Functional interdependence of genes in bacteriophage T2

J . M ol. B iol. (1962) 5, 506-510

Functional Interdependence of Genes in Bacteriophage T2 K.

EBIS UZAKIt

Department of Biological Chemistry, University of M ichigan , Ann A rbor, M ichigan , U.S.A . (R eceived 6 J une 1962, and in revised f orm 2 J uly 1962) 32P-Iabclcd T2 phage cornplexed with bacteria an d t hen frozen loses its abilit y to ma ke the phage-initiated enzymes, deoxycytidine 5'-phospha te hydroxy methylase an d hydr oxymethyldeoxycytidinc 5'-phosphate kinase, at a. rate of 40 to 50 % of th e rate of loss of infectiv e centers. This observation suggests that a very large subunit of phage DNA is involved in the synthesis of these enzym es. The consequences of t his idea lead to some relevant considerations concerning the phenotypic expression of a phage gene.

1. Introduction Infecti on of Escherichia coli with t he T -even bacteriophages lead s t o a series of events beginning with the form ati on of "early protein s" , followed by the appearance of phage-specific DNA a nd culminat ing in t he synt hes is of mature ph age. R ecen tl y it has been established t ha t some of t hese early proteins arc concerne d with nucl eoti de metabolism in t he infect ed host (F la ks & Cohen , 1957 ; K orn berg, Zimmerman , K ornberg & J osse, 1959 ; Bessman, 1959; K oerner , Sm ith & Bu chanan, 1960 ; Fl ak s & Cohen , 1959; K cck , Mahler & Fraser , 1960 ; Somerville, E bisuz aki & Greenberg, 1959). H owever , the mechani sm by which infecting phage DNA initiates the forma. ti on of t hese new enzy mes is not known. This communication is conce rned with one as pect of this problem . Phages whose DNA has been hea vily labeled with 32p lose their abilit y to form plaques at a rate proportional to the specific a cti vity of the isotop e a nd t he amo unt of DNA in the ph age (He rshey, Kamen , K ennedy & Gest , 1951). Furtherm or e. a ll a vaila ble evide nce suggests that the primary lethal action of3 2p decay is the destruction of genetic material (Hershey et al., 1951 ; Stent & Fuerst, 1960 ; Stahl , 1956 ) presumably by scission of the d ouble helix of the DNA molecule (St ent & Fuerst, 1955). One might expect that if new enzyme formation were a property of ph age DNA, it should also be subj ect t o inactivation. and possibly at a rate specific for the size of the genetic unit involved. The present experiments show that the capacity of the cell infected with 32P-Iab eled phage to form som e of these early proteins is strongly affected by decay of the in corporated isotope.

2. Materials and Methods Organism.'! Escherichia coli R2 an d bacteriophage T2(H) were obt ained from Dr. J. Spizizen, Western R eserve University, Cleveland, Ohio. Oompo8ition of media (a) M edium for growth of 32P_labeled bacteri ophage. The basal composit ion of phosphate. free medium included: 2 g NH (Cl; 0·3 g MgSO(, 7H 20 ; 0·0 1 g FeCIs ; 6 g tria; 5·9 g NaCI;

t Presen t address, Bioch emical R esearch Laboratory. Mas sachusetts Gen eral Hospital and Harvard Medi cal Schoo l. B ost on 14, Massachusetts, U .S .A. 506

INTERDEPENDENCE OF GEXES IN BACTERIOPHAGE T2

507

10 ml. glycerol; distilled water to make 1 liter, pH adjusted to 7'5. This medium was supplemented with 2·4 g of Casamino acids (Nutritional Biochemical Corp.), which was prepared by passage through an Amberlite IR4B(H+) column to remove inorganic phosphate and was sterilized separately. For the propagation of 32P-labeled bacteriophage, 1 me of neutralized 32P0 4 (P-32-P-l processed, specific activity 40,000 mc/g of phosphorus (Oak Ridge National Laboratories)) was added per 3 ml. of supplemented medium. (b) Adsorption medium (Benzel', 1952). This contained 0·1 M·NaCl, 0·05 M.phosphate buffer, pH 6'8, and 0-001 M-MgS0 4 • (c) Storage medium. This consisted of 20% sucrose, 2% bovine serum albumin (Armour Laboratories) and 0·005 M-MgS0 4 •

Preparation oj 32P-labeled T2 phage Procedure 1. E. coli R2 was grown to a titer of approximately 9 x 10 7 cells/ml. in a synthetic medium containing high specific activity 321' and infected with a multiplicity of infection of 0·01 (1 phage: 100 bacteria). After 6 hr, the incubation was discontinued and the lysate (with chloroform added) was stored in the refrigerator. Phage titers, determined by the standard soft agar layer technique (Adams, 1950), indicated usual titers of 2 to 4 x 10 10/m!. Lysates were used in the subsequent experiments without further purification. The above procedure was used for experiments I and II. Procedure 2. In experiment III two modifications were introduced to avoid the considerable death of the phage during storage prior to adsorption and also to improve the adsorption. The first modification, which was introduced to avoid loss of phage, was performed simply by using the lysates immediately. Under closely controlled conditions the phage titers varied little, thus multiplicity of infection was easy to estimate. (The actual multiplicity of infection is made by the phage count and the assay of infective centers which arc made with the experiment.) The second modification which improved adsorption utilized a technique based on the observation of Sagik (1954), that the phage titer increases considerably if the phage are diluted into warm distilled water and incubated. Thus, in experiment III, the lysate was diluted 25-fold into distilled water and incubated at 37°C for 30 min. Preparation oj injected cells and enzymes E. coli R2 was grown with aeration at 37°C in nutrient broth to a titer of 4 x 10 8 • The bacteria were then centrifuged, washed with half the original volume of 0-08 M-KaCl and centrifuged again. Finally, the cells were resuspended in the original volume of adsorption media containing chloramphenicol (20 p.g/m!.). These cells were then infected with 32P-labeled or unlabeled phage at a multiplicity of infection of 0·1 and incubated with shaking at 37°C for 5 min, rapidly chilled in ice, centrifuged and suspended in a storage solution in a volume equivalent to 0·01 of the original culture. This was distributed in I ml. amounts and kept frozen in a dry ice-ethanol mixture. At appropriate times, samples were withdrawn and diluted 100-fold into nutrient broth and assayed for infective centers and for enzyme-forming capacity. For the assay of infective centers, the thawed preparations were further diluted in nutrient broth and plated with the soft agar layer technique, with E. coli R2 as indicator (Adams, 1950). For the measurement of enzyme-forming capacity, the preparations were incubated for 10 min at 37°C with shaking. Chloramphenicol (20 p.g/m!.) was then added and the prepara· tions were chilled in ice and centrifuged. The cells were washed with 15 ml. of cold 0·05 M· phosphate buffer (pH 6'8) containing 0·005 M.MgS0 4 , centrifuged again and frozen at - 20°C. All samples were thawed, ground in alumina, eluted with 1·2 ml. 0·05 M.phosphate buffer (pH 6,8) containing 0·008 M.MgS0 4 , centrifuged to remove alumina, and then dialysed overnight against 0·03 M-phosphate buffer (pH 6,8) containing 0·1 M-RaC!. Assay jor enzyme and protein The assay for the enzyme dCMI' hydroxymethylase was performed by following uCH 20 fixation into dCMP (Flaks & Cohen, 1957; Schlesinger, Figueredo & Greenberg, to be published). Another enzyme which is formed upon phage infection, dCMp deaminase (Keck et al., 1960), was assayed by measuring the formation of d"UMp from the substrate tritiated dCMp (gift of Dr. Richard Potter). It was purified on a Dowex-I (Cl-) column prior

508

K. EBISUZAKI

to use. t The assay for both dCMP hydroxyrnethylase and dCMP deaminase showed negligible interference by 32PO, or 32P-containing compounds. This interference was not more than 0'15% of the dCMP hydroxymethylase activity of the control infected cells. There was no measurable formation of enzyme during the adsorption period. The dCMP hydroxymethylase activity formed during the adsorption period was less than 0·1 % of the enzyme activity of the control infected cells. Hydroxymcthyl dCMP (dHMP) kinase was assayed by following the phosphorylation of [14C]dHMP (gift of Dr. Ronald Somerville) in the presence of ATP and assaying the products on a Dowex-50 (H+) column (Somerville et al., 1959). Protein was measured by the procedure of Lowry, Rosebrough, Farr & Randall (1951), using crystalline bovine serum albumin as a standard.

3. Results The results of the experiment I are plotted in the usual form of survival curves (Fig. 1). The enzyme-forming capacity is calculated as specific activity of dCMP hydroxymethylase (fillloies of hydroxymethyl dCMP formedJhourJmg protein). Both infective centers and enzyme production are reduced exponentially with increasing

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FIG. 1. The effect of "P decay on the formation of infective centers and dCMP hydroxymethylase in E. coli infected with SIP-labeled T2 phage. The half-life of "P is 14 days. The zero day levels for enzyme-forming capacity for the control WII.B 1)·010 /Lmoles product/mg proteinfhr and for the I'p sample 0·0018. The corresponding infective centers were 8·3 x lOG/m !. for the control and 1·7 x lOG for tho 31p sample. ...- - - . . . Infective centers in control phage. 6,---6, Enzyme formation in control phage. e---e Infective centers in "P-labeled phage. 0 - - - 0 Enzyme formation in alP-labeled phage.

t The assay conditions were similar to those used by Keck et al, (1960) with the following exceptions. Tritiated dCMP WII.B used and the product dUMP was isolated by passage through a 1 ml, column of Dowex-50 (H+), and the radioactivity assayed on a Tricarb liquid scintillation spectrometer.

IXTERD El' g KD EX CE OF G E N ES IX BA CTERIOPHAGE T2

509

32p decay. With cells infect ed with 32P-Iab eled ph age, the slopes of the enzymeforming capacity and infective centers differ by a fa ctor of 0·5. Th e results of three separate experiments arc summarized in Tabl e 1. This Table also gives th e results of the dHMP kinase assays. It is noted that the loss of dHMP kin ase r oughly parallels the loss of dCMP hydroxymethylase. dCMP deaminase which was assayed in experiment II showed a similar decline. Further assays of deaminase were not perform ed because of the relative insensitivity of the assay. In the controls with unlabeled phage, there was no declin e in infective' centers or enzym e-forming capacity. TABLE

Exper im en t no.

1

R at io of slop es of s urviv al curves. (en zymo-form ing capacit y )j (n umber of infect iv e centers) d CMP hydrox ymethylase dH:\IP kinase

I II

III

0·50 0· 44 0·38

0·50 0·47

4. Discussion Before discussing the results, the assum ptions and possibl e sources of erro r involved in the interpretation of this type of experiment need to be considered . Th e view ha s been implied throughout this pap er th at the formation of the ph ageinduced enzymes is directl y related t o t he ph age genes (or DNA). The sensiti vity of the form ation of the ph age-initiated enzymes t o 32p decay observed in these studies support this view. Also, th e recent data of Wib erg, Dirksen , Epstein, Luria & Bu chanan (1962) on the amber mutants of bacteriophage T4, some of which appea r to caITy mutations affecting the early proteins, pr ovide strong support for the idea. A second consideration involves the kinetics of enzyme form ation at various levels OP2p decay. This has not been studied and if the kin eti cs were greatly altered, clearly the dis cussion which follows requires reconsideration . However , the following evidence suggests that there is no drastic alte ra tio n of the initial kineti cs of enzyme form ation. Stent & Fuerst (1955) ha ve shown t ha t under expe rimental conditions similar to those used here there is no change of the burst size or latent period after th e E . coli and 32P-labeled phage complex had undergone considerable 32p decay. I t has been assum ed here t hat t he effect of 321) decay involves damage or scission of t he DNA molecule, a view genera lly ag reed up on. Nevertheless, in our inte rp retation of the results there should be a caut ious regard for the possibility of unknown side effect s. These results may be interpreted to mean that the DNA unit responsible for enzyme form ation is approxima tely 40 t o 50 % of the size of t he DNA required for viability . This calculation is derived from the pap er of Ste nt & Fuerst (1955) where it was show n that t here is a direct corr elati on of t he slopes of the deat h curve and the am ount of ph osphoru s per infecti ve unit. The very large genetic uni t required for the formation of a specific enzyme suggests t hat it is far t oo large for a single gene't or cistron and a single gene cannot fun cti on ind epend ently und er t hese conditions . Th is idea has been proposed before on studies on enzyme form ation with 32P-Iabeled E . coli (McFall, Pardee & Stent, 1958) alt hough under somewha t more compl ex conditions (McFall, 1961). t A gene is defined her e in t erms of a fuu ctional unit; for instance, s genetic unit that determines the che m ica l make-up of a protein.

510

K. EBISUZAKI

The fact that the enzyme-forming capacity declined at about 40 to 50% of the rate of infective centers may be interpreted as follows. In T2 phage, there are possibly 2 (or 3) functional systems'[ and the loss of infective centers could result from inactivation of any of these functional systems, whereas the loss of enzyme-forming capacity may be due to the inactivation of only one of the functional systems. Furthermore, this idea pre-supposes an all-or-none inactivation of a functional system. This interpretation is not incompatible with the observation of Sekely (1960) and Streisinger & Bruce (1960), who have provided evidence that the genetic markers of T2 and T4 phages are located on one continuous genetic map. If the genes for the early enzymesalso are on this continuous map, we may visualize 2 (or 3) segments corresponding to the functional systems. Additional support for this view will be published in a subsequent paper dealing with phage morphogenesis. (Ebisuzaki, to be published.) Unpublished experiments of Steinberg (Stahl, 1959) have indicated that the function of the TIl cistron was destroyed by 32p decay at a rate about one-third that of the whole phage inactivation. While these various results support the idea of the functional systems and the interdependence of genes, the underlying mechanisms why such large subunits of genetic material are required for phenotypic expression remain unexplained. The author extends his gratitude to the following people who have assisted with the preparation and criticism of the manuscript: Dr. Ronald Somerville, Dr. Nancy Nossal, Dr. Paul Srere, Dr. Herman M. Kalckar, Dr. Frank Stahl, Dr. S. E. Luria and Dr. G. R. Greenberg. REFERENCES Adams, 1\'1. H. (1950). Meth. Med. Res. 2, I-n. Benzel', S. (1952). J. Bact. 63, 59. Bossman, M. J. (1959). J. Biol. Chem. 234, 2735. Flaks, J. G. & Cohen, S. S. (1957). Biochim. biophsts, Acta, 25, 667. Flaks, J. G. & Cohen, S. S. (1959). J. Biol. Ohern: 234, 2981. Hershey, A. D., Kamen, lid. D., Kennedy, J. W. & Gest, H. (1951).J. Gen. Physiol. 34, 305. Jacob, F., Perrin, D., Sanchez, C. & Monad, J. (1960). C.R. Acad. Sci., Paris, 250,1727_ Keck, K., Mahler, H. R. & Fraser, D. (1960). Arch. Biochem. Biophys. 86, 85. Koerner, J. F., Smith, M. S. & Buchanan, J. M. (1960). J. Biol. Chem. 235, 2691. Kornberg, A., Zimmerman, S. B., Kornberg, S. R. & Josse, J. (1959). Proc. Nat. Acad. Sci., Wash. 45,772. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265. ~1cFa.ll, E. (1961). J. Mol. Biol. 3, 219. McFall, E., Pardee, A. B. & Stent, G. S. (1958). Biochim. biophys. Acta, 27, 282. Sagik, B. P. (1954).J. Bact. 58, 430. Sekely, L. I. (1960). Virology, 12, lI8. Somerville, R., Ebisuzaki, K. & Greenberg, G. R. (1959). Proc. Nat. Acad. Sci., Wash. 45, 1240. Stahl, F. W. (1956). Virology, 2, 206. Stahl, F. W. (1959), In The Viru.ses, vol. 2. New York: Academic Press. Stent, G. S. & Fuerst, C. R. (1955). J. Gen. Physiol. 38, 441. Stent, G. S. & Fuerst, C. R. (1960). Advanc. Biol. Moo. Phys. 7, I. Streisinger, G. & Bruce, V. (1960). Genetics, 45, 1289. Wiberg, J. S., Dirksen, M. L., Epstein, R. H., Luria, flo E. & Buchanan, J. M. (1962). Proc. Nat. Acad. Sci., Wash. 48, 293.

t The term functional system is used because it is not possible yet to equate the unit involved in terms of a chromosome, DNA molecule or operon (Jacob, Perrin, Sanchez & Monod, 1960).