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J. gen. Virol. (1969), 4, 489-5o4 Printed in Great Britain 489 Acquisition of Thymidylate Synthetase Activity by a Thymine-requiring Mutant of Bacillus subtilis following Infection by the Temperate Phage ¢3 By R. G. T U C K E R Microbiology Unit, Department of Biochemistry, University of Oxford (Accepted 29 October 1968) SUMMARY Infection of a thymineless strainof Bacillus subtiliswith citherthe temperate phage 43 or its clear plaque mutant ~3c enabled D N A to be formed in the absence of added thymine. Growth of phage ~3c under these conditions was associated with an enhanced rate of D N A synthesis comparcd with uninfected cultures growing in the presence of thymine; when phage 43 became a prophage the lysogcnized cellssynthesized D N A at the same rate as the uninfected bacteria with thyminc. The thymine independence of phage-infected bacteria was due to the acquisition of the cnzyme thymidylate synthctase absent in the uninfected mutant bacteria.Phage ~3c growth caused a rapid risein the specificactivityof the enzyme aftera short lag,and by the time the cellslysed there was approximately ten times more activity than in uninfected wild-type B. subtilis.In thymineless B. subtilislysogenizcd with phage 43 the amount of thymidylate synthetase was the same as in wild-type B. subti[is.Phage D N A was ablc to transform thymine-requiring B. subtilisto thymine independence, showing that it contained the gene for thymidylate synthctasc.Plaquc-forming particlescould not be separated by density gradient centrffugationfrom those possessing the thymidylate synthetase gcne. This result, together with the failure to gct 'thyminelcss' mutants of the phage to regain their abilityto promote thymine synthesis, suggested that phage 43 was a converting phage. However, the rcsults of transformation implied that the phage thymidylate synthctasc gene was closely related to that of the recipientcells,and may have originated from the bacterium. INTRODUCTION The acquisition of enzymic activity as a result of phage infection is a well-recognized phenomenon and is often due to the transfer with the phage DNA of the genetic information for the synthesis of the enzyme. The need for enzyme modification is most evident where the phage contains components that are absent from the uninfected host bacterium (Flaks & Cohen, 1959; Kahan, I963; Roscoe & Tucker, I966) but the reason for other changes is not always clear. Some are presumably the result of subtle metabolic requirements for phage growth, but others seem to be fortuitous, having no role in phage development, though possibly of importance in the broad context of genetic exchange in bacteria (Sneath, I962). Transduction provides an example of the fortuitous acquisition of new enzymes following phage infection. Generalized transduction is widespread among the different genera of bacteria but far fewer examples of specialized transduction are known; there is no reason to suppose that the process is uncommon, merely that by its nature it is more difficult to detect. Similarly, phage Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 49 ° R.G. TUCKER conversion is found in many different bacterial groups, and in the modified somatic antigens of Group e Salmonellae the enzymic changes brought about by phages have been examined in detail (Robbins et al. I965). As with specialized transduction, there is no systematic way of detecting phage conversion, and this probably accounts for the paucity of examples other than the easily recognizable changes due to altered surface properties of cells. I report here the acquisition of the enzyme thymidylate synthetase by bacteria infected with the temperate subtilis phage 43, and discuss the possible relation this phenomenon bears to specialized transduction and conversion. METHODS Bacteria. Bacillus subtilis NCTC 3610 was used as the host for phage growth and as the indicator for the phage assay; B. subtilis L 4 was a derivative of B. subtilis 36Io lysogenized with phage ~3; B. subtilis 168 was the indole-rcquiring, transformable strain described by Spizizen (I958); B. subtilis 168 thy-I, a thymine-requiring mutant of B. subtilis 168 (Farmer & Rothman, 1965) was kindly supplied by Dr J. Farmer, and an independently isolated thymineless mutant, B. subtilis 168 thy-2, was kindly supplied by Dr C. Anagnostopoulos. Stocks of bacteria were maintained on slopes of nutrient agar (Oxoid Ltd, London, S.E. t) which for strains requiring thymine was supplemented with 5 × lO-4 M-thymine. Liquid cultures for most of the experiments were grown in 200 ml. volumes in I 1. conical flasks and aerated at 37 ° on a gyrotary shaker. The number of bacteria in a culture was estimated routinely by measuring the turbidity in an EEL (Evans Electroselenium Ltd, Halstead, Essex) photoelectric colorimeter with a neutral density filter, referring to a standard curve relating turbidity to viable cell count. Media. Nutrient broth for the production of phage lysates and the potato extract for the growth of spores were described by Roscoe & Tucker 0966). Minimal medium was the minimal salts solution of Davis & Mingioli (195o) used at twice the concentration and supplemented with ferric citrate and glucose before use (Haslam, Roscoe & Tucker, I967). This medium was satisfactory for B. subtilis 36Io but for B. subtilis I68 and its derivatives the addition of o.o 4 % acid hydrolysed casein (Oxoid) together with any specific growth requirements was necessary for speedy growth. Solid, defined media consisted of minimal salts solution containing 1"5 % (w/v) Oxoid no. 3 agar which, after autoclaving, was supplemented with 5 × lO-3 M-glucose, 6.6 × Io -5 M-Lasparagine and 6.6 × lO-5 M-DL-glutamic acid. Further additions were made as follows: 5 × io -5 M-L-tryptophan (ST plates); 5 × lO-4 M-thymine (SP plates); 5 × IO-5 M-Ltryptophan plus 5 × lO-4 M-thymine (STP plates). Phage assay. Standard plating procedures were used (Adams, I959). Agar medium for the bottom layer of the plates contained per litre: nutrient broth CM1 granules (Oxoid), 13 g. ; NaCl, 3 g. ; glucose, I g. ; Oxoid no. 3 agar, I5 g. For the overlay the same medium was used but with agar (7 g./1.) Phage suspensions were diluted in nutrient broth and added to tubes of 3 ml. melted overlay medium containing lO8 spores of B. subtilis 361o. Plates were incubated overnight at 25 °. Phage lysates. Preparations of phage 43 were generally obtained as follows. An overnight culture of B. subtilis L 4 in broth was added to I 1. fresh broth and aerated in a rotating 5 1. flask (Mitchell, 1949) at 37 ° until the cell density reached Io 8 bacteria/ml. Four ml. of 2 vol. H20~ were added per 1. of culture and aeration was continued for Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 Phage-induced thymidylate synthetase 49I about an hour when the opacity of the culture had fallen maximally. Crude lysates of phage 43 contained about lO2° p.f.u./ml., though sometimes titres were much lower. In the later stages of the work more reproducible titres were obtained by adding 2 x lO-2 M-3-amino-I,2,4,-triazole, a catalase inhibitor (Margoliash, Novogrodsky & Schejter, I96O), to the cultures induced by hydrogen peroxide. Phage ~3c was a clear-plaque mutant of phage 43, picked from a phage 43 assay plate and taken through three successive single-plaque isolations. Lysates of the phage were prepared by infecting cultures of Bacillus subtilis 36IO, freshly grown to IOa cells ml. in aerated broth, with approximately fivefold excess of phage ~3c. Aeration was continued until lysis was maximal. Crude lysates contained about lOl° p.f.u./ml. Cells and debris were removed from lysates by centrifugation at 2ooo g for 3o min. and the clear supernatant fluid was transferred to a beaker to which solid ammonium sulphate (4oo g./l.) was added with stirring. After standing overnight at 4 ° the precipitated material was collected by centrifuging, and resuspended in a buffered salts solution (Hayes, 1957). The suspension was clarified by centrifugation at low speed, then the phage was sedimented in an ultracentrifuge at 55,0oo g for 6o rain. The pellets were suspended in buffered salts and treated with lysozyme (Armour Laboratories Ltd, Hampden Park, Eastbourne, Sussex), IOO #g./ml. ; ribonuclease (Armour) IO #g.[ ml., and DNase (Calbiochem Ltd, London, W. I), IO #g./ml. at 37 ° for 30 min. The suspension was clarified by centrifuging at IO,OOOg for 15 min., then the particles were sedimented at 5o,0oo g for 6o min. and finally suspended in buffered salts at a concentration of approximately IOTM p.f.u./ml. Preparation of DNA. A culture of B. subtilis, grown with aeration for I6 hr in I 1. nutrient broth, was centrifuged at 2ooo g for 2o rain. and washed once with 40o ml. buffered citrate saline (o.14 M-NaC1; o.or M-sodium citrate; o'ot M-phosphate buffer, pH 7"o). The bacteria were suspended in 30 ml. buffered citrate saline and lysozyme added to a concentration of I mg./ml. After 2o min. at 37 °, sodium dodecylsulphate was added to a concentration of 0.2 % to complete the lysis and the mixture diluted with 30 ml. ice-cold buffered citrate saline. An equal volume of phenol saturated with lO-7 M-phosphate buffer, pH 7"0, was now added and the mixture shaken gently for 2o min. The aqueous and phenol phases were separated by centrifugation and after re-extracting the aqueous layer with fresh phenol the nucleic acid was precipitated by adding two volumes ethanol. The precipitate was dissolved in a solution containing o. 15 M-NaCI and o.o 15 M-sodium citrate (SSC) and treated with RNase (50/zg./ml. of a solution in o.14 M-NaC1 which bad been heated at 80 ° for IO min. to destroy any contaminating DNase) for 45 min. at 37 °. After another precipitation with ethanol the nucleic acid was dissolved in SSC and dialysed overnight against the same solution. For the purposes o f base analysis, traces of R N A that this material contained were removed by treatment with charcoal (Zamenhof & Chargaff, 195 I). Phage nucleic acid was prepared by the method of Mandell & Hershey (I96O), followed by overnight dialysis against SSC. Estimation of DNA in bacterial cultures. Ten ml. volumes of culture were pipetted into I ml. 50 % trichloracetic acid in a centrifuge tube cooled in ice. After storage overnight in ice the tubes were centrifuged and the supernatant fluid removed by decantation and thorough draining. The precipitated material was resuspended in 1.5 ml. 5 % trichloracetic acid and heated in a water bath at 9°o for 30 min. The tubes were cooled and centrifuged and 0"5 ml. volumes of the supernatant fluid used for the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 492 R.G. TUCKER determination of purine-bound deoxyribose by the diphenylamine reaction (Burton I956). 2-Deoxyribose (Calbiochem) was used to make a standard curve. Transformation. The media and procedures were those of Anagnostopoulos & Spizizen (I96i) as modified for B. subtilis i68 thy-i by Farmer & Rothman (t965). D N A was added to competent cultures after 60 min. incubation in the secondary growth medium and exposure was terminated by adding DNase (50 #g./ml.) Io or I5 min. later. The culture was incubated for a further 45 rain. before being plated on suitable selective solid media. Preparation of cell-free enzyme extracts. B. subtilis I68 thy-I was grown with aeration overnight in nutrient broth containing 5 × Io -4 M-thymine. The bacteria were diluted to about 5 × Io7 cells/ml, in 200 ml. volumes of minimal medium supplemented with acid hydrolysed casein (o'04 %, w/v), L-tryptophan (I'25 x lO-4 M) and thymine (Io -4 M), and aerated at 37 ° till the cell count was 3 × Ioa cells/ml. The cultures were cooled by pouring them into half their volume of frozen minimal salts solution and the cells harvested by centrifugation. The bacteria were now resuspended in the original culture volume of minimal medium with acid hydrolysed casein and L-tryptophan as before, but lacking thymine, and distributed in IOO ml. volumes in I 1. conical flasks. The bacterial suspensions were equilibrated at 37 ° for I5 rain., with aeration, phage was added and the aeration continued. Individual flasks were removed at intervals and their contents rapidly cooled by tipping into 50 ml. frozen minimal medium. The cells were sedimented by centrifugation, resuspended in 5 ml. buffer A (Haslam et al. I967) and disrupted at 25 kcyc./sec, for 4 min. in a Mullard Ultrasonic Generator Type E 759oB. Unbroken cells and debris were removed by a preliminary centrifugation at Io,ooo g for 60 rain. Extracts of uninfected bacteria were obtained in a similar manner. The protein content of the extracts was estimated by the method of Lowry et al. (I95 I), using crystalline bovine serum albumin (Armour) for the standard curve. Assay ofthymidylate synthetase. The bacterial extracts, prepared as described above, were used without further purification. Thymidylate synthetase was estimated by a spectrophotometric method (Haslam et al. I967) based on that of Wahba & Friedkin (1962). Antiserum. Purified, concentrated phage suspensions were injected subcutaneously and intravenously into rabbits on three alternate days, and a week later the intravenous series of injections was repeated. After a further week blood was collected and tested for antiphage activity. RESULTS Properties of phage (~3 Phage ~3 was isolated from garden compost by conventional enrichment techniques using Bacillus subtilis 36IO as the host bacterium. Electron microscopy showed tadpoleshaped particles with heads which were hexagonal in outline and 57 nm. in diameter, and slender tails 270 nm. long. Under the plating conditions used the phage formed turbid plaques approximately I mm. in diameter, with an occasional clear plaque on crowded plates. Bacterial growth picked from the centre of a turbid plaque with a sterile needle was streaked on to nutrient agar and several isolated colonies appearing after incubation were purified by streaking on three successive occasions on fresh nutrient agar plates. All the colonies obtained in this way produced peripheral zones of lysis when replica-plated on to a lawn of B. subtilis 36Io, and one of them, designated strain L4, was selected as stock for further study. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 Phage-induced thymidylate synthetase 493 These initial results could be explained if phage ~b3 either formed some carrier-type association with the host or if there was a lysogenic relation between them. Evidence for the latter was obtained from a number of experiments. Thus when isolated colonies of Bacillus subtilis L4 were replica-plated on a lawn of B. subtilis 36IO virtually all of the transferred regions were surrounded by a zone of lysis after overnight incubation. This behaviour persisted after numerous single colony isolations of B. subtilis L 4 and was unaffected either by prior growth of the bacteria in the presence of antiphage serum or by two cycles of growth in the presence of Io -4 M-proflavine. No difference between the rates of growth of B. subtilis 36Io and B. subtilis L4 in broth or minimal medium could be detected. Culture supernatants of the latter organism, however, always contained free phage 43 particles, and the numbers could be greatly increased by a variety of treatments, such as u.v. irradiation, exposure to mitomycin C, addition of hydrogen peroxide, known to induce phage growth in lysogenic bacteria. For routine purposes lysates of phage ~b3 were obtained by hydrogen peroxide treatment of broth cultures of B. subtilis L 4. The rates of growth of strains 36Io, I68 and I68 thy-I of Bacillus subtilis in minimal medium, supplemented where necessary with tryptophan and thymine, were little affected by the addition of phage ~b3, even with a I5-fold excess of phage to cells. In contrast, infection of the same organisms with clear-plaque mutant phage ~b3cresulted in distinct lysis. The growth of cultures of B. subtilis L 4 was unchecked either by phage 43 or by phage ff3c, and neither phage produced plaques on indicator lawns of this bacterium. Formic acid hydrolysis of the nucleic acids extracted from phages ~b3 and ~3c followed by paper chromatography (Wyatt, I95 I) showed that they contained all the normal DNA bases but that the guanine plus cytosine (GC) content of 36 ~o was significantly less than that of the DNAs of B. subtilis 36Io or B. subtilis L4, both of which had a GC content of 43 %- A limited comparison was made between phage ~b3 and two subtilis phages that might have been related to it: phage SP3, which has the same GC content as phage ~b3 (Marmur & Cordes, I963) but is not stably lysogenic, and phage ~kIo5, a temperate phage isolated by Dr B. Reilly. Neither of these phages was inactivated by antiserum against phage ~b3, and both formed plaques on B. subtilis L4, which was immune to phage ~b3. Phage ~b3 therefore was distinct from these other subtilis phages. DNA synthesis in bacteria infected with phage (~3 As with many phages, the growth of phage ~3c in host bacteria, e.g. Bacillus subtilis 36Io or B. subtilis I68, was accompanied by an enhanced rate of DNA synthesis until the culture lysed. Experiments were also done to determine the effect of phage infection on the DNA metabolism of thymineless mutants of B. subtilis. A culture of B. subtilis I68 thy-I was grown to a concentration of 3 x io 8 ceUs/ml, in 4oo ml. minimal medium supplemented with acid-hydrolysed casein (0"o4 % w/v), t.-tryptophan (I'5 x Io -4 M) and thymine (IO -4 M). The organisms were chilled rapidly by pouring them on to frozen double-strength minimal salts solution (Davis & Mingioli, I95O), then centrifuged and washed with fresh salts solution. The bacteria were taken up in the original culture volume of minimal medium, supplemented with acid-hydrolysed casein and tryptophan as before but without added thymine, and the suspension divided into four IOO ml. volumes in I 1. conical flasks. Thymine (I0 -4 M) was added to two of Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 494 R.G. TUCKER the flasks and phage 43c was added to one of these to give approximately tenfold excess of phage to cells. Phage 43c was also added to one of the two remaining flasks lacking thymine. All the cultures were incubated with aeration at 37° and Io ml. samples were withdrawn at intervals for the estimation of DNA. In the presence of thymine the results were no different from those with wild type bacteria. However, there was also appreciable DNA synthesis by the culture infected with phage 43c in the absence of thymine (Fig. r a), suggesting the ability of this phage, like certain coliphages (Barner & Cohen, I959), to promote thymine biosynthesis in thyminerequiring bacteria. Similar burst sizes were obtained in one-step growth experiments in which B. subtilis I68 thy-I was infected in minimal medium with or without thymine, showing that the DNA synthesized in the absence of thymine was fully functional. The clear-plaque mutant phage 43 used in the previous experiments lysed the bacteria it infected, so it was of interest to determine what effect the temperate phage 43 would have on the DNA synthesis of thymineless bacteria. A culture of B. subtilis I68 thy-I was grown, washed and resuspended as above, then infected with a tenfold excess of phage 43. The infected culture synthesized DNA in the absence of added thymine and it continued to do so well beyond the time at which cultures infected with the clear plaque mutant phage 43 lysed (Fig. I b). Thus it may be concluded that the phageinduced synthesis of thymine can be obtained even when the phage enters the prophage state. Acquisition of thymine independence by phage 43-infected B. subtilis Since Bacillus subtilis I68 thy-I infected by phage 43 can form DNA in the absence of exogenous thymine it seemed likely that lysogeny would confer on the bacteria the ability to grow without thymine. A culture ofB. subtilis I68 thy-I, grown to a concentration of 3"5 x ioa cells/ml, in minimal medium with added acid-hydrolysed casein (o'o4 % w/v), L-tryptophan 0.25 × IO-4 M) and thymine 0 o -4 M), was infected with a suspension of phage 43 at a cell:phage ratio of Io: i and incubated at 37°. Samples of the infected culture were removed at intervals and after dilution were spread in duplicate on three sets of minimal agar medium supplemented with tryptophan plus thymine (STP plates), with thymine (SP plates) and with tryptophan (ST plates). The first set permitted the growth of all the bacteria: the other two the growth only of those able to synthesize tryptophan and thymine respectively. All the plates were incubated overnight at 37°. Infection of B. subtilis I68 thy-I with phage 43 resulted in the rapid acquisition by the majority of the cells of the ability to grow on the medium lacking added thymine, but there was no corresponding growth on the plates that lacked tryptophan (Fig. 2). Two consistent features of this type of experiment were the slight initial decrease in numbers of colonies appearing on STP plates and a later decrease, about 5o min. after infection, in colony counts both on ST and STP plates. The reason for these apparently temporary losses of viability are unknown: possibly they were the result of physiological changes caused by plating at these particular times, since the balance between lysogenic and lytic response is often susceptible to environmental changes (Luria et al. 1958). The relation between phage:cell ratio and the number of organisms gaining the ability to synthesize thymine and thereby grow on ST plates is shown in Fig. 3. The loss of viability occurring when the bacteria were plated immediately after infection with phage 43 was a complicating factor, and an exposure time of 2o rain. before plating Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 Phage-induced thymidylate synthetase 0.7 ! 495 (a) ~, 0.6 0.5 0 ,.o 0 ,i 0.4 I 0.3 0 J~ < Z O~ 0 '" 0.1 20 40 60 80 20 40 60 Minutes after infection 80 I00 120 t40 160 Fig. I. Effect of phage infection on DNA synthesis by thymine-requiringBacillus subtilis 168 thy-I. (a) DNA content of control cultures, without ( O - - O ) and with ( 0 - - 0 ) thymine, and of phage ¢3c-infected cultures, without ( A - - A ) and with ( & - - A ) thymine. (b) DNA content of control cultures, without ( O - - O ) and with ( 0 - - 0 ) thymine, and of phage ¢3-infected cultures, with ( A - - A ) and without ( & - - A ) thymine. I I 20 40 I I I I 60 80 1 O0 120 600 ~ 500 400 300 o ~ 2oo! 100 140 Minutes after infection Fig. 2. Acquisition of thymine independence after infection with phage ¢3. STP plates (O--O), ST plates ( Q - - O ) SP plates ( A - - A ) . Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 496 R.G. TUCKER was therefore chosen, since it combined good recovery with least growth of the culture. With a tenfold multiplicity of phage to cells, 8o % of the bacteria became capable of growing in the absence of thymine within 2o rain. Growth of phage ff3-infected Bacillus subtilis 168 thy-I without thymine is a stable property. A colony picked at random from those growing on an ST plate in the previous experiment was transferred to nutrient broth containing 5 x io - t M-thymine plus antiphage serum and incubated overnight at 37 °. The culture was then diluted and suitable volumes were spread on to STP plates to permit the growth of thyminedependent as well as thymine-independent organisms. The colonies appearing after I I I 1 I 0 O 8 .8> 7 I::I 0 ¢,1 0 6 5 i ,,I Log. phage:coil ratio Fig. 3. Relation between phage :cell ratio and growth without exogenous thymine. STP plates (O--©), ST plates (Q--O). Colonies were counted after overnight incubation at 37°. incubation were replica-plated on to ST plates and also on to a lawn of B. subtilis 361o on nutrient agar to detect phage. In all, 45o colonies were examined: all grew in the absence of thymine and all were lysogenic. To determine the proportion of phages able to confer thymine independence on Bacillus subtilis I68 thy-I, stock suspensions of phage 43 were diluted and plated against B. subtilis 168 thy-I as indicator using nutrient agar medium supplemented with 5 x Io -4 M-thymine. Individual plaques were picked off with a sterile needle and transferred to marked positions on fresh nutrient agar containing thymine. After overnight incubation at 37 ° patches of bacterial growth appeared where the plaques had been transferred, consisting of B. subtilis 168 thy-1 lysogenized with phage 43, and these were replica-plated on to ST plates. Any replicated patches that failed to grow on further incubation were presumed to contain 'thymineless' phage, but this was tested by spreading the bacteria from these patches on to ST and STP plates, and also spotting on to a lawn of B. subtilis 36IO to confirm the presence of phage. Out of a total of 1364 plaques examined in this way, 46 (3"4 %) consisted of phage unable to confer thymine independence on B. subtilis 168 thy-i. These 'thymineless' derivatives Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 Phage-induced thymidylate synthetase 497 of phage 43, designated phage ~b3T-, were indistinguishable from the present phage apart from their failure to convey thymine independence. Moreover, crowded assay plates contained clear-plaque mutants (phage ~b3cT-) which lysed growing cultures of B. subtilis and gave similar burst sizes to those of phage $3c in one-step growth experiments. Thymidylate synthetase activity following ~b3 infection The growth and formation of DNA by Bacillus subtilis 168 thy-I in the absence of thymine after phage infection implied that phages 43 and $3c could correct the biochemical defect of this bacterium, namely the absence of the enzyme thymidylate synthetase (Farmer & Rothman, 1965). To confirm this, B. subtilis 168 thy-I was grown in minimal medium containing acid-hydrolysed casein (0"04 %), L-tryptophan (i'25 x lO-4 M) and thymine (lO -4 M) to a concentration of 3 x io s cells/ml. The bacteria were harvested, resuspended in the same medium without added thymine and infected | 300 ! I (a) I I I ~0 O I (b) 2 ~ 200 E 0 Q. 100 ff j 10 20 30 20 40 60 80 100 120 Minutes after infection Fig. 4- Specific activity of thymidylate synthetase in extracts of Bacillus subtilis 168 thy-I followingphage infection. (a) Cultures infectedwith phage ~b3c,(O--O) with L-tryptophan, (A--&), without L-tryptophan, (A--A), with L-tryptophan and 50 #g./rnl. chloramphenicol; (O--O), uninfected culture with L-tryptophan. (b) Cultures infected with phage ~3: (O--O), with L-tryptophan; (Q--O), uninfected control culture with L-tryptophan. with a tenfold excess of phage ~b3c. Samples were removed at intervals and cell-free extracts of the bacteria were assayed for thymidylate synthetase activity by the spectrephotometric method (Fig. 4a). After a lag of at least 8 min. the enzyme was detectable and its specific activity increased rapidly to 30o m/,mole H4PtG oxidized/hr/mg, protein by the time the cells had started to lyse. The validity of the spectrophotometric method for assaying thymidylate synthetase activity under these conditions was confirmed by showing that extracts from infected cells had acquired the ability to incor32 J. Virol. 4 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 498 R.G. TUCKER porate label from [14C]HCHO into thymidylic acid. No increase in enzyme activity was obtained if chloramphenicol (5o/zg./ml.) was added to the medium with the phage or if tryptophan, one of the growth requirements of the bacterium, was omitted from the medium used for phage infection, both results showing that de novo protein synthesis was needed for the appearance of the enzyme. Similar experiments were made with the temperate phage 43 to determine whether it, too, could generate thymidylate synthetase activity in B. subtilis I68 thy-I. The bacteria were grown and transferred to medium lacking thymine as before. A tenfold excess of phage to cells was added, giving approximately 80 % lysogenization, and samples were taken at intervals to determine their enzyme content. The specific activity of thymidylate synthetase in the 300 T T I I i 1 I 10 10 o 2 ? 8~ x 7i 0 r~ ~E 200 80 ~ X 6- 7. I O "6 6~J O = N 10o N O 3- : 4 2- -- 3 1 - .I. I ~ 10 20 Minutes after infection 30 50 60 70 Drop number 80 Fig. 6 Fig. 5 Fig. 5. Specific activity of thymidylate synthetase in cell extracts of Bacillus subtilis 168 following phage infection. (©--©), culture infected with phage ~b3c; (A--A), culture infected with phage ~b3cT-; (@--@), uninfected control culture. Fig. 6. Density gradient centrifugation of phage ~3. (@--@), plaque-forming particles against Bacillus subtilis 36Io; (©--©), particles carrying the thymidylate synthetase gene. extracts increased rapidly from zero at the time of infection to 60 m#mole H4PtG oxidized/hr/mg, protein zo min. later. Thereafter the specific activity declined, its value remaining at about 35 m/~mole H4PtG oxidized/hr/mg, protein until the end of the experiment (Fig. 4b). Infection of wild type Bacillus subtilis 36[o or B. subtilis r68 with phage ~3c also caused an increase in the thymidylate synthetase activity of cell-free extracts. In a typical experiment the specific activity of the enzyme increased from an initial value of 36 to 4o0 m#mole H4PtG oxidized/hr/mg, protein after zo min. The dramatic increase Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 2 Phage-induced thymidylate synthetase 499 in enzyme activity after phage infection, both of the wild type and of thymineless B. subtilis, probably represented an uncontrolled expression of the thymidylate synthetase gene rather than a requirement for thymine that the existing host mechanism was unable to provide. For example, when the 'thymineless' phage ¢3cT- was used to infect B. subtilis I68 the thymidylate synthetase activity remained at about 36 m/zmoles H4PtG oxidized/hr/mg, protein but the yield of phage was the same as with phage ¢3c which did increase enzyme activity (Fig. 5). The origin of the thymidylate synthetase gene of phage ¢3 The appearance of the enzyme thymidylate synthetase after infection of thymineless Bacillus subtilis with phage ¢3 could be readily explained if the DNA injected by the phage contained the genetic information necessary for the formation of this enzyme. Phage ¢3 must therefore have been capable either of lysogenic conversion or of transduction of a portion of the bacterial chromosome. The possibility that the phage was a generalized transducing phage was ruled out by the experiment showing that approximately 97 ~/o of the plaque-forming particles were able to confer thymine independence on B. subtilis 168 thy-I, and also more directly by the failure to transduce a variety of amino-acid auxotrophs of B. subtilis 36IO. If phage ¢3 is a specialized transducing phage it should have been possible, by analogy with phage A, for' thymineless' derivatives of the phage to acquire the thymidylate synthetase gene following growth in wild type bacteria. Attempts to demonstrate transduction of thymine biosynthesis using lysates of B. subtilis 361o lysogenized with phage ¢3T- which were obtained either by hydrogen peroxide treatment or by induction with u.v. irradiation were, however, unsuccessful. Further experiments were done to determine whether it was possible to separate plaque-forming particles from transducing particles by centrifugation in a density gradient (Weigle, Meselson & Paigen, 1959). A suspension of phage ¢3 diluted to 5 x io 10p.f.u./ml, in 1.5 X IO-~ M-tris+HC1, pH 8, was mixed with solid CsCI to give a solution of density 1"511 g./cm8. The phage was centrifuged at 27,0oo rev./min. for 16 hr at 2o ° in the SW 39 rotor of a Spinco Model L ultracentrifuge. The centrifuge tubes were then punctured at the bottom and single drops collected in 2 ml. o.14 MNaC1. Fractions were analysed for plaque-forming particles by mixing with spores of B. subtilis 36IO and plating on nutrient agar, and also for particles conferring thymine independence by mixing with a culture of B. subtilis 168 thy-1 and plating on ST medium after 15 min. incubation at 37°. Coincident peaks of activity were obtained (Fig. 6) showing that there was no difference in density between the plaque-forming particles of phage ¢3 and those carrying the thymidylate synthetase gene. In a further experiment B. subtilis 168 thy-I lysogenized with the 'thymineless' phage ¢3T- was used as the indicator for phage carrying the thymidylate synthetase gene in order to supply functions that might be absent from any defective transducing particles. Again, there was no density difference between the two phage activities, and it must be presumed that the thymidylate synthetase gene is not carried by defective particles. Synthesis of thymine after the uptake of phage nucleic acid Since competent cultures of Bacillus subtilis 168 can incorporate homologous DNA it should be possible to determine whether the thymidylate synthetase gene of phage ¢3 is specific for the phage or whether the gene originally came from the bacterium. Cultures of B. subtilis 168 thy-I were prepared for transformation by conventional 32-2 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 500 R.G. TUCKER procedures (Anagnostopoulos & Spizizen, I96I; Farmer & Rothman, I965). After 60 rain. incubation at 37 ° in the secondary growth medium containing Io -~ Mthymidine, D N A from phage ~b3 was added, followed I5 min. later by DNase (50/~g./ ml.). Incubation was continued for another 45 min. before the cultures were diluted and spread on plates of ST and STP medium. Phage ~b3c D N A was able to transform B. subtilis I68 thy-I to thymine independence, and the frequency of transformation increased linearly with the concentration of D N A up to a plateau (Fig. 7). In control experiments with phage D N A used over the same range of concentration and for the same duration there was neither transformation of B. subtilis 168 thy-t to tryptophan 5 ! I ! I I S I I I I ///y o o 2 ,I I I I ~ ~. ~ Log. DNA (/~g.]ml.) I o 1 I ~ I I I ~ ~ ~ o Log. DNA (#g./rnl.) Fig. 7 Fig. 8 Fig. 7. Transforming activity of phage ~b3cDNA. Fig. 8. Comparison of transforming activity of bacterial and phage DNA. Phage ~b3cDNA ( 0 - - 0 ) and DNA from Bacillus subtilis 36Io (O--O) was added to competent cultures of B. subtilis i68 thy-1. independence nor transfection and phage reproduction. Identical transformation activity was shown by D N A obtained from phage ~b3c and phage ~b3. However, at concentrations below saturation, phage D N A was more active in transforming than D N A from wild type B. subtilis 361o; similar numbers of thymine-independent transformants being given by 25 to 5o times less phage D N A than bacterial. Since the bacterial D N A used in these experiments had been subjected to more vigorous treatment during extraction than the phage DNA it was probably more fragmented and so less active in transformation (Litt et al. 1958). To find out whether this accounted for the greater relative activity of the phage D N A similar comparisons were made with phage D N A Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 Phage-induced thymidylate synthetase 5oi and bacterial DNA isolated by the gentler procedure of Berns & Thomas (I965). Again, the phage DNA was at least ten times more active than the bacterial DNA in the concentration range below o'5 #g. DNA/ml. (Fig. 8). DISCUSSION The association between phage 43 and its host Bacillus subtilis is considered to be of the lysogenic type rather than a pseudo-lysogenic carrier state. The criteria for this view include the failure of the infected cells to lose their phage-producing potential after many single-colony isolations or after growth in phage ¢3 antiserum, but more convincing evidence comes from the phage-induced acquisition by thymineless mutants of B. subtilis of the power to synthesize DNA and grow without the addition of thymine to the medium. Thymine independence is a stable property of these bacteria and is accompanied by the ability to produce phage even after many single colony isolations. A chromosomal location for prophage 43 has yet to be demonstrated but if there is integration into the bacterial chromosome as there is for phage A (Campbell, I962) then some similarity in the sequence of bases along the bacterial and phage chromosomes would be expected. Preliminary experiments using the DNA-agar technique (Cowie & McCarthy, I963) have shown approximately 30 ~/ohomology, giving at least qualitative evidence for similarity which might permit genetic interaction in spite of the difference in the gross DNA base composition of the phage and bacterium. Examination of extracts ofthymineless bacteria showed that they gained thymidylate synthetase activity as a result of phage 43 infection and that this could account for their subsequently being able to dispense with exogenous thymine. The vegetative growth of phage 43 is accompanied by an enormous increase in the specific activity of the enzyme so that by the time the cells start to lyse it is about ten times the value normally found in Bacillus subtilis I68. On the other hand, the enzyme activity after infection of B. subtilis I68 thy-r with the temperate phage ~b3 is regulated to about the level found in the parent organism B. subtilis 168. The loss of specific activity of thymidylate synthetase following its initial increase after phage 43 infection is puzzling; it could be due to direct inhibition of the enzyme (Haslam et al. I967) or to dilution of enzyme, formed in an initially unregulated manner, by cell growth. Another possibility is that the phage grows vegetatively in a small number of infected cells and that enzyme activity is lost when these cells lyse. While the synthesis of thymine accompanying phage 43 growth in thymine-requiring cells might be explained in a variety of ways, the simplest hypothesis is that the phage contains the thymidylate synthetase gene, especially since the enzyme activity of wild-type B. subtilis also increases after infection. Support for this is provided by the experiments showing that DNA from phages if3 and ¢3c can transform B. subtilis I68 thy-I to thymine independence. Since the DNA content of each phage is less than that of the bacterium the phage DNA would be expected to be more active in transformation than bacterial DNA. In practice, IO to 5o times as many transformants were obtained with phage DNA compared with the same amount of bacterial DNA, but no great significance should be attached to the absolute size of this difference because the methods of DNA extraction were not the same and varying degrees of fragmentation of DNA could affect the efficiency of transformation. Also, the efficiency of integration of a given gene seems to depend upon the nature of genes in the vicinity of the particular marker (Schaeffer, I958), and Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 502 R.G. TUCKER these are presumably different in the two cases. A few transformation experiments were made with an independently isolated thymine-requiring strain, B. subtilis t68 thy-2. Identical results were obtained showing that the phenomenon of transformation by phage if3 DNA is not a peculiarity of B. subtilis I68 thy-I. It has been assumed throughout that thymidylate synthetase is the only enzyme responsible for forming thymine after phage infection. From genetic experiments, Anagnostopoulos & Schneider-Champagne 0966) and Wilson, Farmer & Rothman 0966) concluded that there is another, as yet unknown, enzymic mechanism for synthesizing thymidylic acid. No attempt was made to determine whether phage 43 infection leads to the appearance of this second mechanism too. Further study of bacteria infected with phage 43 may shed light on the way the two mechanisms for forming thymidylic acid are interrelated, and the way their control is coordinated with that of other enzymes forming DNA components. Of the two likely methods of carriage of the thymidylate synthetase gene by phage 43, the balance of evidence favours phage conversion rather than specialized transduction. The phage as first isolated possessed the gene and all attempts to get' thymineless' mutants of the phage to regain it failed, though a very low frequency of gene pick-up cannot at the moment be excluded. Defective phage particles certainly play no part in the process, but it is doubtful whether a clear distinction can be made between conversion and specialized transduction of the type reported by Matsushiro, Sato & Kida (I964) for derivatives of coliphage ~8o, where the phage chromosome may acquire host genes without replacement of its own genetic material. Also, Young, Fukazawa & Hartman (I964) showed that phage P22 can lose its converting properties without becoming defective, as if the gene responsible for conversion were not essential for phage development. Such seems to be the situation with the 'thymineless' mutants of phage 43. If the conclusion that phage ~3 is a converting phage is correct, the discovery that phage DNA can transform thymine-requiring strains of B. subtilis has added significance. Marmur, Seaman & Levine 0963) found that there were strict requirements for genetic homology in the B. subtilis transforming system, so the integration of DNA from the phage could mean that a fragment of DNA including the gene for thymidylate synthetase was derived from the chromosome of the wild-type bacterium at some stage in the history of the phage. It may be that some other converting phages have gained their characteristic properties in this way, and should be regarded as extreme forms of specialized transducing phages (Luria, Adams & Ting, I96o; Matsushiro et al. t964). In the particular instance of phage ~b3 a comparison of the properties of the purified enzymes from wild-type B. subtilis and from thymineless bacteria infected with phage ~3 would tell whether the thymidylate synthetase genes are as closely related as the transformation experiments suggest. I am grateful to Professor J. Mandelstam for his interest and helpful advice, and to Mrs J. Newell for expert technical assistance. I also wish to thank Dr D. Kay for electron microscopy, and Drs C. Anagnostopoulos, J.L. Farmer, B. Reilly and W. R. Romig for kindly supplying bacteria and phages. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 14:30:36 Phage-induced thyrnidylate synthetase 503 REFERENCES ADAMS, M. H. (I959). Bacteriophages. New York: Interscience. ANACNOSTOVOOLOS,C. & SCn~aDrR-CrtAMPAO~rr, A.-M. (I 966). D6terminisme g6n6tique de l'exigence en thymine chez certains mutants de Bacillus subtilis. C. r. hebd. S~anc. Acad. Sci., Paris 262, I3II. ANA~NOSTOVOULOS,C. & SV~Iz~N, J. (I96I). Requirements for transformation in Bacillus subtilis. J. Bact. 8x, 741. BAR~rER, H. D. & CoheN, S. S. (I959). Virus-induced acquisition of metabolic tunction. IV. Thymidylate synthetase in thymine-requiring Escherichia coli infected by T2 and T5 bacteriophages. J. biol. Chem. 234, 2987. BERNS, K. I. & TnOUAS, C. A. (I965). 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