exhib i ted the same thermal s t a b i l i t y as the w i l d type. In the case of T5, F i g . 13. Thermal i n a c t i v a t i o n curves of 0W-14a+ i n L u r i a b r o t h . X - X, 50 C; A - A, 55 C; 0 - 0, 60 C; A - A, 65 C. F i g . 14. Arrhenius p l o t of the thermal i n a c t i v a t i o n of 0W-14a+ 71 0.1% of the populat ion were phenotypica l ly r e s i s t a n t , whi le the —7 —8 inc idence of genotypic re s i s tance was of the order of 10 to 10 I t was most probable , there fore , that the heat r e s i s t a n t 0W-14a+ p a r t i c l e s were phenotyp ica l ly rather than genotyp ica l ly r e s i s t a n t . I t would be of i n t e r e s t to inves t iga te the adsorpt ion k i n e t i c s of t h i s r e s i s t a n t f r a c t i o n s ince an almost i d e n t i c a l f r a c t i o n of the phage showed d i f f e r i n g adsorpt ion and heat i n a c t i v a t i o n k i n e t i c s . The a c t i v a t i o n energy c a l c u l a t e d for t h i s phage i s s i m i l a r to those obtained for B a c i l l u s megaterium phage M-1 (76,000), E. c o l i phage T2 (71,700), and Streptococcus l a c t i s phage 122-4 (76,000) ( P o l l a r d , 1953). Sect ion V I . Sonic S e n s i t i v i t y of Phages The sonic s t a b i l i t y of phages i s mainly of t h e o r e t i c a l i n t e r e s t , though s o n i c a t i o n has been used to study the i n t r a c e l l u l a r development of an RNA-containing phage (Paranchych and Graham, 1962). The three phages tested were i n a c t i v a t e d exponent ia l ly by sonic i r r a d i a t i o n ( F i g . 15). Coliphage T l and P_, acidovorans phage 0W-14a+ showed qui te s i m i l a r i n a c t i v a t i o n r a t e s , with k g values of 1.45 min. ^ for T l and 1.74 min.-"*" for 0W-14a+. Phage S13 was f a r more s tab le to sonic i r r a d i a t i o n than e i t h e r of the other phages and gave a k g value of 0.12 min. 1 . The few reports which e x i s t i n the l i t e r a t u r e concerning the sonic 72 / / i - (j — i » ° 0 30 60 90 120 150 SECONDS F i g . 15. Sonic s e n s i t i v i t y of col iphage S13, T l and 0W<L4a+: (A — A ) , S13; ( X — X),.T1; (0 0 ) , 0W-14a+. 73 i n a c t i v a t i o n of bacteriophages i n d i c a t e that the s m a l l , compact v iruses are f a r more s tab le than the l a r g e r ones (Anderson et a l . , 1948). The s p h e r i c a l s ing le - s t randed DNA-containing phages 0X174 and S13 (Minamishima et a l . , 1968; present research) and the small RNA-containing phages (Paranchych and Graham, 1962) are p a r t i c u l a r l y s tab le ; on the other hand, the long f ibrous phage P f l of P_. aeruginosa i s h i g h l y l a b i l e (Minamishima et a l . , 1968). A general r e l a t i o n s h i p ex i s t s between the v i r u s volume and the ra te of sonic i n a c t i v a t i o n ( P o l l a r d , 1953), which i n the case of t h i s study would i n d i c a t e that T l and 0W-14 are quite s i m i l a r i n s i z e . This was not borne out by e l e c t r o n microscopy which showed that 0W-14 possessed a heat 87 my i n diameter, whi le i t i s known that the head diameter of T l i s 50 my (Will iams and F r a z e r , 1953). S e c t i o n V I I . pH I n a c t i v a t i o n A normal i n a c t i v a t i o n curve was obtained f o r the w i l d type phage, 0W-14a+ ( F i g . 16). However, an anomalous i n a c t i v a t i o n curve was obtained for 0W-14 ( F i g . 16): the phage appeared to be ac t iva ted by incubat ion at pH 4-5. Approximately 1.6 f o l d more plaque forming un i t s were found i n the preparat ion at pH 4-5 than were present i n the preparat ion at pH 6-9. S i m i l a r r e s u l t s were obtained with the host range mutant 0W-14h. The r e s u l t s of the pH i n a c t i v a t i o n experiment fur ther d i s t ingu i shed 74 / / the mutant phage from the w i l d type. S e c t i o n V I I I . S e n s i t i v i t y to U l t r a v i o l e t L i g h t and Photoreac t iva t ion M u l t i h i t k i n e t i c s were observed for the U V - i n a c t i v a t i o n of 0W-14a+ and col iphage T l ( F i g . 17). E x t r a p o l a t i o n of the exponential regions of the curves to the ordinate gave values 2.4 and 1.6 times higher than the i n i t i a l t i t r e of the two phages. The ra te constants (k ) c a l c u l a t e d from the exponential regions of the curves were 0.60 m i n . - 1 for T l , and 4.35 m i n . - 1 for 0W-14a+. Therefore , 0W-14a+ was 7 times as s e n s i t i v e as T l to U V - i r r a d i a t i o n . The l e t h a l e f f ec t s of u l t r a v i o l e t l i g h t were reversed to a considerable extent by i r r a d i a t i o n of the overlay p la tes with white l i g h t . Photoreact ivable sectors of 0.35 and 0.71 were c a l c u l a t e d for T l and 0W-14a+, r e s p e c t i v e l y . P r i o r to t h i s research , the u l t r a v i o l e t s e n s i t i v i t y of a number of Pseudomonas aeruginosa phages had been reported (Benzer and Jacob, 1953; Jacob, 1952; M a t s u i , 1952; Holloway and Monk, 1959; Holloway et a l . 1962) . The i r r a d i a t i o n death curves of 0W-14a+ c l o s e l y resembled those obtained for the T-even phages (Harm, 1959) i n comparison to those ob-tained for T l (Dulbecco, 1950; present research , F i g . 17). The UV s e n s i t i v i t y of 0W-14 i s of s p e c i a l i n t e r e s t because the-main l e t h a l e f f ec t of such i r r a d i a t i o n i s the formation of thymine dimers (Wacker, 1963) , and t h i s phage contains a novel base p a r t i a l l y r e p l a c i n g thymine 76 F i g . 17. UV i n a c t i v a t i o n and photoreac t iva t ion of col iphage T l and 0W-14a+. 0W-14a+ was i r r a d i a t e d with UV l i g h t and incubated i n the dark ( © @) or i n the l i g h t ( 0 0 ) . T l was i r r a d i a t e d and incubated i n the dark ( A A); i n the l i g h t (A A). F i g . 17. (see Sect ion I X ) . P_. acidovorans appears to contain a photoreact ivat ing enzyme system s ince a major increase i n the t i t r e of the i r r a d i a t e d phage was obtained by incubat ion of the phage in fec ted c e l l s i n white l i g h t . I t appears that the major i ty (71%) of the l e t h a l e f fec t s of UV l i g h t can be reversed by p h o t o r e a c t i v a t i o n . The • value obtained for the photoreact ivable sector of T l d i d not agree with the value of 0.68 obtained by Dulbecco (1950). The reason for t h i s discrepancy was not apparent. S e c t i o n IX. Phage Res is tant Mutants and the C a r r i e r State 1. Phage r e s i s t a n t mutants S u r p r i s i n g l y , s e l e c t i o n for phage r e s i s t a n t s t r a i n s of P_. acidovorans appeared i n some cases to se l ec t for s t r a i n s with a l t e r a t i o n s i n c e l l d i v i s i o n and/or m o t i l i t y . The phage r e s i s t a n t mutants f e l l in to s i x d i s t i n g u i s h a b l e groups with respect to c e l l morphology at var ious temperatures and to m o t i l i t y . They f e l l into two groups with respect to t h e i r s e n s i t i v i t y to 0W-14h. In a l l , nine groups of mutants were c l a s s i f i e d (Table I X ) . Photomicrographs of the w i l d type and mutant 29-20 are presented i n F i g . 18, F i g . 19. When mutant "snakes" prepared at 30 C were t rans ferred to fresh broth at 22 C, they d iv ided to produce c e l l s of normal s i z e . In many cases these apparently normal c e l l s were nonmotile. Fi lament formation i n b a c t e r i a can be induced by a v a r i e t y of p h y s i c a l and chemical agents, amongst them a n t i b i o t i c s (Hunt and P i t t i l l o , 1968), D-amino acids (Grula and G r u l a , 1962), heavy metal ions (Rosenberg et a l . , 1967), UV i r r a d i a t i o n (Deering, 1958), X-rays (Adler and Hardigree , 1964), increased h y d r o s t a t i c pressure (Zobe l l and Cobet, 1964) and extremes of temperature (Hoffman and Frank, 1963; T e r r y , Gaffar and Sagers, 1966; Shaw, 1968). At e levated temperatures, i . e . those near the maximum temperature for growth, IS. c o l i , C l o s t r i d i u m a c i d i u r i c i , and P_. acidovorans tended to elongate and form fi laments (commonly c a l l e d "snakes"), presumably because of an i n h i b i t i o n of c e l l d i v i s i o n . I f the temperature of incubat ion of the cu l ture i s decreased, normal c e l l s are segmented o f f . The fac t that s i x of our phage r e s i s t a n t mutants i s o l a t e d i s o l a t e d during the course th i s research formed f i laments at a temperature at which the w i l d type c e l l s were "normal" was of considerable i n t e r e s t , as was the observat ion that four of them were nonmotile at 22 C . Golub and Orlova (1968) compared the growth of an phage re leas from lysogenic cu l tures of _E. c o l i C at 37 C and 45 C. Lysogeniza t i o n of IS. c o l i C with a mutant of phage 299 rendered the c u l t u r e more thermosensit ive i n that i t formed f i laments at the higher temperature while E . c o l i C and E , c o l i C(299) were unaffected. 80 Table IX. C h a r a c t e r i s t i c s of Some Phage Res is tant Mutants of P. acidovorans #29. Mutant # Pat tern of Phage Resistance Growth a t : L y s i s by: 22 C 30 C 37 C 0W-14a 0W-14a+ 0W-14h 29-1 + NnM* SnM SnM 29-5 - + N M SnM 29r-6 - + N M SnM 29-7 - + N M SnM 29-8 - + N M SnM 29-11 - _ N M S M SnM 29-12 - - N-M SnM 29-15 - - N M SnM 29-16 - - NnM SnM 29-18 - + NnM SnM SnM 29-20 - - NnM SnM SnM 29-21 - - N M SnM SnM .29-22s - - N M PNG 29-221 - - N M SnM 29-23 - + N M PNG 29-24 - - NnM SnM SnM Wild type. + + + N M N M SnM * N (normal c e l l morphology); S ( snake- l ike c e l l morphology); M (mot i le ) ; nM (nonmotile); PNG (poor or no growth) P. acidovorans #29 grown at 30 C, x 4,400 F i g . 18 b . P. acidovorans #29 grown at 18 C, x 4,400 2. C a r r i e r s tate Attempts at the p u r i f i c a t i o n of the phage in fec ted cu l tures by p l a t i n g them on L u r i a agar p la tes f a i l e d i n that a l l of the co lonies which arose on the p lates were phage d e f i c i e n t . The existence of c e l l s prev ious ly in fec ted with phage was ind ica ted by a number of smal l v iscous co lonies which appeared on some of the p l a t e s . Kawakami and Landman (1968) used minimal medium supplemented with 0.01% casein hydrolysate and sodium succinate (0.48M) or sucrose (0.8M) to s t a b i l i z e c a r r i e r c e l l s of 13. s u b t i l i s . Supplementation of minimal medium with 70 ml of L u r i a broth (per l i t r e ) d id not s t a b i l i z e P_. acidovorans c a r r i e r c e l l s , ne i ther d i d the a d d i t i o n of sodium succinate (0.48M), sucrose (0.8M) or g l y c e r o l (5%, w/v) . Streaking the cu l tures on overlays containing 0W-14 appeared to be the only means of p u r i f y i n g these s t r a i n s , though i t had obvious disadvantages. When washed, phage in fec ted c e l l s were p la ted to determine the t o t a l number of v i a b l e c e l l s and the number of phage in fec ted c e l l s , a discrepancy was always noted. The percentage of in fec ted c e l l s i n the cu l tures v a r i e d from 0.2 to 37% of the t o t a l c e l l number, a fac t which confirmed the suspected c a r r i e r s ta te . C a r r i e r cu l tures conta in ing a lower percentage of in fec ted c e l l s appear to be unstable , as the phage i s r e a d i l y l o s t on t rans fer i n b r o t h . One example of a h y p e r - c a r r i e r cu l ture was found but t h i s was h i g h l y unstable and "grew" i n a p a r t i a l l y lysed c o n d i t i o n . The c a r r i e r cu l tures grew at rates comparable with that of P_. acidovorans #29, from which they were der ived . Phage was re leased only during the logar i thmic phase of growth. E i t h e r of the two phages 0W-14a+ or 0W-14a, could enter in to a c a r r i e r s tate and be re leased from in fec ted c u l t u r e s . The c a r r i e r s tate i s a phage-bacterium r e l a t i o n s h i p i n t e r -mediate between l y s i s and lysogeny, hence the synonyms "pseudo-lysogeny" (Lwoff, 1953) and " l a s t i n g semitemperate complex" (Frazer , 1957). This r e l a t i o n s h i p , which w i l l be described i n greater d e t a i l l a t e r , occurs i n many b a c t e r i a e . g . : _E. c o l i B with phage T3 #40 (Frazer , 1957); E . c o l i K12 with phage T 7 s h ( L i , Barksdale , and Garmise, 1961); E. c o l i C3000 with phage HR (Hsu, 1968); B r u c e l l a abortus 544A phage P19 (Jones, McDuff and Wi lson , 1962); Proteus m i r a b i l i s with phage 57 (Coetzee and Hawtrey, 1962); S h i g e l l a dysenteriae cu l tures with phage T7 ( L i et a l . , 1961); Salmonella typhimurium LT22 with phage PLT22 ( L i et a l . , 1961). Nor i s th i s phenomenon r e s t r i c t e d to Gram-negative organisms s ince c a r r i e r condit ions have been reported i n IJ. s u b t i l i s in fec ted with SP13 (Romig and Brodetsky, 1961) and SP-10 (Kawakami and Landman, 1968). When p l a t e d , c a r r i e r c e l l s segregate at a high frequency into normal, phage s e n s i t i v e , c e l l s and into c a r r i e r c e l l s capable of producing phage. This i s d i f f e r e n t from the lysogenic s ta t e , i n which the frequency of segregation of nonlysogenic c e l l s i s low. In a d d i t i o n , the phage genome i n a c a r r i e r c e l l ex i s t s as a plasmid rather than as an integrated prophage (Takahashi, 1964); and, c a r r i e r cu l tures grown i n the presence of antiphage serum are r a p i d l y converted to a phage s e n s i t i v e s ta te , i n d i c a t i n g that phage r e i n f e c t i o n i s necessary for the maintenance of t h i s s t a t e . The experiments conducted with P_. acidovorans and phage 0W-14 suggested the occurrence of a c a r r i e r s tate i n t h i s system. Development of t h i s aspect of the problem depends upon a s u i t a b l e s o l i d medium being found to s t a b i l i z e the phage in fec ted c e l l s . S e c t i o n X. Recombination Only one previous case ex i s t s for recombinational experiments being run on Pseudomonas phages (Egan and Holloway, 1961). In the case of the present research , the pre l iminary experiments i n d i c a t e that i t was p o s s i b l e to carry out the fo l lowing cross : a+h+ x ah ——•> a+h+, ah, a+h, and ah+. Using mixed i n d i c a t o r s i t was pos s ib l e to i d e n t i f y the a+h recombinant, but ah+ resembled a+h+ too c l o s e l y to be d i s t i n g u i s h e d . The former recombinant appeared at a frequency of approximately 6%. S e c t i o n X I . Phage 0W-14 DNA 1. General proper t i e s The p u r i f i e d n u c l e i c a c i d extracted from 0W-14 formed a viscous s o l u t i o n when d i s so lved i n SSC. The spectrum of t h i s s o l u t i o n exh ib i ted a X max. at 258 mp and a X min. at 232 mu when d i l u t e d in to 0.1 x SSC. Upon the a d d i t i o n of NaOH to 0 .25 M, the X max. s h i f t e d to 263 my and the X min. to 237 my and the chromici ty at 260 my increased by almost 30 percent . The s p e c t r a l r a t i o s 1: 0.46: 0.51 (260 my: 230 my: 280 my) agreed w e l l with Marmur"s (1961) va lue of 1: 0.450: 0.515 for pure DNA. The n u c l e i c a c i d was diphenylamine p o s i t i v e and o r c i n o l nega-t i v e . I t was s e n s i t i v e to DN'ase d iges t ion but r e s i s t a n t to RN'ase. Therefore , the n u c l e i c a c i d i s o l a t e d from 0W-14 was DNA and not RNA. 2. M e l t i n g temperatures The Tm values i n 0.1 x SSC for the DNA preparat ions from P_. acidovorans #14 and from 0W-14 were 80.7 and 83.0 C , r e s p e c t i v e l y . As the Tm of DNA i s 15.4 C lower i n 0.1 x SSC than i n SSC the fo l lowing equation was used to c a l c u l a t e the moles percent guanine plus cytos ine i n the phage n u c l e i c a c i d (Mandel and Marmur, 1968): Moles % GC = 2.44 (Tm - 53.9) The GC content of the host DNA was c a l c u l a t e d to be 65.4% and that of the phage DNA 71.9%. 87 T E M P E R A T U R E ' C F i g . 20. M e l t i n g p r o f i l e s of 0W-14 DNA (0 0) and P. acidovorans #14 DNA ( X X) i n 0.1 x SSC. Upon heat ing the hyperchromia s h i f t at 260 my for the host DNA was 31%, that for 0W-14 DNA 37%. 3. Buoyant densi ty The densi ty of the phage DNA, on the bas i s of three determina-3 t ions us ing two d i f f e r e n t preparat ions was 1.666 g/cc , a value which corresponded to about 6% GC. The densi ty of heat-denatured 3 phage DNA was 1.681 g/cc . A l k a l i n e denaturat ion increased the 2 densi ty to 1.680 g/cc . There was no i n d i c a t i o n of m u l t i p l e densi ty bands i n the denatured DNA preparat ions (see F i g . 21). , 3 P_. acidovorans #14 DNA banded at a densi ty of 1.723 g/cc ( F i g . 21). This value corresponds w e l l to the l i t e r a t u r e values 3 of 1.724 g/cc ( C o l w e l l , C i t a r e l l a and Ryman, 1965) and 1.7255 (Mandel, 1965) for t h i s DNA. Though the densi ty of the phage DNA was unusual ly low, the DNA behaved normally on denaturat ion by heat or a l k a l i . A densi ty 3 increase of 15 mg/cc agreed w e l l with those values obtained upon 3 the denaturat ion of E_. c o l i DNA - 15 mg/cc - (Lee and Boez i , 1966). The lack of m u l t i p l e densi ty peaks upon denaturat ion i n d i c a t e d that the m a t e r i a l was homogeneous with respect ,to i t s buoyant dens i ty , i . e . each s trand of the double h e l i x was of s i m i l a r dens i ty . 89 F i g . 21. Microdensitometer scans of C s C l densi ty gradients . A . 0W-14 DNA (p 1.666)., denatured 0W-14 DNA (p 1681), and E_. c o l i DNA marker (p 1.710). B. _E. c o l i DNA marker (p 1.710) and P_. acidovorans #14 DNA (p 1.723). Samples were centr i fuged using Beckman Model E a n a l y t i c a l u l t r a c e n t r i f u g e at 44,000 rpm. for 22 h r . p r i o r to photography using the UV o p t i c s . 4. Chemical composition of 0W-14 DNA Hydro lys i s of the phage DNA with formic a c i d l i b e r a t e d f i v e bases. Four of the bases were i d e n t i f i e d by paper chromatography (Table X b) and t h e i r s p e c t r a l proper t i e s i n a c i d i c and bas i c s o l u t i o n as adenine, guanine, cytos ine and thymine (Table X I ) . The f i f t h base, which migrated slower than guanine i n the i sopropanol -HCl-water solvent system of Bendich (1957), had the s p e c t r a l proper t i e s described i n Table XII and F i g . 22. This base was present i n the formic a c i d hydrolysates and i n a hydro-c h l o r i c a c i d d igest but not i n a p e r c h l o r i c a c i d hydro lysate . Further evidence for the existence of another base was suggested by the fac t that [A + G] / [T + C] ^ 1. The concentrat ion of thymine i n three d i f f e r e n t experiments was always about one-hal f the concentrat ion of adenine. The exact nature of the other base was not c l e a r s ince i t s spectrum d i d not correspond to any of the common minor bases found i n the l i t e r a t u r e . The 0W-14 DNA preparat ions a lso gave a .very strong anthrone r e a c t i o n , the coloured product of which had a A max. at 624 mu, and a spectrum i d e n t i c a l to that of the r e a c t i o n product obtained with g lucose . A number of saccharides were separated by paper chromatography fo l lowing a c i d h y d r o l y s i s of the DNA. However, i f the DNA was p r e c i p i t a t e d with TCA and then hydrolyzed , no saccharides could be detected i n the hydrolysate by paper chroma-tography. Table X (a) . Paper Chromatographic Separation of Purine and Pyr imidine Bases. Standards Solvent System Fluorescence* A B C (Rf x 100) Adenine • 55 43 30 pu Guanine 37s** 11 20s b l Cytos ine 50 34 44 pu 5-Hydroxymethylcytosine (HMC) 47 23 45 pu Thymine 74 56 71 pu 5-Hydroxymethyluraci l (HMU) 55 32 59 pu U r a c i l 64 40 61 pu * f luorescence of the spots on the paper under UV l i g h t : pu (purple) and b l (blue) * * s (streaked) Table X (b) . Separation of the Bases i n a 0W-14 DNA Hydrolysate (Rf x 100) and Fluorescence ( i ) Solvent System A 38s (bl) 51 (pu) I d e n t i f i c a t i o n Guanine Cytosine ( i i ) Solvent System B 13 (bl) 34 (pu) 42 (pu) 56 (pu) I d e n t i f i c a t i o n Guanine Cytosine Adenine Thymine ( i i i ) Solvent System C 21 (bl) 31 (pu) 44 (pu) 71 (pu) I d e n t i f i c a t i o n Guanine Adenine Cytosine Thymine or HMC 92 Table X I . Spectra Proper t i e s of the Bases I so lated from 0W-14 DNA. Base* pH** Amax.(my) Xmin.(my) Absorbance Ratios 250/260 280/260 Adenine 1 263(262)*** 229.5(229) 0.76 0.41 13 269(269) 238(237) 0.58 0.62 Cytosine 1 276(275) 239(238.5) 0.47 1.57 13 282(281.5) 250.5(250.5) 0.67 2.20 Guanine 1 249(248) 224(224) 1.31 0.78 13 275(274) 240.5(239.5). 0.84 1.24 Thymine 2 262.5(264.5) 233.5(233) 0.73 0.50 12 289(291) 244(244) 0.79 1.12 * t e n t a t i v e l y i d e n t i f i e d by paper chromatography * * pH 1 (0.10 N HC1); pH 2 (0.01 N HC1); pH 12 (0.01 N NaOH); and, pH 13 (0.1 N NaOH) * * * values i n parentheses are from - the l i t e r a t u r e 93 Table X I I . S p e c t r a l Propert i e s of a Novel Base Found i n 0W-14 DNA pH Araax. (my) Xmin. (my) Absorbance Rat ios R e l a t i v e Absorbance 250/260 280/260 290/260 E257my/E281my 1 257 228.5 0.91 0.47 0.18 0.80 13 281 246.5 0.80 1.49 1.39 94 225 250 275 300 325 W A V E L E N G T H (rnp) F i g . 22. Spectra of the Novel base i n 0W-14 DNA. -spectrum i n 0.1 N NaOH; . _ . spectrum i n 0.1 N HC1. The p o s s i b i l i t y that th i s m a t e r i a l , though not cova lent ly bonded to the phage DNA, might have af fected the buoyant densi ty of the DNA was r u l e d out i n the fo l lowing manner. The TCA p r e -c i p i t a t e was d i s so lved i n 0.2 M T r i s - H C l buf fer pH 8.5 with gentle heat ing , and subjected to Cs CI densi ty gradient a n a l y s i s . Two predominant bands were formed corresponding to the double-stranded and denatured forms of the phage DNA. The presence of unusual bases found prev ious ly i n DNA tends to a l t e r the p h y s i c a l proper t i e s of the DNA from some phages. The presence.of 5-hydroxymethylcytosine i n the DNA of the T even co l iphages , and the g l u c o s y l a t i o n of t h i s base r e s u l t s i n a decrease i n the expected densi ty but has l i t t l e e f fec t on the Tm ( S z y b a l s k i , 1968). A number of 15. s u b t i l i s phages conta in e i t h e r u r a c i l (phage PBS2-Takahashi and Marmur, 1963) or 5-hydroxymethyl-u r a c i l (phage SP8-Kal l en , Simon and Marmur, 1962) the presence of which markedly increases the buoyant densi ty and decreases the Tm. The a r t i f i c i a l replacement of 66% of the thymine residues i n T3 DNA by 5 - e t h y l u r a c i l res idues was shown to lower the Tm by 4.5 C (Pietrzkowska and Shugar, 1967). The presence of a f i f t h base i n bacteriophage DNA appears to be r a r e . In B a c i l l u s phage Vx the presence of a f i f t h base, a pyr imid ine , was suspected s ince the r a t i o of adenine to thymine w a s ' l : 0.22. However, no unusual base was observed i n hydrolysates of the DNA and i t was concluded that the f i f t h base was e i ther a c i d l a b i l e or non-UV 96 Table X I I I . Base Composition of 0W-14 DNA as Determined by Three Methods. Buoyant Density Thermal Spectro-chemical p(g/c.c ) %GC Tm %GC Base Moles % %GC 1.666 98.4 C 71.9 Adenine Cytosine Guanine Thymine Unknown 21.8 26.6 28.2 11.1 12.3* 54.8 * based on the r e l a t i o n s h i p : [ A + G ] = 1 [ T + C + Unknown ] adsorbing (Ikeda et a l . , 1965). Pons (1967) reported that the DNA from S e r r a t i a marcescens phage 7 contained f i v e bases. In t h i s case, the f i f t h base subs t i tu ted f o r a de f i c i ency of guanine res idues . I t was i s o l a t e d and i t s s p e c t r a l proper t i e s l i s t e d but i t was not i d e n t i f i e d . The unknown pyr imidine base i s o l a t e d from 0W-14 DNA, though present i n a f a i r l y low concen-t r a t i o n , appears to have a marked e f fec t on the buoyant densi ty and the Tm of the DNA (Table X I I I ) . I t s presence poses a number of i n t e r e s t i n g problems, not the l e a s t of which are i t s s t r u c t u r e , b i o s y n t h e s i s , and what contro l s are employed to regulate i t s i n c o r p o r a t i o n in to DNA i n the presence of thymine. GENERAL DISCUSSION Further research on 0W-14 by way of extending or exp la in ing the present r e s u l t s could inc lude the fo l lowing items: The development of methods f or the preparat ion of non-aggregated phage p a r t i c l e s would enable ana lys i s of h igh ly p u r i f i e d preparat ions o f the phage. This i s necessary to al low fur ther p h y s i c a l c h a r a c t e r i -z a t i o n of the phage. A thorough i n v e s t i g a t i o n of the adsorpt ion of 0W-14a+ and 0W-14a u s i n g inorganic buf fers rather than L u r i a broth as the d i l u e n t should be c a r r i e d out . The e f f ec t of- temperature and of var ious a d d i t i o n s , such as s a l t s and proteinaceous m a t e r i a l s , could then be s tud ied . A fur ther attempt to demonstrate l y s i s i n h i b i t i o n i n th i s phage system, and an e l ec t ron microscopic study of i n t r a c e l l u l a r phage m u l t i p l i c a t i o n i n s i n g l y and m u l t i p l y in fec ted c e l l s , which might supply some explanat ion for the lower burs t s i zes from m u l t i p l y i n -fected c e l l s . An attempt to obta in l y t i c transduct ion of genetic markers with QfW-14 using the method of Gunsalus et a l (1967). Though the biochemical c a p a b i l i t i e s of P. acidovorans have been inves t iga ted i n d e t a i l (S tan ier , P a l l e r o n i , and Doudoroff, 1966) the genet ics of t h i s important group have rece ived no a t t e n t i o n . A more d e t a i l e d study of the phage r e s i s t a n t mutants, i n c l u d i n g an e l e c t r o n microscopic study of the f i laments to determine whether or not they are septate , and a search for a poss ib le explanat ion for the unusual temperature s e n s i t i v e mutant s t r a i n s . A d e t a i l e d i n v e s t i g a t i o n of the phage DNA and the d i f f erence between the wi ld - type and 0W-14a. Thi s would inc lude: buoyant densi ty i n C s C l ; sedimentat ional a n a l y s i s ; ana lys i s for s ing le - s tranded breaks; viscometry; and chemical ana lys i s - t o t a l P, deoxyribose, and bases. I t would a l so e n t a i l the i d e n t i f i c a t i o n and s p e c t r a l propert ie s of the unusual base. An attempt at obta in ing t r a n s f e c t i o n with 0W-14 DNA, and competent J3. s u b t i l i s c e l l s or P_. acidovorans spheroplasts . 100 BIBLIOGRAPHY Adams, M.H. 1953. 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