Role of gal repressor depletion in λdg transduction escape synthesis

J. Mol. Biol. (1970) 47, 121-135

Role of gal Repressor Depletion in Xdg Transduction EscapeLSynthesis

GREG R.GERMAINE~-ANDPAWR ROGERS

Department of Microbiology, University of Minnesota Minneapolis, Minnesota 55455, U.X.A.

(Received 20 Januury 1969, and in revised form 22 September 1969)

Transduction escape synthesis of galactokinase in Escherichia coli has been studied with the following results. (1) Escape synthesis of host-directed galactokinase is observed upon infection of a X-sensitive E. coli with phage XdgA-J, containing a deletion of the galactokinase region. (2) When a X-lysogen of a galactokinase-negative E. coli is infected with purified Adg, escape synthesis of galactokinase is observed. The differential rate of enzyme synthesis is shown to be dependent upon the multiplicity of infecting hdg. The date describe B hyperbolic curve without added inducer and a strictly linear relationship is found with inducer added. (3) Simultaneous infection of the h-lysogen with both hdg and XdgA-J reveals an enhanced escape synthesis of hdg-directed galactokinese, while infection with h together with Mg did not enhance escape synthesis. (4) gal repressor concentration end an apparent repressor-operon binding constant are estimated from these data by applying a simple model relating the rate of enzyme synthesis to the numbers of operons and repressors per cell. Approximate values of 17 gal repressors per cell end a dissociation constant, Ka = 6 x lo-%I, for the gal repressor-operon system sre obtained.

The data support the hypothesis that the event of prime importance in transduction escape synthesis is depletion of cellular repressor.

1. Introduction

Transduction escape synthesis is the uninduced derepressed formation of gene products by a phage-carried repressible bacterial operon as a result of infection with an appropriate transducing bacteriophage. Following infection of Escherichia coli with hdg (Buttin, Jacob & Monod, 1960; Yarmolinsky & Wiesmeyer, 1960; Buttin, 1963c) or Pldlac (Revel, Luria & Rotman, 1961; Revel 8-z Luria, 1961; Revel, Luria C% Young, 1961) and @O&c (Beckwith, Signer & Epstein, 1966; Epstein, 1967), the gaZ$ and lac operons are derepressed in the absence of exogenously supplied small molecule effecters (inducers). Escape synthesis in both the Zac (Revel & Luria, 1963) and gal (Willard & Echols, 1968) systems has been shown to require phage DNA synthesis. Recently, it was shown that gal (Germaine & Rogers, 1967; Willard & Echols, 1968)

i Present address: North Star Research and Development Institute, Division of Biosciences, Minneapolis, Minnesota, U.S.A.

$ Abbreviations used : gal + and gal-, ability or inability respectively to utilize galactose for growth; K, T and E, the structural genes for galactokinase (kinase), g&lactose-l-phosphate uridyl transferase (transferase) and uridine diphosphogalactose-4-epimerase (epimerase) respectively; these three linked structural genes constitute the gal operon. Mutations in K, T and E which lead to loss in enzyme activity are designated K- , T- and E- . The normal alleles are designated K+, T+ and E+.

9 121

122 G. R. GERMAINE AND P. ROGERS

and lac (Epstein, 1967) operons that reside on the bacterial chromosome are derepressed by infection with Xdg or $80&c, respectively. This phenomenon has been termed tra?zs-derepression by Willard & Echols (1968) and interpreted in terms of a repressor depletion model to account for escape synthesis (Germaine & Rogers, 1967; Epstein, 1967 ; Willard & Echols, 1968), supporting the orginal interpretation of escape synthesis suggested by Buttin (1963c) and by Revel & Luria (1963). Thus, the related phenomena of escape synthesis and trans-derepression suggest that phage-carried bacterial operons effect the removal of cellular repressors, perhaps by binding of repressor directly to specific operons on DNA (Jacob & Monod, 1961; Gilbert & Miiller- Hill, 1966, 1967).

We report here studies on Adg-initiated escape synthesis and .&an.+derepression of galaotokinase in lambda lysogens. The data are used to estimate the concentration of gal repressor per cell and the dissociation constant of the repressor-operon complex.

Our findings provide support for the explanation of gal escape synthesis and trans- derepression in which derepression results from a depletion of free repressor by the presence of excess gal operator regions provided by hdg. Further, the results indicate that the extent of derepression observed is a function of the relative number of gal operons and repressors, and their affinity to one another in the host cell. Preliminary reports of some of these findings have been presented elsewhere (Germaine & Rogers, 1967; Rogers & Germaine 1968).

2. Materials and Methods

(a) Bacteria, bacteriophage and media

The strains of Escherichia coli used in these studies were originally described by Morse, Lederberg & Lederberg (1956): W3110, agal+ strain; W3350, gal l-, 2-, a gal- (K- and T-) strain; W3350 (X); and W3350 (hh)(r\dg), were all kindly supplied by Dr Kenneth Paigen. W4221, gal 16-, a gal- (E-) strain; and W3805 (X) (hdgA-J), gal 22-, a gal- (E-) strain, carrying hdgA-J, a defective h deleted for the h-genes A through J and for gene K and part of gene T of the gaZ operon (identical with hdg C described by Adler & Templeton, 1963), were kindly supplied by Dr Julius Adler.

The following media were used. TB medium: 1 y0 Bactotryptone (Difco), 0.5% N&l, 0.7 y0 K,HP04 and 0.2% KH,PO, ; TB agar : TB medium with 1.5% agar (D&o) ; soft agar : TB medium with 0.7% agar ; EMB-gal agar : complete EMB agar (Lennox, 1955) with 1 o/0 D-galactose ; H-medium : O-6 M-potassium phosphahe buffer (pH 7-O), 0.001 M-ikfgso,, 0.015 M-(NH&SO~ and 2 x 10V6 M-FeSO,.

Bacteriophage stocks were kept or diluted in phage suspension medium containing 0.006 M-Tris-HCl buffer (pH 7*5), 0.085 M-NaGI, 0.001 M-MgSO, and 50 pg gelatin/ml.

(b) Bacteriophage YeparaGon and assay

The lysogenic strains of E. coZi were grown in B-liter batches of H-medium containing 1 yo galactose at 37°C to late exponential phase (5 x lo* cells/ml.). After cooling to lO”C, the cultures were induced by ultraviolet light, an equal volume of 4% Bactotryptone was added, the cultures were warmed to 37°C and growth continued until maximum lysis was observed to occur (optical density at 660 mp). Alternatively, smaller batches of cells grown to 5 x lo8 cells/ml. in TB medium were induced with 1 pg mitomycin C/ml. The remaining cells and debris were removed from the culture fluids, and bacteriophage were concentrated and purified by (NH,),SOB precipitation, dialysis and differential centrifugation according to the method of Kaiser & Hogness (1960). Suspensions of r\ and hdg or X and I\dgA-J were further purified and separated by CsCl density-gradient centrifugation. The less dense band (either Adg or XdgA-J) contained 2 to 8 x 1012 bacteriophage with less than 4% normal X. The denser X band usually contained 1.5 to 1.8 times as much bacteriophage as the hdg or XdgA-J band in all our preparations, as determined by optical

REPRESSOR DEPLETION IN ESCAPE SYNTHESIS 123

density at 260 mp. The purified bacteriophage were dialyzed against phage suspension medium and stored at 4°C over a drop of chloroform.

The number of active X phage was determined by the agar layer technique described by Adams (1959).

Assay of Xdg and hdgA-J depended upon transduction of W3350 and W4221, respectively. The standard transduction procedure was as follows. Cells were grown in TB medium to 2 x lo8 cells/ml., centrifuged and suspended in 0.02 M-MgSC,. Appropriate dilutions of hdg (or XdgA-J) were added to the cell suspension together with puri6ed X (multiplicity of three) as helper. After incubation for 20 min at 37”C, the infected cells were diluted into TB medium and incubated another 10 min at 37°C. Cultures were finally

diluted and plated on EMB-gal agar and gal+ colonies were scored after a 4%hr incubation period at 37°C. The number of Xdg particles was obtained by multiplying the number of gal + colonies by 5. The results from this procedure were confirmed by a second method. The optical density (O.D. 260 rnp) of each band from CsCl gradients of mixed lysate of X and hdg or XdgA-J was determined; and the ratio of O.D. units per 1012 active X varied from 3.7 to 7 for the h-band. Using the value obtained for the h-bands, the number of hdg or hdgA-J particles in the transducing phage-bands from the same gradient was estimated. As shown previously (Kaiser & Hogness, 1960) the number of active hdg or XdgA-J particles estimated by the two methods described above agreed well.

(c) Infection and incubation cotiditions

Bacterial strains growing exponentially in TB medium to 5 x 10s cells/ml. were pelleted by centrifugation and immediately suspended in warm (37°C) sterile 0.02 M-MgSO, to 2 x lo9 cells/ml. One volume of phage suspension medium containing the desired number and type of bacteriophage particles was mixed with one volume of the cell suspension and the mixture was incubated at 37°C for 20 min. Adsorption of bacteriophage was found to ‘be 98% complete within this 20-mm period. Three volumes of warm TB medium were added to each tube or flask together with other compounds such as D-fUCOSe or [%?]leucine as desired and the cultures incubated with aeration at 37°C. In all experiments, zero time refers to the time of addition of TB medium. Samples were withdrawn for assays and the turbidity of the oultures monitored at the times shown for each experiment.

(d) Assay of galactoh&ase

I-O-ml. samples of the cultures were pipetted into iced test tubes containing 0.2 ml. EDTA (0.01 M), 0.05 ml. /I-mercaptoethanol (1.4 M), and 0.05 ml. toluene (Buttin, 1963a). At the end of the experiment, the culture samples were incubated at 37°C for 20 min with oocasional rapid shaking. To 0.2 ml. of toluene-treated cells was added 0.1 ml. of a reaction mixture containing, per ml.: ATP, 10 pmoles; MgCI,, 2Opmoles; Tris-HCl buffer (pH 7-S), 400 wales; n-[l-14C]galactose (0.7 PC), 15 pmoles; NaF, 4 pmoles and /I-mercaptoethanol, 30 pmoles. Reaction tubes were incubated at 37°C and Osl-ml. samples were withdrawn at 0 and 20 min and applied to DEAE-cellulose discs (DEAE-81, Whatman) to adsorb the galactose-l-phosphate formed during incubation (Sherman, 1963). The discs were washed in 4 changes of ice-cold water to remove unreacted galactose, air dried, and the radioactivity measured by liquid-scintillation speotrometry. 1 unit of galaotokinase activity was defined as that amount of enzyme causing the formation of 1 pmole galactose- 1 -phosphate/hr.

Where indicated in the following sections, the relative rate of galactokinase synthesis was determined by dividing units galactokinase/ml. by either the culture optical density or the L-[i4C]leucine cts/min/ml. incorporated into cellular protein.

(e) Assay of growth and protein synthesis Throughout all experiments, growth was monitored with a Klett-Summerson colori-

meter at 660 rnp. A calculated optical density of 0.2 was equivalent to 5 x 108 cells/ml. for bacteria grown in TB medium.

For estimation of the rate of protein synthesis, L-[14C]leucine was added at zero time to cells suspended in TB medium to about 1 to 2 &ml. At the appropriate intervals, 0.2 ml. of culture was pipetted into 5 ml. cold 10% trichloroacetic acid and kept in ice for the duration of the experiment. Acid-insoluble material was collected on membrane


filters (Millipore, 0.45 p, H.A.) which were washed with 10 ml. loo/, trichloroacetic acid, dried and assayed for radioactivity by liquid scintillation spectrometry.

(f) Cheticals and radioisotopes

Special chemicals were obtained as follows: n-fucose (6-deoxygalactose), Calbiochem, Los Angeles, Calif.; n-galactose, essentially glucose free, Sigma Chemical Company, St. Louis, Mo. ; o-[ I-‘“C]galactose (3.75 c/mole), New England R’uclear, Boston, Mass. ; CsCl, American Potash and Chemical Corp., Los Angeles, Calif. ; Al’P, P-L Biochemicals, Inc., Milwaukee, Wise * ., L-[U.I. 14C]leucine (180 c/mole), Schwarz BioResearch, Inc., Orangeburg, N.Y.

3. Results

(a) Derepression of the cellular gal operon after infection with a kinaseless Xdg

.Infection of a gal + strain of E. coli with K- hdg (Mg A-J) results in an uninduced derepression of galactokinase (Fig. 1 and Table 1). Similar infection of a K-strain

Optical density

FIO. 1. Dereprossion of the host gal operon by hdgA-J. E. co& strain W3110 was treated as follows: uninfected control, -e-a--; infectud with

hdgA-J (mult,iplicity of infection := 3) -O-O--; infected with hdgA-J and n-fucose ($qoles/ ml,) added at zero time, --A-A-. After adding TB broth, the cultures were incubated at 37”C, turbidity was monitored, samples were withdrawn at 30-min intervals and assayed for galactokinase act.ivity.

(W3350) did not result in galactokinase synthesis; thus t,he hdgA-J prcparat,ion was free of K+ hdg (Table 1). Wit.h this syst.em of trans-derepression, wo never observed full derepression by phage infection alone. However, it is apparent that multiplication of phage-carried K- ga2 operons in E. wli (Joyner, Issacs, Echols & Sly, 1966) permits derepression of the host K +gal operons in the absence of an externally supplied inducer.

(b) Gductokinase escape synthesis in a lysogen dependent on multiplicity of infecting hdg

To investigate the dependence of the extent of derepression on gene dosage, superinfection experiments were done. Superinfection of a lysogen results in a condition in

REPRESSOR DEPLETION IN ESCAPE SYNTHESIS

TABLE 1

Derepression of the host gal operon by Xdg A-J

126

Experiment Units galactokin~se/ml.

-Fu +Fu

w3110 0.21 1.28 W3110 + XdgA-J 1.05 1.64 w3110 + x O-30 l-25 w3350 0.11 0.11 W3350 + XdgA-J 0.10 0.11 W3350 + Xdg 5.2 7.2

Strains W3110 and W3350 were infected with XdgA-J, hdg or h at a multiplicity of about 2. Following an adsorption period, TB broth was added, the cultures were divided, and incubsted 2 hr at 37°C with n-fucose (2 pmoles/ml.) (+Fu), and without n-fucose (-Fu). Galactokinase activity/ml. cell suspension was determined.

which the virus genome neither replicates nor is destroyed (Wolf & Meselson, 1963). Thus, the number of Xdg chromosomes per cell (and thus gal operons) may be ap- proximated by the multiplicity of superinfecting Adg.

Both a sensitive (non-lysogenic) and an immune (lysogenic) strain of E. coli, K-T-, were infected with increasing multiplicities of hdg. Two hours after infection, galactokinase was determined ‘(Fig. 2). Escape synthesis in either the presence or absence of D-fUCOSe was independent of the multiplicity of hdg infecting the non-lysogen. In

m.o.i. hdg

FIG. 2. Effect of multiplicity of hdg on escape synthesis of galactokinase in lysogenic and non- lysogenic E. coli.

E. co& (K-T-) strains W3350 and W3350 (X) were infected with hdg at the multiplicities shown on the abscissa. Each culture w&s divided and n-fucose (2 pmoles/ml.) was added at zero time as follows: W3350, -a-*--; W3350 plus fucose, -o-o-; w3350 (A), --A--A---; w3350 (X) plus fucose, ---A-A--. Cultures were incubated for 2 hr in TB broth and assayed for increase in galectokinase activity and turbidity. (a) Increase in galactokinase activity (units/ml.). (b) Increase in galsctokinase activity per 0.2 O.D. unit turbidity increase (&V/&f). m.o.i., multiplicity of infection in this and following graphs.


the lysogen, however, synthesis of galactokinase was markedly dependent on multiplicity of infection in either the presence or absence of fuoose.

Xdg infection of lysogenic cultures decreased growth dependent upon the multiplicity of infection of hdg. Although the rate of protein synthesis was unaffected by less than about 10 Adg per cell, a lag in initiation of protein synthesis was found which was proportional to the multiplicity of infection. At higher multiplicities, the lag remained essentially constant but the rate of protein synthesis decreased in proportion to the multiplicity. Terzi & Levinthal(l967) reported similar findings upon h infection of either lysogenic or sensitive cells. Thus, superinfection of the lysogenic strain, W3350, (X), with Adg was repeated and the differential rate of galactokinase synthesis determined (Fig. 3(a)). In the presence of fucose, a linear relationship with multiplicity

m.o.i. Adg GT

FIG. 3. (a) Effect of multiplicity of Xdg on the differential rate of escape synthesis of galactokinase in E. coli (h).

E. coli strain W3350 (X) was infected with hdg at the multiplicities shown on the abscissa. Each culture was partitioned into 3 separate cultures and incubated as follows: without inducer, -O--O---; with n-fucose (2 pmoles/ml.) -O-e-; and n-[14C]leucine (2 @/ml.) was added to the remaining culture. Galaotokinase activity was determined on samples withdrawn from the first two cultures at 15-min intervals from 0 to 90 min. These values were plotted against the amount of r,-[14C]leucine incorporated into protein at each time in the third culture. Differential rates of galactokinase synthesis shown in the Figure were determined from the slopes of the isometric plots for each multiplicity of infection with and without fuoose.

(b) Theoretical curves of V plotted wers2cs cfT according to equation (2) for various values of KG at RT = 20. The straight line, (+E), is a plot of equation (3) for saturating concentration of inducer present.

(c) Same as (b) but for various values of R, at KG = 1.

of Xdg was found. Each doubling of the multiplicity resulted in a doubling of the differential rate of galactokinase synthesis. In the absence of inducer a more complex relationship existed. A curvilinear response was found in which the slope at high multiplicity of infection approached that obtained in the presence of fucose. The results with the lysogen were interpreted to reflect the removal of cellular gal repressor by h-carried gaE operons through formation of repressor-operon complexes. Thus, in the absence of repression (inducer present), the rate of enzyme synthesis


was apparently directly proportional to the total number of gal operons present in the cell; whereas in the presence of repression (no inducer), the rate of enzyme synthesis was probably dependent upon the number of free (unrepressed) gal-operons (see Results Section (d)).

One might argue that the escape synthesis observed in the lysogen was due to a breakdown of superinfection immunity by the superinfecting hdg (Wiesmeyer, 1966). If this were the case, infection of the lysogen with a low multiplicity of Xdg together with increasing multiplicities of h should result in increased escape synthesis of galactokinase. In the absence of fucose, no increase in the differential rate of galactokinase synthesis was observed with 10 hdg and 20 h added per cell (Fig. 4). The fucose- induced activity, similarly, failed to increase.

m.0.i.

FIG. 4. Effect of h on hdg-directed escape synthesis of galactokinase in E. coli (A). E. coli strain W3350 (h) was simultaneously infected with hdg and h at the multiplicities shown

on the abscissa. The cultures were partitioned into 3 cultures and the differential rates of galactokinase synthesis were determined as for Fig. 3. -O-O---, No inducer; -•--a--, with fucose.

(c) Enhancement of Xdg-induced galactokinase escape synthesis by Xdg A-J

On the basis of the foregoing results, it might be expected that )\dg A-J and Xdg would compliment each other in the lysogenic strain, since the gal operons of the K- (hdg A-J) genomes would bind to the gal-repressors that ordinarily combine with the K+ (h dg) genomes. Thus, we would expect that hdgA-J would produce an increased escape synthesis of galactokinase for a given number of Xdg per cell in the absence of added fucose. Infection of E. coli strain W3350 (h) with 5 XdgA-J per cell and from 0 to 40 Xdg per cell revealed that, indeed, hdgA-J enhanced the expression of hdg in the absence of fucose (Fig. 5(a)). Since repression is relieved by inducer, enhancement in the presence of fucose was not expected and was not observed. Also, at a high multiplicity of infection of Xdg, the effect of XdgA-J should be relatively smaller since the ratio, hdgA-Jjhdg, decreases. This was also observed.

In a second type of experiment, the number of superinfecting Xdg was held constant and varying multiplicities of XdgA-J were added to E. COG strain W3350 (X), and the rates of galaotokiuase synthesis were determined with and without added fucose (Fig. 6). The data plotted shows the ratio of the rate of galactokinase synthesis with AdgA-J + hdg to the rate with hdg only: (hdgA-J and hdg)/(Xdg only). An increase in the ratio with increasing multiplicity of infecting hdg A-J was found in the absence


6-

5-

4-

3-

2-

l-

.

oc

m.o.i. tldg

IC

1 L

C

(b) I I

5 IO

Ga,

FIG. 5. (a) Enhancement of hdg-directed escape synthesis of galactokinase by XdgA-J in E. coli (X). E. co& strain W3350 (h) was infected with hdg at the multiplicities shown on the abscissa. Each culture was divided and hdgA-J was added to a multiplicity of about 6 to one set of the cultures. Again each culture was divided and n-fucose (2 ~moles/ml.) W&S added to one set. Following 2-hr incubation, the increase in galactokinase units per O-2 O.D. unit increase in turbidity was determined (dE/dM). With hdg only, -O--O-; hdg plus hdgA-J, -O--O--; hdg plus fucose, -A--A--; hdg plus hdgA-J and fucose, -A-A-.

(b) Theoretical plot of trams-derepression by inactive operons. V versus Ga, plotted according to equations (4) and (5) at KG = 1 and RT = 5. Curves: inducer added (curve A); no inducer, Gi, = 5 (curve B); no inducer, Gi, = 0 (curve C).

2-

a /

./ 0

.

0 0

1 00 0 0 0 0

0

m.o.i. cldg A-J

ha. 6. Enhancement of Xdg-directed esoape synthesis of galaotokinase in E. COG (A) by increasing multiplicities of r\dgA-J.

E. coli strain W3350 (h) was infected with a multiplicity of 5 Xdg and from 0 to 12. XdgA-J/cell. All the cultures were divided and D-fUCOSe (2~moles/ml.) was added to one set. Following a SO-min incubation, turbidity and galactokinase activities were determined. The data shown are plotted as the ratio of the increase in galactokinase activity per 0.2 on. unit increase with and without added XdgA-J: without inducer, -O-e-; with fucose -O-O-.


of fucose, whereas an increase was not observed in the presence of fucose. Thus, in the lysogen, a multiplicity-dependent derepression of hdg-directed galactokinase synthesis by AdgA-J occurred. Finally, recall that similar mixed infection of IV3350 (h) with hdg and X did not yield derepression (Fig. 4). Thus, the trans-derepression of galactokinase synthesis depended upon the gal-operon carried by h.

As shown in the next section, when the ratio of rates of galactokinase synthesis without and with added fucose (Fu-/Fu+) are plotted versus the reciprocal of the

I I / 1 I I I h 1

0.02 0.04 0.06 0.08 0.1 04 0.5 " 2.0 1

m.& cldg+ndgA-I ---L-- mm I. ddg

FIG. 7. Tram-derepression of hdg-directed galaotokinase synthesis by /\dgA-J. E. co& strain W3350 (h) was infected as follows: (a) with hdg at a constant multiplicity of about

10 virons/oell added together with varying amounts of XdgA-J from 0 to 30/oell; (b) with varying multiplicities of Xdg added (same preparation as in (a)) and no hdgA-J. The cultures were treated and galaotokinase activities determined as in Fig. 5(a). The ratio of rates of galaotokinase synthesis without uers~ with added n-fuoose (Fu-/Fu+), is plotted ‘uer~ZLS the reciprocal of the multiplicity of added bacteriophage (see equation (6)).

I I I I I I I

0 0.05 0.1 o-2 0.05 01 0.2 m.o.i ndg

FIG. 8. Effect of multiplicity of Adg infecting E. co&i (h) on the relative rate of galaotokinase synthesis wrth and without inducer.

Experimental data showing the ratio of rates of galaotokinase synthesis with and without added n-fuoose (2 pmoles/ml.) (Fu-/Fu+), are plotted vera~~ the reciprocal of the multiplicity of Adg superinfecting E. co& W3350 (X). Data points in (a) and (b) are from experiments similar to those shown in Fig. 3 and Fig. 2, respectively. Curves are calculated from equation (6) using the following values of R, and Ko respectively: 18 and 3.6, (a); 15.5 and 2.1, (b).


multiplicity of infection of Xdg, the shape of the curve suggests removal of gal repressor with increasing doses of gal operons (Fig. 8). When E. coli strain W3350 (X) was infected with 10 Xdg together with increasing doses of hdgA-J, an increase in the ratio Fu-/Fu+ was seen (Fig. 7(a)). The data from Figure 7(a) fall on the linear portion of the control experiment (Fig. 7(b)), indicating that the action of hdgA-J in binding gal repressor is essentially identical to that of hdg containing the intact operon.

(d) Estimation of repressor concentration and repressor-operon binding

The preceding results suggest that the operons carried into the cell by a transducing phage are most likely equivalent to the same operons of the host bacterium both with respect to their capacity to code for enzyme synthesis and to their ability to bind to cellular repressor. Beginning with the assumptions outlined by Koch (1967), a simple equation can be derived relating the velocity of enzyme synthesis to the total number of operons present in the cell in the presence of a finite number of repressor molecules :

v = k G T _ [RT + GT + Kdl + EdKdI 2

(VT + c;, + K,(l + %/Kd12 - 4R&W’2 - El 1 (1) a I

where V, R,, GT and E, are respectively the velocity of enzyme synthesis, the total number of repressors per cell, the total number of operons per cell and the total inducer or effector molecules per cell, and where KG and K, are dissociation constants dependent upon the affinities of R for G and E for R respectively. In the absence of inducer (E, = 0), equation (1) simplifies to:

v = k G T

_ VT + G, + Ksl - [Ph + (A + Kd2 - 4Wh11’2 2 I

(2)

and in the presence of excess inducer it becomes:

V = kG,. (3)

The data shown in Figures 2 and 3(a), for the rate of galaotokinase synthesis versus number of hdg infecting a lysogen, take the same form as plots of equations (2) and (3) shown in Figure 3(b) and (c).

In the case where both hdg and hdgA-J infected the lysogen, only hdg contributed operons active in coding for enzyme synthesis, whereas added XdgA-J apparently combined with cellular repressor, allowing enhanced expression of Xdg operons (Figs 5(a), 6 and 7). Assuming the same dissociation constant for the binding of active operons, G,, and inactive operons, G,, for cellular repressor, equations (2) become :

v = kG a.T

1 _ (RT + GT + Kc+) - [(RT + GT + Kc12 - ~RTGT~~'~

3% 1

and: V = kG,,

and (3)

(4)

(5)

where G,, is the concentration of total active operons per cell and G, = G,, + GiT, and thus is equivalent to all operons in the cell capable of binding repressor. Again plots of equations (4) and (5) show the same form as the data obtained (cf. Fig. 5(a) and (b)).


Estimates of experimental values for K, and R, can be derived from our superinfection experiments by fitting the data to the simple model outlined above. The ratio of rates of enzyme synthesis without and with inducer were plotted versus the reciprocal of the number of infecting Xdg per cell (Fig. 8). The data showed that the ratio of galactokinase synthesis in the absence of inducer to that with inducer (Fu-/ Fu+ ), increased with increasing dose of hdg infecting the lysogen.

The significance of this method of displaying the data is shown by combining equations (2) and (3):

V (no inducer) equat. (2) I-

‘v (inducer) = equat. (3) = TT

I- ---=1-

( R, + 6 + K, - Wh + GT + &I2 - 4R&.J1’2 1

I+ 2 I :,’ (6)

The curves drawn through the data in Figure 8 are plots of equation (6) using the values of R, and KG indicated. Since the slope of the line at high G, approaches R,

5 IO 20

m.o.i. Adg (I- LC ) F”f

FIG. 9. Slope-intercept analysis of the effect of multiplicity of superinfecting Xdg on galactokinase synthesis in E. COG (X). Data from an experiment of the type described in Fig. 3 treated according to equation (7). The ratio of rates of galactokinase synthesis with and without o-fucose (2 pmoles/ml.) (Fu+/Fu-), are plotted ver8’sus multiplicity of infecting hdg times the term (1 - Fu-/Fu+). K, = 5.5 and R, = 22 in this experiment.

TABLE 2

Estimation of the concentration of gal-repressor and the dissociation constant for the gal-operon-gal-repressor complex

Experiment RT KG (per cell) (molarity x lo*) (per cell) (molarity X 109)

1 18 3 3.6 6 2 14 2.3 3-6 6 3 22 3.7 5-5 9.1 4 15 2.5 2.1 3.5

Average 17.3 2.9 3.7 6-l

R, and K, were determined by the curve-plot method (equation (6)) for experiments 1 and 4, and by the slope-intercept method (equation (7)) for experiments 2 and 3. Molarities were approxi- mated assuming a value of 1Ol2 cells/cm3.


and the horizontal asymptote approaches K,/(K, + RT), the plot provides a rapid method for graphic estimation of K, and RT. By rearrangement and squaring equation (6), a simplified linear form is obtained:

&(l --I-/I+) = 1+/I-( -KG) + (RT + KG). (7) A straight line drawn through experimental data points plotted in this manner also

allows estimation of K, and R, from the slope of the line (-KG) and the intercepts

CR, + KG) ana KGz RT (Fig. 9). Table 2 is a summary of values of R, and K, G

obtained from our data for the gal operon-gal repressor system in E. coli.

4. Discussion

Our results demonstrate that the essential requirement for transduction escape synthesis by hdg is that the number of operons in a cell exceed the number which can be effectively repressed by the supply of cellular repressor. We originally reported (Germaine & Rogers, 1967) that derepression of galactokinase synthesis directed by the gal operon of the host genome occurred as a result of replication of hdgA-J (K-) in the absence of inducer. This result was recently confirmed by Willard & Echols (1968) who coined the term trans-derepression. Our results reported here on host- directed galactokinase escape synthesis after infection with XdgA-J) led us to examine escape synthesis of galactokinase following superinfection of a lysogenic bacterium with purified Xdg. Many other workers have shown that transduction escape synthesis does not occur when reproduction of the Xdg genome is repressed after infection of a X-lysogenic cell (Buttin et al., 1960; Yarmolinsky & Wiesmeyer, 1960; Buttin, 1963c) ; and Willard & Echols (1968) demonstrated that hdg, carrying mutations in N, P and 0, preventing DNA synthesis, resulted in greatly reduced escape synthesis of epimerase either after infection at a multiplicity of 3 or after induction of lysogens with mitomycin C. Our data support the results of others in that, at low multiplicities of infection of lysogens with hdg, very little escape synthesis of galactokinase was noted. However, as the dose of Xdg was increased, a greater rate of enzyme synthesis was observed. More important, when rate of enzyme synthesis is plotted versus dose of Xdg, the curves described fit the shape of theoretical hyperbolic curves (Fig. 3) based on a repressor-depletion model (equation (2)). The model simply predicts that as the number of operons added to the cell exceeds the number which can be effectively repressed by the cellular supply of repressor, the derepressed level of operon products increases. Such an explanation has been offered for the escape synthesis observed during the multiplication of transducing phages in the host (Revel & Luria, 1963 ; Buttin, 1963e; Yarmolinsky, 1963; Epstein, 1967; Willard & Echols, 1968). Further, we observed that the rate of galactokinase synthesis was a simple linear function of the dose of Xdg infecting the lysogen in the presence of the inducer, n-fucose. Thus, our data fit the concept that the rate of operon product produced is directly proportional to the total number of actively expressing operons when the inducer concentration is adequate completely to prevent significant binding of cellular repressor to any operon (equation (3)).

Our data suggest that appreciable Xdg DNA synthesis is probably not an essential prerequisite to escape synthesis of galaotokinase coded for by the phage genomes. The role taken by breakdown of superinfection immunity is relatively minor, since


added X phage did not enhance the expression of Xdg gal operons in the absence of inducer (Fig. 4). On the other hand, added XdgA-J enhanced transduction escape synthesis by Xdg in the lysogen. The curves described by the experimental data (Fig. 5(a)) fit the prediction of the model presented (Fig. 5(b) and equation (4)). Also, the experiment showing that the ratio of rates of galactokinase synthesis without versus with inducer (Fu - /l?u + ) increased with increasing doses of XdgA- J added with 10 hdg, suggests that hdgA-J is equivalent to hdg (Fig. 7). Thus trans-derepression of Xdg- directed escape synthesis depends upon removal of cellular repressor by “inactive” operons carrying a functional operator (XdgA-J), resulting in derepression of the freed “active” operons (xag).

In our superinfection experiments, we observed that the ratio of the rates of galaotokinase synthesis without ver.sm with added inducer (Fu-/Fu+) increased with increasing dose of Xdg. Fitting this data to a repressor-depletion model (equation 6) allowed a tentative estimate of the gal repressor concentration per cell (I&) and a dissociation constant (KG) for binding of gal repressor to the gal operon. Table 2 is a summary of values of R, and K, obtained by treating data from four experiments by the two methods outlined. The average value of 17-3 gal repressor per cell is similar to values estimated for the lac repressor (Gilbert & Miiller-Hill, 1966) and the C, immunity substance (Wiesmeyer, 1966). The value of KG for binding of repressor to the gal operon (6 x 10ms M) is higher than lac repressor binding to lac operon DNA (lo-l1 to lo-l2 and 2 to 4 x lo-lo M) reported by (Gilbert &Miiller-Hill, 1967) and by Riggs, Bourgeois, Newby & Cohn (1968), respectively. However, the dissociation constant for binding of the h repressor to hDNA ( 10ms to lo-lo M) estimated by Ptashne (1967) is somewhat closer to the value of KG determined for the galactose operon- repressor system. The values for KG and R, obtained appear reasonable, since their substitution into equation (6) at G, = 2 predicts a sixfold amplification of enzyme synthesis due to induction (I’/I-). Though somewhat low, this is within the range of six- to tenfold observed when induction of galactokinase with and without D-fUCOSe

was determined in E. COG W3110 in our growth medium (Table 1). There are a number of difficulties in using our data for quantitative estimates of

KG and R,. First, during the course of our studies we observed retardation of growth and protein synthesis in Xdg-infected cells. Similar effects of X infection of both lysogenic and non-lysogenic E. coli have been reported previously (Terzi & Levinthal, 1967). Because of this complication, the differential rate of galactokinase synthesis was determined at different multiplicities of infecting Xdg and hdgA-J. Second, we have assumed that the calculated multiplicity of infecting Xdg or XdgA-J per cell is directly related to the average number of gal operons per cell under our conditions, where 98% adsorption of virus was obtained. We observed that in the presence of inducer the rate of galactokinase synthesis for the culture was linearly related to the dose of added Xdg. However, it is not possible to determine whether the full expressed potential of the gal operon per hdg adsorbed or some constant fraction thereof was responsible for the linear relationship found. Thus, the data appear to allow only an approximation of the average number of gal operons per cell for an infected culture. Third, we assume that during the time of measurement of galactokinase synthesis following infection, the concentration of gal repressor per cell remains constant. During this time there is a substantial increase (50 to 100%) in protoplasmic mass, and since the gal R locus, presumably controlling gal repressor synthesis, is located in a region of the host genome distinctly separate from the gal operon (Buttin, 19633), there is


reason to suppose that gal repressor synthesis occurs at a rate commensurate with protein synthesis regardless of the number of Xdg genomes per cell. Thus, the ratio of gal operons to gal repressors may change with time after superinfeotion with hdg. However, our isometric plots of galactokinase synthesis versus protein synthesis remained linear for up to 105 minutes after superinfection with hdg. Thus, either gal repressor synthesis is slowed down following infection or alternatively, perhaps some gal repressor is rendered unstable after association with gal operons. The possi- bility that the luc repressor in E. coli was growth unstable (Sadler & Novick, 1965) was recently ruled against, since derepression of an iTSS mutant at 42°C was found to be dependent upon DNA synthesis (Barbour, Gross & Novick, 1968). These authors interpret their results to indicate that derepression during growth occurs as the number of lac genes present begin to exceed the number of luc repressors. Therefore, with these considerations, the value of about 17 gal repressor molecules per cell derived from these experiments (Table 2) may be high by as much as twofold.

Subject to the limitations described above, our findings support the hypothesis that the extent of gal escape synthesis observed is a function of the number of gal operons relative to the number of gal repressors per cell as well as a function of the affinity of binding of repressors to the gal operator regions. The technique described for estimating both the apparent binding constant and the effective number of repressor units per cell within an intact bacteria, should lend support to estimates of these values made in vitro by widely different test-tube conditions.

This work was supported by research grant AM-09782 from the National Institute of Arthritis and Metabolic Diseases, U.S. Public Health Service. We thank Ted Kaden for excellent technical assistance and Dr John E. Gander and James Drake for advice in developing the mathematikal treatment.

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Role of gal repressor depletion in λdg transduction escape synthesis

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