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Mixed bacteriophage infection of Escherichia coli : exclusion of superinfecting T4 by T7 Loewen, Ronald R. 1978

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V MIXED BACTERIOPHAGE INFECTION OF ESCHERICHIA COLI: EXCLUSION OF SUPERINFECTING T4 BY T7 by Ronald R. Loewen B.Sc.(Hons.), University of Manitoba, 1975 A thesis submitted in part ia l f u l f i l lment of the requirements for the degree of Master of Science in . The Faculty of Graduate Studies The Department of Microbiology We accept this thesis as conforming to the required standard The University of B r i t i s h Columbia September, 1978 © Ronald R. Loewen 1978 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i i ABSTRACT E. co l i was infected with T7 as primary infector and T4 as secondary infector. When the time between primary and secondary infect ion was 5 minutes or more, most of the result ing plaques were T7. Varying m u l t i p l i c i t y of T4 and T7 from 3 to 9 did not affect exclusion e f f i c iency . Genomes of excluded T4 phages were injected but did not repl icate or break down extensively. No T4 proteins were detected in superinfection exclusion. RNA synthesis upon injected T4 genomes was p a r t i a l l y inh ib i ted. T7 0.7amJS62a as primary infector gave a reduced ef f ic iency of exclusion. T7 laml93 as primary infector abolished exclusion. i i i Table of Contents page Abstract i i Table of Contents i i i L i s t of Figures v L i s t of Tables vi Acknowledgement v i i Introduction 1 Materials and Methods 5 Bacteria 5 Bacteriophages 5 Chemicals 5 Radiochemicals 5 Media and Buffers 6 Preparation of Labelled Bacteriophages 7 DNA Extraction and Pur i f i ca t ion 8 Cesium Chloride Density Gradient Centrifugation 8 SDS Polyacrylamide Gel Electrophoresis 9 Preparation of DNA F i l t e r s for Hybridization 9 Incorporation of 5-( J H)-uraci l 10 RNA Extraction Procedure 10 RNA-DNA Hybridization 11 Plat ing of Bacteria and Infective Centres 11 Results 12 Kinetics of Superinfection Exclusion 12 Effect of M u l t i p l i c i t y of Infection on Super-infection Exclusion 17 Depression of T7 Infective Centres During Superinfection Exclusion 19 Lack of Inhibit ion of T4 Injection By Pr ior Infection with T7 21 Fate of T4 DNA Injected During Superinfection Exclusion 25 iv Protein Synthesis During Superinfection Exclusion 28 Superinfection Experiments Using T7 , T7am 0.7, T7aml.O 31 RNA Synthesis During Superinfection Exclusion 33 Conclusions References n L i s t of Figures Page Figure 1. Kinetics of exclusion of T4 by T7 Figure 2. K i l l i n g of c o l i by T7 Figure 3. Density transfer experiment Figure 4. Autoradiogram of polyacrylamide gel electrophoresis of infected ce l l s 13 15 26 29 Effect of moi on y i e l d of T4 and T7 infect ive centres after superinfection Depression of in fect ive centres in superinfection exclusion Lack of i nh ib i t i on of T4 inject ion by pr ior infect ion with T7 Superinfection of T4 upon T7 + , T7am0.7, T7aml.O RNA-DNA hybridization vi i Acknow!edgement I thank Bob M i l l e r for his encouragement and assistance through-out the course of th is project. I thank Tony Warren, Kirk Maltman and Vikram Misra for helpful discussions and proofreading of the manuscript. F i na l l y , I would l i k e to thank Kirk Maltman for being a good person to share a lab with. 1 INTRODUCTION Exclusion among bacterial viruses is a phenomenon in which two d i f ferent bacteriophages infect the same bacterium, with only one virus infect ing successfully and producing progeny. Exclusion was f i r s t formally described in 1945 (Delbruck and Luria 1945). Since then a number of bacteriophage pairs have been examined with respect to exclusion. In the i n i t i a l mixed infect ion experiments Delbruck and Luria (1945) studied the phages T l and T2. When Tl and T2 were mixed with bacteria T2 produced infect ive centres; no T l in fect ive centres were detected. In order for T l to dominate T2 in a mixed infect ion i t required at least a 7 minute temporal advantage, otherwise T2 would dominate. A control experiment showed Tl and T2 did not interfere with the adsorption rate of the other phage. Mixed infect ion would not result in any single bacterium producing both T l and T2 part ic les (Delbruck and Luria 1942). U l t rav io le t l i ght - inact i vated T2 would s t i l l exclude T l (Luria and Delbruck 1942). In mixed infections of T l and T4, T4 behaved in qua l i ta t i ve ly the same way as T2 in T l and T2 mixed infections (Delbruck 1945). Delbruck (1945) defined the "depressor e f fect " of mutual exclusion: in a mutual exclusion experiment, the burst s ize of the excluding phage i s reduced from the burst s ize of the same phage in a single infect ion experiment. Hershey (1946) observed that mixed infections with T4r + and T4r~ gave r i se to mottled plaques, indicating that infect ive centres yielded 2 types of phage. T2r + _ j2r' mixed infections (Delbruck 2 and Bailey 1946) resulted in a higher than expected number of non-mottled plaques; a result which indicated genetic exchange was taking place. Exchanges of genetic information were also noted between pairs of T6 and T4 r + and r~ phages (Delbruck and Bailey 1946). Exclusion among d i f ferent T even phages is a part ia l exclusion. Part ia l exclusion of T2 by T4 was examined by Streis inger and Weigle (1956). T2 progeny of a T2-T4 mixed infect ion were not excluded when used for a subsequent T2-T4 mixed infect ion (Streis inger and Weigle 1956). At least 2 points on the early region of the T2 genome determine the sen s i t i v i t y of T2 to exclusion by T4 (Russell and Huskey 1974). Russell and Huskey (1974) postulate that exclusion phenomena provide a mechanism for speciation of bacteriophages. They suggest regions of exclusion sen s i t i v i t y on the T2 map may correspond to loops of nonhomology in the T2-T4 heteroduplex map. Analysis of pulse label led proteins from superinfection exclusion of T3 by T7 on SDS polyacrylamide gels provides no evidence for expression of T3 genes in the exclusion (Hirsch-Kauffmann et al 1976). Analysis of superinfecting pairs of T7 which d i f fered by a single amber mutation infect ing a supressor posit ive host has been performed (Hirsch-Kauffmann 1976). Functions of the superinfecting virus were not expressed. In a genetic analysis of superinfection exclusion of T7 by T7 exclusion occurred i f the T7 RNA polymerase gene (and therefore the late genes), protein kinase gene, 1.1 gene, or DNA l igase gene were inactivated in the excluding virus (Hirsch-Kauffmann et al 1976). By the process of elimination this l e f t gene 3 0.3 or an as yet un ident i f ied, small early protein. A T7 mutant which was 1 am would exclude T7 whereas a mutant which was lam, 0.3am would not (Hirsch-Kauffmann et al 1976). This result seems to point to the 0.3 gene product as being responsible for exclusion but a T7 phage which was 1 am 0.3am would exclude, suggesting a late function is also involved in superinfection exclusion (Hirsch-Kauffmann et al 1976). Despite the fact that exclusion occurred even i f the infect ion was carr ied out in the presence of r i fampicin or chloramphenicol, the 0.3 gene product could not be detected in SDS polyacrylamide gel electrophoresis of the T7 v i r ion (Hirsch-Kauffmann et al 1976). Gene 0.3 functions in overcoming host re s t r i c t i on and modification (Studier 1975). As a result of these experiments the view has been expressed that superinfection exclusion of T7 by T7 is due to a membrane event which prevents inject ion of DNA of superinfecting T7 (Schweiger et al 1975). This view is in concord with the results of Benbasat, Burck and M i l l e r ( in press) showing primary infect ing T7 f a c i l i t a t e s inh ib i t i on of inject ion of DNA of T7 phages which attatch at a la ter time. Two other results are related to the membrane a l terat ion hypothesis. F i r s t , inact ivat ion of receptors for T5 (which is related to T7) during infect ion of E. co l i takes 30 minutes at 30° (Dunn and Duckworth 1977). Second, the gene 0.3 product has been linked to T7 induced eff lux of potassium ions from T7 infected ce l l s (Ponta et al 1976), indicating T7 infect ion has an effect on bacterial membranes. Shut-off of E. co l i by T7 is relevant to superinfection exclusion since expression of phage early genes is largely dependent on host functions. Inactivation of host RNA polymerase during T7 infect ion is primari ly due to binding of T7 gene 2 product to the polymerase 4 (Hesselbach and Nakada 1977a) and is enhanced by the T7 qene 0.7 product. The T7 gene 2 product has been pur i f ied (Hesselbach and Nakada 1977b). T7 gene 0.7 gene product phosphorylates the beta subunit of E. co l i RNA polymerase ( Z i l l i g 1975). Phage lambda excludes T l . Two lambda gene products required for exclusion of Tl have been ident i f ied (Christensen, Gawron and Hal pern 1978): N and Q gene products. The suggestion has been made that lambda N gene-mediated exclusion of T l is due to N gene product interfer ing with T l rep l i ca t ion . From th is overview of exclusion studies of bacteriophages of d i f fe r ing relationships 2 points emerge: 1) Exclusion sen s i t i v i t y may be due to a defined genetic locus of the excluded phage in addition to functions of the excluding phage 2) Exclusion may have 2 temporal modes, late and ear ly. The study reported here examined the exclusion of T4 by T7. In par t i cu la r , attention was focussed on macromolecular synthesis during exclusion. The molecular events studied in the course of superinfection were: phage attatchment and DNA in jec t i on , DNA synthesis, RNA synthesis and protein synthesis. MATERIALS AND METHODS 5 Bacteria Escherichia c o l i B e and B23 were used as the nonpermissive hosts for amber mutants in a l l experiments. Both are su~. O i l 1 i s a su +  E. c o l i B derivative which was used to prepare amber phage stocks. Cultures were grown to between 2 and 3 x 10 cells/ml in H broth before resuspension and growth in defined media. Bacteriophages The following T7 strains were from the co l lect ion of F. W. Studier: T7 + , T7 laml93, T7 0.7amJS62a. T4D was from the co l lec t ion of A. H. Doermann. Chemicals 5-Bromo-2'-deoxyuridine (5-BrUrd) and 5-fluoro-2'-deoxyuridine (5-FdUrd) were purchased from Sigma Chemical Company. Tetra-methylethylenediamine (TEMED), N,N'-bis-methylene acrylamide (bis) and ammonium persulphate (AP) were from Biorad. Radiochemicals 5-( 3 H)-uraci l was purchased from Amersham. ( 3 2 P) cs H 3 3 2 P 0 4 and uniformly label led ( 1 4C)-amino acids were from New England Nuclear. 6 Media and Buffers K G was composed of 0.1 M T r i s - C l , 0.16 mM Na 2S0 4, 1.0 mM MgS04, 0.05% NaCI, 0.1% glucose, 0.1 mM CaC l 3 , 3 juM FeC l 3 , 0.05% vitamin free casamino acids, f i na l pH 7.4 (Kozinski and Szybalski 1959). TCG was supplemented with phosphate by the addition of 0.1 M KH 2P0 4 as 0.65 ml per 100 ml TCG for normal P0 4 TCG, 0.128 ml per 100 ml TCG for 1/5 P0 4 TCG and 6.4 ml per 100. ml TCG for lOx P0 4 TCG. lOx P0 4 TCG contained 250 ug of P0 4 per ml. 5-BrUrd TCG was lOx P0 4 TCG supplemented with 100 jug 5-BrUrd per ml, 25 jug per ml urac i l and 10 jug per ml Fudr. H broth contained per lOOOvml of d i s t i l l e d water: 5 g peptone (Difco), 1 g glucose, 5 g NaCI, 8 g nutrient broth (Difco), pH adjusted to 7.0. TN was 0.01 M T r i s - C l , 0.15 M NaCI, f i na l pH 7.4. TNE was TN supplemented with 0.015 M EDTA. T7 Tr is Sa lt was 0.01 M T r i s - C l , 1.0 M NaCI, pH 7.4. T4 Tr is Sa lt was 0.01 M T r i s - C l , 0.15 M NaCI, pH 7.35. M9S medium was a modification of that o f Bol le et al (1968) and was: 0.04 M Na 2HP0 4, 0.022 M KH 2P0 4, 0.05% NaCI, 0.02 M NH4C1, 0.01 M FeCl-j, 1% vitamin-free casamino acids (Difco) and 0.4% glucose, pH 7.2. M9S.1 was the same as M9S except the concentration of vitamin-free casamino acids was 0.1%. Top layer agar for phage plating contained, per 1000 ml: 7 g Bacto agar, 10 g Bacto peptone, 8 g NaCI, 1 g glucose, pH 7.0. Bottom layer agar contained, per 1000 ml: 11 g Bacto agar, 10 g Bacto tryptone, 8 g NaCI, 1 g glucose, pH 7. RNA extraction buffer was 0.05 M T r i s - C l , 2 mM EDTA, 1% SOS, pH 6.8. Shearing medium contained 1 mM MgS0A, 0.1 mM CaCl_, 0.1% ge la t in . 7 TAG was the same as TCG except that the vitamin-free casamino acids were supstitured with the twenty amino acids, each at 10 /ig/ml. Sample so lub i l i za t ion mixture for SDS polyacrylamide gel electrophoresis was 0.125 M T r i s - C l , 4% SDS, 20% g lycero l , 0.01% bromphenol blue, 105 mercaptoethanol, pH 6.8. Preparation of Labelled Bacteriophages 3 32 H- P-T4 - E. co l i B23 was grown at least 2 generation to 3 x g 10 cells/ml in H broth. The bacteria were sedimented, washed and 32 resuspended in 1/5 P0^ TAG without leucine and containing P (as 32 P0^) to a spec i f i c a c t i v i t y of 1 uCi/ug. The ce l l s were grown 2 generations to 3 x 10 8 cells/ml at 37° and infected with T4 at a m u l t i p l i c i t y of infect ion (moi) of 10. Ten minutes af ter in fec -t ion ( H)-leucine was added to 5 >jCi/ml and the culture was i n -cubated at 37° unt i l l y s i s . The lysates were treated with chloro-form and DNase, and the phage were pur i f ied by d i f f e ren t i a l cent-r i fugat ion. An al iquot of the phage preparation was transferred to a glass f iber f i l t e r , dr ied, overlayed with toluene-based 1,4-bis-(2)-(5-phenyloxolyl)benzene and 2,5-biphenyloxazolyl s c i n t i l l a n t and assayed for rad ioact i v i ty with a Nuclear Chicago Isocap 300 s c i n t i l l a t i o n spectrophotometer. 3 2 P-T4 - 3 2 P-T4 was prepared as described for 3 H- 3 2 P-T4 3 except that ( H)-leucine was not added and the medium was 1/5 P0 4 TCG. 8 DNA extraction/and pur i f i cat ion Infective centres or bacteria in TNE were treated with 1% sodium dodecyl sulphate (SDS) for 20 minutes at 37° and incubated with se l f digested pronase (30',37°) at 1 mg/ml for 4.5 hours or over-night at 30°. An equal volume of water-saturated phenol was added to the samples which were then placed in a tube r o l l e r for 1 hour at room temperature. The phenol layer was removed and the samples washed 3 times with water-saturated ether. Phage DNA was extracted d i r e c t l y with phenol followed by 3 ether washes. Cesium Chloride Density Gradient Centrifugation Native DNA was treated as fol lows: 0.3 ml of sample was mixed with 0.5 ml of TNE and transferred to a polyallomer tube containing 3.2 ml satured CsCl solution (in d i s t i l l e d water at room temperature). The tube contents were mixed, overlayed with para-f f i n o i l , placed in a SW 50.1 rotor and spun at 10° for 64-72 hours at 30,000 revolution per minute in a Spinco model L u ltracentr i fuge. The gradient was dripped in 8 drop fract ions onto squares of What-man 3MM f i l t e r paper 2 cm by 2 cm. The squares were dr ied, over-layed with toluene-based s c i n t i l l a n t and counted in the s c i n t i l l a t i o n spectrophotometer. 9 SDS Polyacrylamide Gel Electrophoresis Samples of infected and uninfected ce l l s which had incorporated (^C)-amino acids were sedimented and resuspended in 0.1 ml of sample so lub i l i za t ion mixture and the sample tubes were placed in a boi l ing water bath for 2 minutes. The samples were applied to 0.4 x 1 cm wells in a polyacrylamide gel 14 x 11 x 0.16 cm. The stacking gel (0.125 M T r i s - C l , 0.1% SDS, 3% acrylamide, 0.08% Bis, 0.1% TEMED, 0.07% ammonium persulphate, pH 6.8) occupied the top 2 cm of the ge l . The running gel (0.375 M T r i s - C l , 0.1% SDS, 10% acrylamide, 0.26% B is , 0.1% TEMED, 0.07% ammonium persulphate, pH 6.8) occupied the lower 12 cm of the ge l . The running buffer was composed of 0.025 M T r i s - C l , 0.192 M glycine, 0.1% SDS, pH 8.8. The gel was run with cooling at about 7 watts power unt i l the tracking dye reached the bottom of the ge l . The gels were stained (2% TCA, 1% Coomassie B r i l l i a n t Blue, 15% g lac ia l acetic ac id, 47.5% ethanol) overnight at room temperature. The gels were destained (2% TCA, 1% Coomassie B r i l l i a n t Blue, 15% g lac ia l acetic acid) for 1 hour and washed (7.5% g lac ia l acetic acid) for about 0.5 hours. The gels were dried with heat and vacuum onto a sheet of Whatman 3MM f i l t e r paper and exposed to Kodak XR-2 X,ray f i l m . Preparation of DNA F i l t e r s for Hybridization 90 ml of 6x SSC was combined with 720 pg DNA. NaOH was added to 0.33 N and the sample was s t i r red for 1 hour at room temperature. The DNA solution was brought to neutra l i ty by adding 6 N HC1 and applied to a Mi H i pore HAWP 142 50 f i l t e r . The f i l t e r was washed with 100 6x SSC. The f i l t e r was dried in a part ia l vacuum over dessicant for 2-3 hours at 80°. The f i l t e r was cut into 9 cm c i r c l e s containing 6 ug of DNA per f i l t e r . 10 Incorporation of 5-( H)-uraci l 20 JJI of a sample which had been extracted for RNA was placed into a tube containing 1 ml of 1 M KOH and l e f t overnight. 1 ml of 1 N HC1 was then added, followed by 50% t r i ch loroacet ic acid (TCA) and 50 mM sodium pyrophosphate (PPi) to give a f i na l concentration of 10% TCA, 10 mM PPi. After 1 hour 0.1 ml of E. co l i B e at 3 x 10 8 cells/ml was added as a ca r r i e r . The sample was f i l t e r e d through a Whatman GF/A glass f i be r f i l t e r . The f i l t e r was washed 3 times with 5 ml of 5% TCA, 10 mM PPi and 2 times with ethanol. The f i l t e r was dr ied, placed into a v ia l containing s c i n t i l l a n t and was counted 3 for H in the s c i n t i l l a t i o n spectrophotometer. Samples for which a lkal ine degradation was not performed were treated as above except the 1 M KOH and 1 N HC1 were not added. RNA Extraction Procedure 3 Samples of infected and uninfected ce l l s which had incorporated 5-( H)-urac i l were sedimented and resuspended in RNA extraction buffer and the sample tube was placed in a boi l ing water bath for 2 minutes. Then 0.2 ml of 2 M sodium acetate, pH 5.2 was added to each sample, after which 2.2 ml of water-saturated phenol was added. The samples were agitated for 5 minutes and the aqueous phase was removed to a tube containing a further 2.2 ml of phenol. After agitat ion the aqueous phase was extracted twice with an equal volume of chloroform. Ethanol was added to the aqueous phase to a f i na l concentration of 80% and the sample was placed in the cold for at least 4 hours to prec ip i tate the RNA. The RNA was sedimented in the cold, washed with cold 80% ethanol and resuspended in 0.8 ml of 0.01 M T r i s - C l , 5 mM MgCl 2, pH 7.4. RNase free DNase was added to f i na l concentration of 0.025 mg/ml and 11 the samples were incubated 10 minutes at 37°. EDTA was added to a f i na l concentration of 5 mM and 2 ml of chloroform was added. The aqueous phase was removed and the OD^Q of each sample was determined with a Unicam SP800 double beam spectrophotometer. RNA-DNA Hybridization RNA samples were hybridized in 3x SSC, 0.1% SDS. Hybridization v ia l s contained 8 f i l t e r s : 2 f i l t e r s each with E. c o l i DNA, T7 DNA, T4 DNA and 2 f i l t e r s without DNA. The tota l amount of each type of DNA per v ia l was 12 jug. Not more than 2.7 yg of RNA was added to each hybrid-izat ion v i a l . After overnight hybridization at 65° the f i l t e r s were washed 2 times with 3 ml of 3x SSC. F i l t e r s were incubated in 3x SSC with 1 wg/ml RNase for 10 minutes at 37°. The f i l t e r s were washed 3 times with 3 ml of 3x SSC, placed into separate s c i n t i l l a t i o n v i a l s , dr ied, overlayed with s c i n t i l l a t i o n f l u i d and assayed for radio-a c t i v i t y . Plating of Bacteria and Infective Centres Platings were performed according to methods outlined by Adams (1959). 2 minutes af ter phage infect ion or superinfection an al iquot of infected culture was transferred to combined T4 and T7 antiserum and incubated at 30° for 5 minutes. The sample was d i luted out of antiserum and d i lut ions were plated on overlays for in fect ive centres and surviving bacteria. 12 RESULTS Kinetics of Superinfection Exclusion Effectiveness of exclusion was examined as the time of superinfec-tion was varied. The results are shown in Figure 1. The results indicate that T4 is only excluded when T4 superinfection occurs 5 minutes or more after T7 in fect ion. The kinet ics of k i l l i n g of E. c o l i by T7 are presented in Figure 2. T7 requires 5 minutes to k i l l E. c o l i and form maximal numbers of in fect ive centres. The conclusion is that when T7 does not form infect ive centres T4 is not excluded by T7. 13 ^ I I 1 I 0 2 4 6 8 Time of superinfection (minutes) Figure 1 14 Figure 1 Legend o Kinetics of exclusion of T4 by T7. E. co l i was grown to 2 x 10 ce l l s per ml in H broth and T7 was added at an moi of 10. At 1, 2.5, 5 and 9 minutes after T7 infect ion aliquots of infected culture were transferred to premeasured volumes of superinfecting T4 at an moi of 10. Two minutes after superinfection samples of infected cultures were transferred to T4 and T7 antiserum, incubated for 5 minutes at 30° and di luted out of antiserum. T4 and T7 infect ive centres and surviving bacteria then were scored. Survivors were less than 2%. Total phage Q y ie lds for each sample were on the order of 2 x 10 phage per ml. ( ) - T7 i n fec t i ve , ( ) - T4 infect ive centres. Figure 2 Figure 2 Legend K i l l i n g of E. co l i by T7. Bacteria at 2 x 10 ce l l s per ml H broth were infected with T7 at an moi of 10 at zero time. Surviving bacteria were plated at 1, 2.5 and 4 minutes after in fect ion. 17 Effect of M u l t i p l i c i t y of Infection on Superinfection Exclusion An experiment was performed to examine the effect of varying the m u l t i p l i c i t y of infect ion (moi) on re la t i ve y ie lds of T4 and T7 infect ive centres from superinfection. When T4 infects 5 minutes after 17 in fect ive centres of both phages can be detected. This was chosen as the superinfection time, because the re la t i ve number of T4 and T7 infect ive centres is of prime interest. The results are displayed in Table 1. Over the range of mo.is the re lat i ve y i e l d of T4 and T7 infect ive centres is constant. 18 moi T4, T7 Yield of in fect ive centres T4 T7 3 2.42 x 10 8 8.30 x 10 7 6 2.95 x 10 8 7.80 x 10 7 9 2.68 x 10 8 7.85 x 10 7 Table 1 Effect of moi on y i e l d of T4 and T7 infect ive centres after superin-fect ion. T7 was added at given moi at time zero. T4 was added at an ident ica l moi 5 minutes l a te r . Infective centres and survivors were plated as described in Methods and Materials. Survivors were less than 1%. 19 Depression of T7 Infective Centres During Superinfection Exclusion A consistent observation made throughout the course of this study was that although T4 was excluded i t coused a depression in the y i e l d of T7 infect ive centres. A typical result i s shown in Table 2. When T4 and T7 infected separate cultures the number of in fect ive centres equalled the number of infected bacter ia. In the superinfection T7 c lear ly dominated - less than 3 per cent of the infect ive centres were T4 - but the number of in fect ive centres did not equal the number of infected bacteria. This depressor ef fect was also noted by Delbruck (1945) in superinfection exclusion of T2 by T7. 20 Yield of in fect ive centres T7 T4 T7 1.5 x 10 ,8 T4 1.8 x 10' 8 superinfection 5.4 x 10 7 1.5 x 10 6 Table 2 Depression of in fect ive centres in superinfection exclusion. o Infected culture contained 1.4 x 10 ce l l s per ml. Bacteria were infected with phage at an mo8 of 10. Superinfection with T4 was performed 5 minutes after T7 in fect ion. In a l l three samples in fect ive centres and surviving bacteria were monitored at 7 minutes after i n i t i a l in fect ion. Surviving bacteria were less than o . l % . 21 Lack of Inhibit ion of T4 Injection by Pr ior Infection With T7 T7 might exclude T4 by a c e l l surface phenomenon. To test this hypothesis an experiment s imi lar to that of Hershey and Chase (1952) was performed to examine phage attachment and DNA inject ion during 32 superinfection. T4 was label led with ( P) orthophosphate which 3 label led phage DNA and ( H)- leucine which label led the phage protein coat. After infect ion cultures were sedimented and resuspended to remove unadsorbed phage and then were sheared in a Waring blendor. Aliquots of each culture were set aside as nonsheared controls to monitor spontaneous desorption of phage. After shearing infected ce l l s were sedimented and resuspended. The supernatants and re-32 3 supended pel lets were analyzed for P and H. 32 3 Absence of either P or H in the pe l le t would mena that either phage attachment did not take place or that the phage attached but 32 did not in ject i t s DNA and was sheared o f f . Association of P with 3 the pe l le t and H with the supernatant would mean the phage attached, the DNA was injected and the empty protein coat was sheared o f f . The control for inject ion of DNA by T4 alone is shown in Table 2. In the 3 32 nonblended T4 alone sample H and P were associated with the pe l le t which is the result expected i f attachment took place and 32 no shearing was performed. In the T4 alone blended sample P was 3 associated with the pe l le t while the H was associated with the super-natant. This result indicates attachment of the phage, in ject ion of DNA and removal of the empty phage head by shearing. The data for 3 32 infect ion with T7 and superinfection with H- P-T4 is shown in Table 2. The nonblended control shows that attachment occurred, 3 32 as both H and P were associated with the pe l l e t . The blended sample 22 3 32 shows H associated with the supernatant and P associated with the pe l l e t , indicating inject ion of T4 DNA . The conclusion to be made from the 2 experimental series is the same: When T4 alone infects bacter ia, i t injects i t s DNA and when T4 infects bacteria 7 minutes after T7 (a superinfection time at which T4 is excluded) T4 injects i t s DNA. 23 Table 3 Distr ibut ion of recovered rad ioact i v i ty (%) 3 R 32f T4 alone Unblended Supernatant 14.5 11.5 Pe l le t 85.5 88.5 Blended Supernatant 72.7 12.3 Pe l le t 27.3 87.7 Superinfection Unblended Supernatant 12.3 7.9 Pe l le t 87.7 92.1 Blended Supernatant 81.3 35.8 Pe l le t 18.7 64.2 24 Table 3 Legend 0 Superinfection samples: Bacteria were grown to 2 x 10 ce l l s per ml in lOxPi TCG. At zero time ce l l s were infected with T7 at an moi of 10. At 7 minutes af ter infect ion the culture was infected with 3 H- 3 2 P-T4 at an moi of 10. T4 alone: Bacteria at a density of 2 x 10 ce l l s per ml in lOxPi TCG were infected with 3 H- 3 2 P-T4. Moi of T4 was 0.5 in order to prevent T4 superinfection exclusion of i t s e l f . T4 alone and superinfection: Thirteen minutes after i n i t i a l infect ion unattatched phage were removed by sedimenting the infected ce l l s and the pe l le t was resuspended in shearing medium with chloramphenicol added to 0.1 mg/ml. The samples were sheared for 5 minutes in a Waring blendor at 1 x 10 revolutions per minute. An al iquot of culture was set aside as nonsheared contro l . The samples were sed-imented and the supernatant and resuspended pe l le t were analyzed for rad ioact i v i ty . In the superinfection sample the y i e l d of in fect ive centres was 1.86 x 10 8 IC/ml for T7 and 5 x 10 7 IC/ml for T4. Q Exclusion did occur. Infected bacteria were at 2 x 10 ce l l s/ml. The tota l y i e l d of in fect ive centres re lat i ve to infected bacteria indicates that blending did not result in extensive bacterial l y s i s . For a l l samples between 54% and 71% of input rad ioact iv i ty was re-covered. 25 Fate of T4 DNA Injected During Superinfection Exclusion Given that T4 injects i t s DNA during superinfection exclusion, an experiment was performed to determine i f the injected T4 DNA repl icates or is degraded. The experiment was a density transfer experiment. The transfer of density was followed for T4 infect ing alone and a superinfection sample as shown in Figure 3. Unreplicated 32 32 P-T4 DNA served as a l i gh t density reference. P which banded at a density greater than the reference provided evidence of T4 rep l i ca t ion . The T4 rep l icat ion control exhibited 2 bands: a sharp one near the top of the gradient and a broader dense band further down the gradient. 32 The sharp peak is l i g h t , unreplicated P-T4 marker DNA. The broad, 32 heavy peak is Pfrom input T4 which has shifted to heavier density due to rep l i ca t ion . The superinfection sample shows no sh i f t in the density of T4 DNA indicating f a i l u re to repl icate during superinfection 32 exclusion. The shape and position of the P Band in the superinfection sample gives clues as to whether the injected DNA i s degraded. Mild degradation would make the band wider. Extensive degradation would 32 result in the P being distr ibuted throughout the gradient. In-act ivat ion by nicking cannot be detected on neutral CsCl gradients. When T4 DNA is injected during superinfection exclusion i t is neither re-pl icated nor extensively degraded. 27 Figure 3 Legend Density transfer experiment. E. co l i was grown in H broth to 3 o x 10 ce l l s/ml, ice c h i l l e d , sedimented, washed with TCG, re-suspended in 5-BrUrd TCG medium and grown for 90 minutes at 37° in the dark. Ten minutes before infect ion the ce l l s were shifted to 30°. At zero time the superinfection sample was infected with T7 at an moi of 10. At 5 minutes the superinfected sample and the 32 T4 alone sample were infected with P-T4 at an moi of 5. At 10 and 15 minutes 2 ml aliquots of infected culture were transferred to a tube containing 5 ml of ice cold TNE. At 16 minutes chlor-amphenicol was added to both samples at a concentration of 100 jug/ml. At 20 and 25 minutes 2 ml al iquots were transferred to a tube containing 5 ml of ice cold TNE. The ce l l s in a l l samples were sedimen-ted and resuspended in TNe. DNA extract ion, cesium chloride density gradient centrifugation and gradient fract ionation were performed as described in Methods and Materials. I l l u s t rated in Figure 3 are the superinfection and T4 samples from the 20 minute time point. The f igure is representative of a l l time points in the experiment. 28 Protein Synthesis During Superinfection Exclusion Examination of proteins synthesized during superinfection exclusion could provide clues as to the nature of superinfection exclusion. Pulse label led proteins from ce l l s infected with 11, T4 and T4 infect ing after T7 were separated by SDS polyacrylamide gel electrophoresis. The result ing gels were autoradiographed. The autoradiograms are presented in Figure 4. No T4 bands can be detected in the superinfection lanes. i Figure 4 29 30 Figure 4 Legend E. co l i B e was grown to 2 x 10 8 cells/ml in M9S medium at 30°. The ce l l s were sedimented and resuspended in M9S.1 medium. The re-suspended ce l l s were s p l i t into 4 equal parts. The 4 cultures were incubated for 5 minutes. At zero time cultures A and C were i n -fected with T7 at an moi of 10 and culture B was infected with T4 at an moi of 10. At 5 minutes after zero time culture C was in fected with T4 at an moi of 10. At 6, 12, and 18 minutes after zero time 1 ml of each of cultures A, B and C was transferred to a tube containing 1.5 uCi of uniformly label led (^C)-amino acids. Four minutes after each transfer 0.1 ml of 10% casamino acids was added to each labe l l i ng tube., Eight minutes after each transfer the labe l l ing tubes were c h i l l e d . At 6 minutes af ter zero time 1 ml of culture D was l abe l led , .chased and ch i l l ed as described above, A l l samples were sedimented and prepared for SDS poly-acrylamide gel electrophoresis as described in the methods. A - T7, B - T4, C - T7 followed by T4, D - uninfected. Assignment of protein bands was made according to Studier (1972, 1973) for T7 and 0 ' Fa r re l l and Gold (1973) and 0 ' F a r r e l l , Gold and Huang (1973) for T4. 31 Superinfection Experiments Using T7+!,T7am0.7, T7aml.O In an attempt to ident i fy the T7 function responsible for exclusion of T4 2 amber mutants of T7 were used in superinfection experiments. 11 gene 0.7 specif ies a protein kinase. The kinase phosphorylates about 40 E. c o l i proteins including the beta subunit of E. c o l i RNA polymerase. As shown in Table 4 T4 superinfected 2 d i f ferent cultures for each type of T7 - one at 6 minutes and one at 9 minutes. The re lat i ve numbers of in fect ive centres gave a measure of e f f i c iency of exclusion. T7 + i s seen to exclude T4 when T4 superinfects at 6 and 9 minutes. The exclusion shows character i s t ic depression of T7 infect ive centres. T7am0.7 does not exclude com-plete ly when T4 superinfects at 6 minutes or 9 minutes but T7 dominates at the la ter time. Although an amber mutation in T7 gene 0.7 reduces the e f f i c iency of exclusion i t does not eliminate i t . Un-l i k e T7 gene 0.7, T7 gene 1.0 is an essential gene for expression of T7 infect ive centres. Despite the fact that when using T7aml.0 superinfected by T4 no T7 infect ive centres w i l l be observed, i f gene 1.0 is not involved in superinfection exclusion of T4 a s i g -n i f i cant exclusion should occur. This was not the case. The data shows that T4 infect ive centres are not much below the value o of 2.1 x 10 per ml expected i f T4 was responsible for the k i l l i n g of 100% of the ce l l s in the infected culture. Two po s s i b i l i t i e s ex i st - 1) T7 RNA polymerase has a d i rect function in excluding T4 2) a late gene product is responsible for exclusion. 32 I n i t i a l Infector Y ie ld of in fect ive centres (IC/ml)  Superinfection at 6' Superinfection at 9' T7 T4 T7 T4 T7 + 1.1 x 10 8 <0l 2.9 x 10 7 1.5 x 10 6 T7am0.7 4.1 x 10 7 9.1 x 10 7 7.8 x 10 7 2.7 x 10 7 T7aml.O <O.I 1.8 x 10 8 <O.I 1.6 x 10 8 Table 4 Superinfection of T4 upon T7 + , T7am0.7, T7aml.O. Yield of in fect ive o centres per ml. Bacteria at a density of 2.1 x 10 in H broth in three d i f ferent f lasks were infected with T7 + , T7am0.7 and T7aml.O at an moi of 7. At 6 and 9 minutes after infect ion aliquots of each of the infected cultures were transferred to tubes containing T4 at an moi of 7. Infective centres were monitored at 2 minutes after superinfection. RNA Synthesis during superinfection exclusion 33 In an attempt to ident i fy the mechanism by which T7 blocks superinfecting T4, RNA synthesis during superinfection exclusion was examined. The technique used was to perform infections in 3 3 the presence of 5-( H)-uraci l and hybridize the result ing H- label-ed RNA to T7 DNA and T4 DNA. The results are shown in Table 5. Infection with T7 results in ( H)- label led RNA hybridizing only to T7 DNA and infect ion with T4 results in ( 3 H)- label led RNA hybrid-iz ing only to T4 DNA. When T7 infect ion is followed at 7 minutes by T4 infect ion hybridization to both T4 DNA and T7 DNA i s noted. Does the ( H)- label led RNA hybridizing to T4 DNA represent the RNA required to produce the 8% nonexcluded T4 phages in th is sample or does the injected T4 genome of excluded T4 encounter a post-transcr ipt ional block? To answer this question the rat io of RNA hybridizing per in fect ive centre was calculated for : a) T4 at 6 minutes and b) T4 in the superinfection at 12 minutes. 3 The amount of ( H)- label led RNA hybridizing to T4 DNA per T4 infec-t i ve centre in the superinfection is over twice that in the T4 infection.This suggests that injected T4 genomes do encounter a transcr ipt ional block but that transcr ipt ion may also take place to a small extent on excluded genomes. 34 3 3 Infector Time (min) Input H (cpm) % input H hybridized to T7 DNA T4 DNA T7 6 13,262 2.4 <0.1 26,254 3.1 <0.1 12 3,259 44.5 <9.1 6,518 45.0 <0.1 T4 6 5,204 <0.1 32.3 10,408 <0.1 24.2 12 14,024 <0.1 24.1 28,048 <0.1 24.0 Super- 6 16,018 5.6 <0.1 infect ion 32,036 6.9 (0.1 12 6,591 36.8 2.0 13,182 36.4 1.8 Table 5 35 Table 5 Legend RNA-DNA hybr idizat ion. E. c o l i B e was grown to 2 x 10 8 cells/ml in H broth and s p l i t into 3 equal volumes. At zero time flasks A and C were infected with T7 at an moi of 10 and f lask B was i n -fected with T4 at an moi of 10. At 7 minutes f lask B was infected with T4 at an moi of 10. At 3 minutes a l l f lasks received 5-( H)-urac i l to a f i na l concentration of 10 jjCi/ml. At 6 and 12 minutes 2 ml al iquots of each f lask were transferred to a tube containing 2 ml of ice ch i l l ed H broth. A l l 6 samples were sedimented, re -suspended in RNA extraction buffer and extracted for RNA as descr ib-ed in Materials and Methods. Evaluation of incorporation of 5-( H)-uraci l into acid insoluble and a l k a l i insoluble material was per-formed as described in Materials and Methods. At least 91% of 3 acid insoluble H was a l k a l i l a b i l e . In the superinfection sample 8% of in fect ive centres were T4 and 92% were T7. Preparation of f i l t e r s binding E. c o l i DNA, T7 DNA and T4 DNA and hybridization of RNA samples to the f i l t e r s was performed as described in Materials and Methods. Background hybridization to E. co l i DNA f i l t e r s was 3 0.22% of input H or less. Hybridization to blank f i l t e r s was 0.2% of input 3H or less. 36 CONCLUSIONS T7 excluded T4 when T4 superinfected E. c o l i 5 minutes or more after T7 in fect ion. In the range of moi from 3 to 9 no change in ef f ic iency of exclusion could be seen. The number of T7 in fect ive centres in an exclusion experiment was lower than the number of T7 infect ive centres in a single T7 infect ion experiment Excluded T4 injects i t s DNA Injected T4 DNA is neither repl icated nor extensively broken down. There i s l i t t l e or no T4 protein synthesis during exclusion. A low level of t ranscr ipt ion occurs on excluded T4 genomes. Transcription i s depressed on primary infect ing T7 genomes. T7 protein kinase i s involved in exclusion of T4 as i s T7 RNA polymerase or a T7 late gene product. 37 REFERENCES Adams, M. 1959. Bacteriophages. New York: Wiley Interscience Publishers Inc. Bo l l e , A., R.H. Epstein, W. Salser, and E. P. Geiduschek. 1968. Transcription During Bacteriophage T4 Development: Synthesis and Relative S t a b i l i t y of Early and Late RNA. Journal of Molecular Biology 31;325-348. Christensen, J . R., M. C. Gawron, and J . Halpern. 1978. Exclusion of Bacteriophage T l by Bacteriophage Lambda. 1. Early Exclusion Requires Lambda N Gene Product and Host Factors involved in N Gene Expression. Journal of Virology 25:527-534 Delbruck, M. 1945. Interference between Bacterial Viruses. I l l The Mutual Exclusion Effect and the Depressor E f fect . Journal of  Bacteriology 50:151-170 Delbruck, M. and W. T. Bai ley, J r . 1946. Induced Mutations in Bacter ial Viruses. Cold Spring Harbour Symposium on Quantitative Biology ll_:33-37 Delbruck, M., and S. E. Lur ia. 1942. Interference between Bacterial Viruses. I Interferince Between Two Bacterial Viruses Acting Upon the Same Host and the Mechanism of Virus Growth. Archives of Biochemistry 1_: 111-141 Dunn, G. B., and D. H. Duckworth. 1977. Inactivation of Receptors for Bacteriophage T5 During Infection of E. c o l i B. Journal of Virology 24:419-421 Her r l i ch , P., H. J . Rahmsdorf, S. H. Pa i , and M. Schweiger. 1974. Translational Control Induced by Bacteriophage T7. Proceedings of the National Academy of Science USA 71:1088-1092 Hershey, A. D. 1946. Spontaneous Mutations in Bacter ial Viruses Cold Spring Harbour Symposium on Quantitative Biology 11:67-77 38 Hershey, A. D., and M. Chase. 1952. Independent Function of V i ra l Protein and Nucleic Acid in Growth of Bacteriophages. Journal of General Physiology 36:39-56 Hesselbach, B. A., and D. Nakada. 1977a. "Host Shutoff" Function of Bacteriophage T7: Involvement of T7 Gene 2 and Gene 0.7 in the Inactivation of E. co l i RNA polymerase. Journal of  Virology 24:736-745 Hesselbach, B. A., and D. Nakada. 1977b. I Protein: Bacteriophage T7-coded Inhibitor of Escherichia co l i RNA Polymerase. Journal of Virology 24:746-760 Hirsch-Kauffmann, M., M. Pfennig-Yeh, H. Ponta, P. Her r i i ch , M. Schweiger, 1976. A Virus Specif ied Mechanism for the Prevention of Mult iple Infection - 17- and T3-mutual and Superinfection Exclusion. Molecular and General Genetics 149:243-249 Kozinski, A. W., and W. Szybalski. 1959. Dispersive Transfer of the Parental DNA Molecule to the Progeny of Phage 0X 174. Virology 9_: 260-274 Lur ia , S. E., and M. Delbruck. 1942. Interference Between Inact ivat-ed Bacterial Virus and Active Virus of the Same Strain and of a Different St ra in. Archives of Biochemistry 1:207-218 0 ' F a r r e l l , P. Z., and L. M. Gold. 1973a. The Ident i f icat ion of Pre-rep l i ca t i ve Bacteriophage T4 Proteins. Journal of B io log ica l  Chemistry 248:5499-5501 0 ' F a r r e l l , P. Z., and L. M. Gold. 1973b. Bacteriophage T4 Expression -Evidence for Two Classes of Prerepl icat ive Cistrons. Journal of Biological Chemistry 248:5502-5511 Russel l , R. L., and R. J . Huskey. 1974. Par t ia l Exclusion Between T Even Bacteriophages; An Incipient Genetic Isolation Mechanism. Genetics 78:989-1014 39* Schweiger, M., M. Hirsch-Kauffmann, H. Ponta, M. Pfennig-Yeh, P. Herr i i ch . 1975. Biochemistry of T7 Development in Organization and Expression of the V i ra l Genome. FEBS  Symposium 39:55-68 Amsterdam: North Holland Stre is inger, G., and J . Weigle. 1956. Properties of Bacteriophages 12 and T4 With Unusual Inheritance. Proceedings of the  National Academy of Science USA 42:504-510 Studier, F. W. 1972. Bacteriophage 11. Science 176:367-376 Studier, F. W. 1973. Analysis of Bacteriophage 11 Early RNAs and Proteins on Slab Gels. Journal of Molecular Biology 79: 237-248 Studier, F. W. 1975. Gene 0.3 of Bacteriophage 11 Acts to Overcome the DNA Restr ict ion System of the Host. Journal of Molecular  Biology 94:263-295 Z i l l i g , W., H. F u j i k i , W. Blum, D. Janekovic, M. Schweiger, H. J . Rahmsdorf, H. Ponta, and M. Hirsch-Kauffmann. 1974. In vivo and in v i t ro Phosphorylation of DNA Dependent RNA Polymerase of Escherichia c o l i by Bacteriophage-T7-induced Protein Kinase. Proceedings of the National Academy of Science USA 72:2506-2510 

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