Packaging of coliphage lambda DNA

J. Mol. Biol. (1977) 117, 733-759

Packaging of Coliphage Lambda DNA II. The Role of the Gene D Protein N.STERNBEROt

AND R.WEISBERQ

Laboratory of Molecular Genetics National Institute of Child Health and Human Developmend National Institutes of Health Bethesda,Mel 20014, U.S.A. (Received 5 April 1977) The gene D protein (pD) of coliphage A is normally an essential component of the virus capsid. It acts during packaging of concatemeric h DNA into the phage prohead and is necessary for cutting the concatemers at the cohesive end site (cos). In this report we show that cos cutting and phage production occur without pD in h deletion mutants whose DNA content is less than 82% that of h wild type. D-independence appears to result directly from DNA loss rather than from inactivation (or activation) of a phage gene. (1) In cells mixedly infected with undeleted h and a deletion mutant, particles of the deletion mutant alone are efficiently produced in the absence of pD ; and (2) D-independence cannot be att,ributed to loss of a specific segment of the phage genome. pD-deficient phage resemble pD-containing phage in head size and DNA ends; they differ in their extreme sensitivity to EDTA, greater density, and ability to accept PD. pD DNA by the normally absence implies head suggest

appears to act by stabilizing the head against disruption by overfilling with rather than by changing the capacity of t’he head for DNA. This is shown observation that the amount of DNA packaged by a “headful” mechanism, in excess of the wild-type chromosome size, is not reduced in the of PD. In fact, pD is required for packaging headfuls of DNA. This that a mechanism exists for preventing the entry of excess DNA into the during packaging of concatemers formed by deletion mutants, and we that t,his is accomplished by binding of cos sites to the head.

The above results show that pD is not an essential component of the nucleate that cuts X concatemers at co8 during packaging, and they imply that 82% of a wild-type chromosome length can enter the prohead in the absence of PD. Yet, pD is needed for the formation of cohesive ends after infection with undeleted phage. We propose two models to accomit for these observations. In the first, cos cutting is assumed to occur early during packaging. The absence of pD leads to release of packaged DNA and the loss of cohesive ends by end-joining. In the second, cos cutting is assumed to occur as a terminal event in packaging. pD promotes cos cutting indirectly through its effect on head stability. We favor the second model because it better explains the asymmetry observed in the packaging of the chromosomes of COB duplication mutants (Emmons, 1974). t Present address: Basic Research Frederick, Md 21701, U.S.A. 48

Program,

NC1 Frederick 733

Cancer

Research

Center,

P.0.

Box

B,

734

N.

STERNBERG

AND

R.

WEISBERG

1. Introduction The product of gene D of bacteriophage X is a major structural protein of the capsid, each virion containing about 420 molecules (Casjens & Hendrix, 1974). pD acts at a rather late stage in capsid assembly: it appears to be incorporated into sites that appear in the protein shell of a head precursor known as petit h upon enlargement of this precursor to full capsid size (Wurtz et al., 1976). Enlargement is thought to be triggered by the binding or entrance of concatemeric phage DNA into pht (seeHohn et al., 1977). After pD acts, the concatemeric DNA can be cut at a site called cos to generate the cohesive ends of the mature phage chromosome(Dove, 1966; MacKinlay & Kaiser, 1969; Skalka et al., 1972; Wake et al., 1972). What is the role of pD in cos cutting! It might act directly at cos. This appears unlikely, however, becausea low level of cos cutting occurs in the absenceof pD (Wake et al., 1972; Wang L%Kaiser, 1973; McClure et al., 1973). Alternatively, it might stabilize or otherwise activate the DNA-prohead packaging complex so that coscutting can occur. If the DNA-prohead complex is indeed unstable without pD, overfilling with DNA might be the cause. We know, for example, that extra DNA destabilizes even pD-containing phage capsids: wild-type h and addition mutants are unstable in the presence of chelating agents, and the particles are stabilized by deletion mutations (Parkinson & Huskey, 1971). We therefore guessedthat if the chromosome size were reduced, pD might become dispensible. We report here that pD is indeed dispensiblein deletion mutants that lack 18% or more of the wild-type h chromosome.The resulting D-deficient particles contain DNA of the expected size and form. These and other observations suggest an explanation of the coupling between DNA packaging and coscutting.

2. Materials The bacterial

and Methods

(a) Bacterial and phuge strains and phage strains used in this report are described

in Table

1.

(b) Norner&ature The following conventions have been adopted for convenience. pD, pA, pE, etc. refer to the gene products of phage genes D, A, E, etc. hDam*supand hDam*sup+ phage refer to ADam phage last grown, respectively, either in a aup- or a “cp+ host. For simplicity hcIts857 is referred to as A wt. (c) Media Tryptone and Luria broth, tryptone top and bottom agar and TMG buffer are described by Sternberg BE Weisberg (1975) and Sternberg (1976) except that MgSO, (10 mM) was added to broth and top agar to ensure stability of AD- phage. Three types of minimal media were used. All three contain M9 salts (Shimada et aE., 1972) supplemented with MgSO, (2 mM) and glucose (0.2%). Medium I contains 2% Casamino acids (Difco vitaminfree), medium II contains 20 rg of 20 amino acids/ml and medium III contains 50 pg of 19 amino acids/ml but not methionine. ts, temperature-sensitive mutation; t Abbreviations used: pX, petit X; am, amber mutation; suppressor-negative; sup + , suppressor positive; supE, suppressor mutation E ; supF, sup-9 suppressor mutation F; wt, wild-type; PEG, polyethylene-glycol; X.sup-, h grown on a sup- host; h grown on a sup+ host; TB, tryptone broth; TMG, Tris/magnesium/gelatin dilution h*.mp+, buffer.

THE

ROLE

OF

pD

IN

THE

PACKAGING

TABLE

OF

735

h DNA

1

Bacterial and phge strains Bacteria

Pertinent

Reference

genotype

594 NS 1020 NSlOOO NS1008 NSlOll NS1060 NS650 YMC NS62 NS617 C600 N205 NS490 NS432 NS444 NS443

sup-, g&K-T594 (XWam403Eam4cIts857Sam7) 694 (L4amllcIts8678am7) 694 (U)am15cIts857Sam7) 594 (hEam4cIts857Sam7) 594 (himm434) 594 (XDamlScIts857) supF YMC (X) YMC (/\imm434) supE sup - , recA N205 (Xb2red3cIts857Sam7) N205 (XDamlSb2red3cIts857Sam7) gaZP180, supgalTam28, sup-

HW842 LE289 NS420 NS377 NS554 NS504

HfrH sup- (X [int-PII]d),,,, YMC (A [int-BIIIA),,,, supF, ligts7 sup-, nwA-1, rip-2 NS377 ($80 psupF) a?%4 - ) pro -

12875

bioB17

Phage

DNA

XDam15cIts857 XDam1562cIts857 mamlSb2cIts857nin5 ~am15b538cIts857 XDamlSb221cIts857 XDam15b22lcIts857Sam7 ,WamlSb221c126 XDam15b638imm434c ~am166538imm434cnin5 XDaml5b2imm2lcIts hDam15b221imm2lcIt~s hbZcIts857cIts857 &mm434 Aimm434atP Xb1451imm21b3000

(d)

size (relative

Campbell (1965) Sternberg & Weisberg (1975) This report This report This report This report This report Dennert 85 Henning (1968) This report This report Appleyard (1954) Sternberg & Weisberg (1975) Stemberg & Weisberg (1975) This report Strain Stz1031, from 8. Adhya Strain SA624 (Adhya & Shapiro, 1969) Enquist & Weisberg (1976) Enquist & Weisberg (1976) Gottesman et al. (1973) Sternberg (1976) Sternberg (1976) Strain AB478 (Sternberg & Weisberg, 1975) Sternberg & We&berg (1975)

to wt)

1.0 0.87 0.816 0.836 0.777

Davidson Davidson Davidson Davidson Davidson

& & & & &

0.805 0.75 0.82 0.727

Davidson Davidson Davidson

& Szybalski & Szybalski & Szybslski

(1971) (1971) (1971)

0.974 1.06

Davidson Fiandt Enquist

& Szybalski (1976) & Weisberg

(1971)

General

phage

Szybalski Szybalski Szybslski Szybalski Szybalski

(1971) (197 1) (1971) (1971) (197 1)

et al.

(1977)

methods

XdocR and AdocL phage were assayed, respectively, by their ability to transduce lmst gal and bio mutations (Sternberg & Weisberg, 1975). Lambda docR was also assayed by it,s ability to plaque on a heteroimmune lysogen (Little & Gottesman, 1971). In general, phagc deletion mutants were detected by their increased resistance to the chelating agent EDTA (Parkinson & Huskey, 1971) ; see section (f), below. Phage deleted for all or part of int and zis genes were detected by the red plaque test (Enquist & Weisberg, 1976) and thoscb delet,ed for the red OL and/or fi genes by their inability to plaque on strain NS420 (t.hr feh

736

N.

STERNBERG

AND

R.

WEISBERG

phenotype ; Zissler et aZ., 1971; Gottesman et al., 1973). Spot complementation phage crosses were performed as described by Sternberg (1976). Preparation stocks, phage crosses, and single-cycle growth experiments were performed by Sternberg & Weisberg (1975) and Sternberg (1976). (e) Mized

tests and of phage as described

infections

Exponentially growing cells in TB broth at 2 x lOa cells/ml were concentrated B-fold in a 10 mivr-MgSG, solution by low-speed centrifugation (6000 revs/min in a Sorval SS34 rotor at 4°C). A portion of the cell suspension was then infected with phage at a multiplicity of 10 phagejcell (when 2 phage were used each was added at a multiplicity of infection of 5). Adsorption was allowed to occur for 5 min at 38°C and then h antiserum (K = 1 min-‘) added and incubation continued for an additional 5 min. The infected cells were then diluted lOOO-fold in TB broth with 10 mm-MgSG, at 38°C incubation was continued at this temperature for 90 min, and lysis completed by vortexing the cells with several drops of chloroform. (f) EDTA inactivation. of phage Phage were diluted at least loo-fold from the original plate lysate into (10 mM-TrisaHCl (pH 7*4), 10 mM-EDTA) or TMG (10 mM-Tris*HCl MgSO,, 0.1% gelatin) and incubated at either 4°C or 32°C. Inactivation diluting a portion of the incubation mixture IOO-fold into TMG at 4°C. (g) CsCl

e.quilibrium

density

gradient

either Tris/EDTA (pH 7*4), 10 mMwas stopped by

analysis

CsCl gradients were prepared by adding 2.7 g solid CsCl to 3.5 ml buffer (10 mMTriseHCl (pH 7*4), 10 mM-MgSG,) containing the phage of interest. Generally, marker phage of known density were added at about IO6 phage per gradient. The CsCl solutions were centrifuged to equilibrium in a Spinco SW56 rotor at 30,000 revs/mm (24 h, 5’C). The distribution of phage in the gradients was determined by puncturing the bottom of the centrifuge tube and collecting ‘l-drop fractions (60 to 70 fractions) into 0.1 ml TMG buffer. (h) Preparation of labeled phage (i) hdocL The NS432 (ii)

preparation, are described

Phage

with

and

purification and quantitation by Sternberg & Weisberg withoti

of labeled XdocL (1977, accompanying

from

strains paper).

NS490

and

pD

A 30 ml culture of strain 594 (sup-) was grown to 2 x lo* cells/ml in minimal medium II at 37°C and the cells then pelleted by centrifugation at 10,000 revs/min for 10 min at 4°C in an 5534 Sorval rotor. After resuspending the cells in 1.5 ml of a 10 mM-MgSG, solution, 0*5-ml portions were infected with 1 of 3 phages: ADam15b221imlla2lcItsSam7, hb221cIts857Sam7, or ~Dam15b221cIts857Sam7, at a multiplicity of 5 phage per cell. Phage were adsorbed for 5 min at 37°C. The infected cell complexes were then diluted 20-fold into minimal medium II at 37°C containing 0.4 #X/ml of a i4C-labeled reconstituted protein hydrolysate (Schwartz-Mann, 3122-09). The culture was vigorously aerated for 90 min, the cells then pelleted by centrifugation as described above and the pellet resuspended in 05 ml TMG containing 1 pg DNase I/ml (Worthington, RNase-free). One drop of chloroform was added, the cells were vortexed for 5 s and lysis completed by incubating the cells an additional 5 min at 37%. During the 90 min labeling period incorporation of radioactivity into trichloroacetic acid-insoluble material increased as an exponential function of time. (i) Pur$cation

of labeled

phage

for

electrophoresiis

Prior to purification of the labeled phage a 20-fold excess (based on plaque-forming units) of hcIts857 phage was added to the above ,W + and hD - lysates to measure recovery and facilitate purification of the labeled phage. Cell debris was then removed by low-speed centrifugation (8000 revs/min for 10 mm at 4°C in an SS34 Sorval rotor) and the phage purified by centrifuging the supernatant into a CsCl step gradient for 60 min at 4°C in a

THE

ILOLE

OF

pD

IN

THE

PACKAGING

OF

h 1)X-A

737

Beckman SW56 rotor at 30,000 revs/min (4 steps of densities 1.7, 1.55, 1.45 and 1.38 g;cnl” were used). The visible phage band was removed with a Pasteur pipette aud dialyzt,d against 1000x vol. 10 mM-TrissHCl (pH 7*4), 10 mM-MgSO,. The phage were concentrated by centrifugation in an SW56 rotor at 30,000 revs/min for 50 min at 4”C, the pell(lt sulfate, 1 y. (v/v) /3-mercaptoethanol, 0.05 Mresuspended in 30 ~11% ( w / v ) so di urn dodecyl l’ris*HCl (pH 6.8), 10% (v/v) glycerol, O*OOl% (w/v) b romophenol blue, and tllen boilccl for 2 min. (j) Preparation of phage and ph particles for electron microscopy (i) f’hage

with

and

without

pD

Five different phages (XcIts857, hb221c126, XDam15b221cI26, hb221ivnm21clts, hDaml5b22limm2lcIts) were prepared as described in section (h), above except that tllo growth medium was tryptone with 10 mM-MgSo, and the cells lysed about 60 miu aftc\r illfection. The phage from 200 ml of lysate were purified by polyethylene glycol precipitation, and CsCl equilibrium density gradient centrifugation as described by Sternbcrg et al. (1977). (ii) pX particles X were prepared and purified as described by Hahn & Hohn (1974). Briefly a 25%ml culture of strain NSlOOO was grown to 2 x 10’ cells/ml in Luria broth at 32°C. The cultllre was then induced by shifting the temperature to 42°C for 15 min and growth allowed to cont)inue for 45 min at 38°C. The cells were then pelleted by centrifugation in a GSA Sorv& rotor at 8000 revs/min for 10 min at 4°C and they were then resuspended in 4 ml Worccl buffer A (10 mnn-TrisaHCl (pH &O), 10 mM-NaN,, 100 mM-NaCl, 20% sucrose). ‘Tilt> following additions were then made to the cell suspension. First 1 ml of Worcel buffer B (120 mlvr-TrisaHCl (pH 8.0), 50 mM-NaEDTA, 4 mg lysozyme/ml (Sigma; egg white)) was added and after incubation for 30 min at 4”C, 5 ml Worcel buffer C (loo/, Brij-58, 0.4°0 sodium deoxycholate, 2 M-NaCl, 10 m&I-NaEDTA) was added and incubation was continued for 10 min. The cells were then centrifuged for 20 min at 15,000 revs/ml11 in all 5534 Sorval rotor at 4°C and the upper phase (containing the ph) removed and oentrifugcsd at 45,000 revs/min for 60 min at 4°C in an SW56 Sorval rotor. The ph pellet was resuspended in 0.4 ml 10% sucrose, 0.1% /&mercaptoethanol, 10 mM-Tris*HCl (pH 8.(b), 1 rnM-MgSO,, 10 mM-NaN,. As judged by electron microscopy the p/\ prepa,rat,ion was contaminated with phage tails (about 20% by number). The latter structures did notJ interfere with the use of the preparation for head size det,ermination (see section, (11). helms). (k) Preparation of pD + and pull - extracts (i ) I :nlabeled ez-tracts Cultlmxs (50 ml) of strains NS1008 and Xi101 1 \vere grown ill minimal tnttlium I ilt 32°C to 2 x 108 cells/ml and then induced by shifting the temperature of the culturc~ to 42“(!. Tile cnlt,ures Lvcrc vigorously aerated for 15 min at tllis temperature, then slliftt~d to 38°C: for 75 mill. After chilling them to 4°C the cells were pelleted by centrifugatiorl ;\t 8000 revs/min for 10 min at 4°C in a GSA Sorval rotor. The pellet was resuspended in I 1111 TM(: and the cells lysed by adding 1 drop of chloroform and vortexirlg for 5 s. (ii)

Labeled

extracts

These extracts were prepared in the same way as the unlabeled extracts except for tile following modifications: (1) minimal medium III was used rather than minimal medium I; (2) 35 min after lysogenic induction L-[35S]methionine (NEN; spry. act. 338, 1 ~n( ‘ii 0.28 ml) was added to a final activity of 3 &i/ml and the cells lIarvested 5 min 1atPI.. During the labeling period the incorporation of [35S]metllionine intro t,ricllloroacetic acitl. insoluble material was complete within 2 min. (1) In vitro

addition

of pD

to phage

witho?Lt

pll

Lambda delet,ion mutants lacking pD were added t.o pD + and p D - extracts immc~diatcly after the extracts were prepared. Incubation was for 10 min at 37”C, a time period sufficient to allow complete addition of pD to Dphage (Sternberg & Weisberg, manuscript itI

N. STERNBERG

738

AND

R. WEISBERG

preparation). The pD addition reaction was stopped by diluting the extract lOOO-fold into TMG at 4°C. Reactions with labeled pD+ or pDextracts were not diluted after 10 min but were simply chilled to 4°C. Phage from such reactions were purified as described in section (i) following the addition of a 20-fold excess of hcIts857 phage. (m)

Sodium

dodecyl

gel electrophoresis

suljate/acrylamide

of labeled phage

Labeled phage preparations were subjected to electrophoresis for 3 h at 10 V/cm at room temperature on slab gels (Studier, 1972) composed of 13% (w/v) acrylamide and O*O44o/o (w/v) bisacrylamide. The gels were stained with Coomassie blue, destained and dried as described by Maize1 (1971). Dried gels were autoradiographed using Kodak SB54 film. (n)

Electron

microscopy

of phage

The procedure for negative staining described by Leonard et al. (1972) was used. Briefly, a carbon film was partially floated from the surface of freshly cleaved mica onto a phage solution (a mixture containing 2 x lOi pA particles and 0.5 x 101’ to 2 x lOi purified phage in 10 mM-TrisaHCl (pH 7*4), 10 mivr-MgSO,). A copper microscope grid was then placed on the floating carbon film for 30 s and both picked up from the liquid surface with tweezers and floated onto a 2% glutaraldehyde solution (made up in 5 mm-KHsPO,, 5 mM KsHPO,, 1 m&r-MgSO,, pH 7.0) for 1 min. The grid was then floated for 20 s onto the staining solution (1% uranyl acetate, pH 7*0), picked up, and dried with absorbent paper. Photographs were taken in a Philips EM300 electron microscope at 60 kV accelerating voltage at a magnification of 27,000 x . The area of the ph and phage head was determined by measuring head circumference from a projection of the negative at an additional magnification of 22.5 with a Numonics electronics graphics calculator. The data were tabulated and represented graphically by a Hewlett-Packard 982111 calculator and

plotter. (0) Heteroduplex

analysis

of

DNA

The technique was that of Westmoreland et al. (1969) as adapted by Enquist & Weisberg (1977). Briefly, phage DNA was extracted and denatured by treatment of purified phage with NaOH. Appropriate mixtures were reannealed by incubation in 50% formamide. The reannealed DNA was spread over a hypophase droplet asdescribed by Lis & Schlief (1975), picked up on parlodian-coated grids, stained with uranyl acetate, shadowed with platinum/ palladium, and coated with carbon. DNA heteroduplexes were photographed in a Phillips EM300 electron microscope, and the lengths of the molecules measured from a projection of the negative with a Numonics graphics calculator interfaced with a Hewlett-Packard 9821A calculator. Circular PM2 DNA, a gift of Dr John Richardson, was used as a length

standard. 3. Results (a) pD dispensible

in

certain

deletion

mutants

To see if D function is dispensible in mutants with less DNA, we crossed a D amber mutant with a series of EDTA-resistant deletion mutants of decreasing chromosome size and determined the proportion of suppressor-sensitive, EDTA-resistant phage among the progeny. We found that more than 99% of the EDTA-resistant progeny of the hDaml5 x hb221 (22% deletion) cross-plated on a sup- host. In contrast, 15 to 30% of the EDTA-resistant progeny of the hb2 and hb538 crosses (13 and 16.5% deletions, respectively) failed to plate on the sup- host. Further tests confirmed that these suppressor-sensitive phage were indeed recombinants carrying the deletion and the Dam15 mutation (see Materials and Methods, section (d)). The failure to find suppressor-sensitive b221 recombinants suggested that plaque formation by Xb221 but not by Xb2 or hb538 was D-independent. This was confirmed as follows. Twelve per cent of the EDTA-resistant progeny of the ADam x X6221 cross formed smaller plaques on a non-permissive than on a permissive host, a difference that disappeared

THE

ROLE

OF

pD

IN

THE

PACKAQING

OF

h DNA

739

when the top agar was supplemented with 10 mM-MgSO,. These small plaque-forming phage were found to carry a cryptic Dam15 mutation (that is, an amber mutation not detectable by differential plaque formation on sup+ and sup- hosts). (1) When a putative XDaml5b22lcIts857 recombinant was crossed with xEts2lcI+ (Fig. I), six of 12 ts + CI + progeny phage tested failed to make plaques on a sup - host and failed to complement hDaml5. (2) When radioactively labeled proteins from putative hDaml5b221 *sup- and hDaml5b22limm21 ‘sup- recombinants were fractionated b> electrophoresis on a sodium dodecyl sulfate/polyacrylamide gel, no band corresponding to pD was seen(Fig. 2, channels 1 and 5). Radioactively labeled hD+b221 proteins and unlabeled h wt proteins are shown for comparison (Fig. 2, channels2, 3, 4 and 6). The bands corresponding to pD, pV, pE, pB*, pH*, and pJ were identified by their mobility (Hohn et al., 1974; Hendrix & Casjens,1975). Except for pD, there was no difference between the proteins of hDaml5b221 *sup- and those of the other straills. We conclude that pD is dispensible for plaque formation by hb221. In section (r)% below we show that the smallest deletion mutation that’ allows D-independent plaque formation removes 18% of the h wt DNA content. The difference between ADam156221 .
L-----Ets21

J

XDaml5622lcIts857

L._---~

I/v

c1+

Mts2lcI+

FIG. 1. A cross between hDam16b221cIts867 and Mts2lcI+. The cross procedure is described by Sternberg (1976). The phage were plaqued on strain YMC (wp,F) at 42°C and turbid plaques (hcI+ ts +) purified and tested for the Dam15 marker. Six of 12 plaques tested failed bot,h to form plaques on strain 594 (sup-) and to complement xDam15. All 6 of these phage are int + and &a+ by the red plaque test (Enquist & Weisberg, 1976) and presumably arise by crossover (1). The remaining six phage are int- and/or xis- defective by the red plaque test, presumably carry the b221 deletion, and probably arise by crossover (2).

(b) The requirement

for pD is determined

by chromosome

size

Phage with large deletions might grow in the absence of pD either because the deletions remove a gene encoding a diffusible inhibitor or becausepD is dispensible when the chromosome size is lessthan 82% that of wild-type h. In the first case, the Dam deletion mutant would not grow in a cell mixedly infected with a non-deleted Dam phage; in the second case, it would. We therefore mixedly infected a nonpermissive cell either with hDaml5b221 and XDaml5 or with hDaml5b538imm434 (D-independent) and hDaml5b538immh (D-dependent). In agreement with the second hypothesis, the D-independent phage grew well in both of these mixed infections (Table 2). The results also rule out two lesslikely hypotheses. (1) The D-independent phagesmight have acquired the ability to sUynthesize a diffusible product that obviates the requirement for PD. If so, the D-dependent phagesshould also have grown in the rnixedly infected cells, and they did not. (2) The D-independent phages might have lost a gene encoding a non-diffusible (“c&s-acting”) inhibitor. This gene cannot be

740

N.

STERNBERG

AND

R.

WEISBERG

DJ-

pw*pB*PC-

P”--

PO-

3

4

5

6

FIG. 2. An analysis of the protein composition of pD-deficient and pD-containing deletion phage by acrylamide gel eleotrophoreses. Phage were labeled by growth in strain 694 with a ‘%-labeled reconstituted protein hydrolysate (Schwartz-Mann; 3122-09, 0.4 &i/ml), purified, and subjected to gel electrophoresis. The gels were stained with Coomassie blue and autoradiographed (see Materials and Methods, sections (g), (h), (i), and (m)). Prior to phage purification a lo-fold excess of unlabeled kItsEEi7 was added to each of the labeled lysates. Slots 2, 4 and 6 are the stained protein bands of the unlabeled hcIts867 phage. Slots 1, 3 and 6 are labeled protein bands, respectively, from ~8mlSb221cIts857~am7~sup-, hD+b221cIts867Sam7.sup-, andXDamlSb22limm2lcItsSam7~sup-.

THE

ROLE

OF

pD

IN

THE

TABLE

Nixed

infections

(a)

warn15

(h)

XDaml5b221

(c)

ADamIS+ hrhd5hm

LCxperiment

OF

741

X l)NA

2

between p,D-dependent

Phagc

Experiment

PACKAGING

sup-

and pD-independent Yield per host

infwted

phage

crll sup + host

I
30

43

47

23 b221 <0.2 tj221+

26 6”“lai “0 b221’

II

(a)

hDam15b538

(b)

hDam15b538imm434

(c)

XDam156538+ hDam15b538imm434

2.5

70

50

95

48 imm434 2 immX

40 imm434 50 immA

These infection experiments were performed as described in Materials and Methods. section (e). The .wp host, was 594 and the aup + host used was C600. The phage in experiment I carry the cIts857 mutation and were assayed with strain LE289 on TR-TTC agar plates at 32°C (Enquist & Weisberg, 1976). Under these conditions non-deleted phage form red plaques and hb221 forms colorless plaqws. The immX phage in experiment II carry the cIto857 mutation and km434 phagv carry an uncharactcrized clear mutation. These phage wc~c assayed, rwprctively. on stmirl-: SW617 and NS62.

located in the immh region as Xh221 immh and several other imm.h deletion pha,ges ar(’ D-independent. Neither can it be located anywhere else, however, as hh538imm434 but not Xh538immh is D-independent. The simplest conclusion is that the requirement for pD is determined by chromosome size. (c) Phage production

without pD increases graduall!! chromosome size

with dPcreasirq

Measurement of phage production after a single cycle of infecbion by XDam phagca gives a more quantitative indication of D dependence tha.n does plaque formation. Accordingly. we measured the production of phage after infection of sup- and supE + hosts wit,h various hDam15 deletion mutants. The results (Table 3) show a progressive increase in phage production with decrease in genome size from 87 to 780;. The kinetics of phage production of hh221Dam15 (780;6 genome size) were independent of pD (Fig. 3). The maximum chromosome size for D-independent plaque formation is between 82qg and 84% of wild type. D amber mutant)s with 827; chromosome size form small plaques on a non-permissive host; mutants with larger deletions form normal plaques on such a host provided the top agar is supplemented with MgSO,. (d) Pseudorevertants

of XD - are deletion

We predict that selection of suppressor-independent should yield deletion mutants that have lost 18% or contiguous stretch of DNA that long, lacking genes equal to about 30% of the )\ chromosome and is located (Fig. 4). The int and xis genes lie near the center of

mutants

variants in a stock of hDarn more of their DNA. The only essential for lytic growth. is between phage genes J and X this interval. Therefore most

N. STERNBERG

142

AND

R. WEISBERG

TABLE 3 The yield of Dam phage in sup + and sup - hosts Deletion size (% hwt)

Phage

sup-

Yield per infected cell host wp+ host

-

-

AD&In15 xDamlbb2 XDamlbbb38 ,WDam1bb2imm21

0

< 0.02 <0.02 1.1 6.5 29 66 58 50

13 16.6 18 19.5 22 25

hDamlbbb38imm434 hDamlbb221 ~amlbbb38imm434ninb ADamlbb221imm21

27

30 45 60 65 75 70 52 42

These single-cycle growth experiments were performed as described by Sternberg (1976). The multiplicity of infection was 0.1 phage/cell, incubation was at 38”C, and the phage yields were measured 90 min after infection. The sup- host was 594 and the sup+ host was C600. All of the immh phage carried the cIts857 mutation, the imm21 phage carried a cIts mutation, and the imm434 phage carried an uncharacterized clear mutation.

I

25

I

35

I

45 Time after

I

55 infection

I

70

1

90

(min)

FIG. 3. The kinetics of hb221cI26 phage production in the presence and absence of PD. The experiment was performed as described by Sternberg (1976). Briefly, strains 694 (-a-@--) and C600 (-- 0 -- 0 --) growing logarithmically at 2 x lo* cells/ml were centrifuged (6000 revs/min in an SS34 Sorval rotor for 6 min at 4°C) and the cells resuspended in l/5 vol. 10 mnr-MgSC4 and infected with hDamlbb221oI26 at a multiplicity of infection of 0.1. Phage adsorption was allowed to occur for 5 min at 38°C and the infected cells were then diluted lOOO-fold into TB with 10 marMgSO+ Portions were removed at designated times after dilution, the cells lysed by vortexing a portion with 1 drop of chloroform and phage assayed.

THE

ROLE

OF

pD

IN

THE

PACKAGING

OF

X DNA

743

-6221 -

6530 -62

Heads

Tais

EEslnMJ5

rbx-csscntlal genes

FIG. 4. A gen&ic map of phage X. The phage DNA is designated by the solid line, the approximate positions of phage genes by the symbols above the line, and the location of head, tail, and non-essential gene clusters by the brackets below the line. The cross-hatched bars above the linv give t,hs location and extent of deletion mutations.

suppressor-independent variants that arise by DNA deletion should be int - xis -. We induced non-permissive lysogens of Dam15 and ADam and plated the lysates on lawns of strain RW842 (sup-) on galactose-tetrazolium plates. In these conditions, hint + xis + makes red plaques while hint - and hxis - make colorless plaques (Enquist & Weisberg, 1976). We found for both Dam lysates that 20 to 50% of the plaques were colorless as expected for deletion mutants. The phage forming red plaques are presumably true revertants to D+ ; in any case, their sensitivity to EDTA (data, not, shown) shows they are not large deletion mutants. The yield of colorless plaqurforming phage per cell was approximately 10m6 for the hDaml5 lysogen and 10e7 for the hDam123 lysogen. We have no explanation of this difference. To confirm the above results and to estimate the range of chromosome sizes that can be obtained in this way, we isolated 20 colorless plaque mutants from a lysatc of 2 x 1011 cells of an induced hDaml5 lysogen. The large number of cells was used to ensure that most of the mutants arose independently. After isolation the mutants were grown on a supF host to ensure that the phage particles would contain PD. All of these phage lines were EDTA-resistant and were both int- and zis- as proved by complementation with Xint6 and Axis1 (Enquist & Weisberg, 1976). Four were either exe- or bet- and gam+ as judged by their inability to plaque on a ligts host (Zissler et ccl., 1971; Gottesman et al., 1973) and their ability to plaque on a recA host (Zissler et al., 1971). When the original lysate was fractionated on an equilibrium CsCl density gradient, the colorless plaque-forming phage had densities corresponding to DNA contents of 73 to 82% h wild-type (Fig. 5 ; see Fig. 6 and Table 4 for the relationship between density and DNA content of pD-deficient phage). A more extensive analysis of the use of D mutants for generating specific classes of deletions is presently in progress (Sternberg, Enquist & Weisberg, manuscript in preparation). (e) Loss ofpD

increases the density of the phage

Phage h is about 500/, protein (Hershey & Dove, 1971) of which about 14% is pD (Murialdo & Siminovitch, 1971; Casjens & Hendrix, 1974). We therefore expect that loss of pD should appreciably increa,se the density of the phage. This prediction was confirmed by equilibrium density gradient centrifugation of /\Dam15b221 (22.3?,, deletion), ADam15b538imm434nin5 (25.0% deletion), and hDam15b221imm21 (27.3”,, deletion) (see Fig. 6 for hDam15b221). The increase in buoyant density caused by growth in a sup- host, and therefore by loss of pD, is given in Table 4. This increase can be converted to the number of copies of pD lost per virion by a suitable modification of equation (5) of Davidson t Szybalski (1971) and by assuming that the monomer molecular weight of pD is 11,000 (Murialdo & Siminovitch, 1971). The values thus

744

N.

STERNBERG

AND

WEISBERG

AD+bZZl

A62

I 17

R.

I

I

I

I

I

21

25

29

33

37

Fractm

no.

5. The distribution of pD-independent deletion phage in a CsCl equilibrium density gradient. A 500-ml exponentially growing culture of strain NS660 at 2 x 10s cells/ml in TB with 10 mMMgSO, was induced by shifting the temperature from 32’C to 42°C. After 16 min at 42°C the culture was shifted to 38°C and aerated until cell lysis occurred (60 min after induction). The titers of the pD-independent deletion phage and Dam+ revertant phage (colorless and red plaques on strain RW842; Enquist & Weisberg, 1976) were, respectively, 1 x lo2 and 3 x 102 phage/ml. These phage were purified by PEG precipitation followed by oentrifugstion in a CsCl step gradient as described in Materials and Methods, section (i) and by Sternberg et al. (1977). Three marker phage (Ximm434Sam7, hb2cIts857Sam7 and hb221cIts867Sam7) were then added in lOOO-fold excess to the purified phage preparation and this phage mixture centrifuged to equilibrium in a CsCl gradient as described in Materials and Methods, section (g). The marker phage (-•-•--) were assayed on strain YMC and their positions assigned on the basis of their expected relative densities The density profile of the himm434SSam7 and hb2cIts867Sam7 marker phages are shown and the peak positions of all 3 marker phages are indicated by the open arrows. The XDamlS deletion phage (-- 0 -- 0 --) were assayed as colorless plaques on strain RW842. The expect,ed positions of hDam16b221cIts867.supand XDam16b221imm21~supin this gradient were determined by centrifuging these pl)-deficient phage with the above 3 marker phage in a parallel gradient. The peak positions of these 2 pD-deficient phages are indicated by the closed arrows. Pm.

obtained (424 to 449 copies of pD per virion; Table 4) are in reasonable agreement with those estimated from a more direct radiochemical determination (390 to 440 copies per virion; Casjens & Hendrix, 1974). (f) pD stabilizes In the Introduction we proposed that complex against disruption by overfilling form of abortive release of DNA from the disruption of the head shell. We now wish pD acts by enlarging the prohead so that

the head

pD stabilizes the DNA-prohead packaging with DNA. Disruption could either take the maturing head or the more drastic physical to consider the alternative hypothesis that it can accommodate more than 82% of a

THE

ROLE

OF

pD

IN

THE

PACKAGING

loL----30

34

38

Fraction

42

OF

46

h I)NA

715

5C

no

FIG. 6. CsCl equilibrium density gradient analysis of r\Dam15b221cI26 phage grown in sup+ and sup- hosts. Plate lysates of mam15b221cI26 were prepared on strains 594 (sup-) and YMC (sup+) starting from a single plaque grown on strain C600. Approximately lo8 phage from either of the 2 lysates were mixed with 10s ,!b2cIts857Sam7 and 10s hb221cIts867Sam7 marker phage and the solution centrifuged to equilibrium in a CsCl density gradient as described in Materials and Methods, section (g). U)Darn phage were assayed as clear plaque-formers on strain YMC at 32°C and the marker phege as turbid plaque-formers at this temperature. The Figure is a composit,e of 2 gradients, 1 containing hDamlSb22lcI26.sup(-@--a--) and the otherhDemlSb22lcI26. sup+ (-O-O-), aligned by the marker phages in each gradient. The density profile of the hb221cIts867Sam7 marker phage (-A-A-) is presented for comparison with hDam156221cT26~ szcp + . The peak position of the hb2 marker phage is indicat,ed by the arrow.

wild-type chromsome length. We can measure the capacity of a head that lacks pII by asking it to package a headful of DNA ( see below). If pD enlarges the head, a headful of DNA packaged without pD will be about 82% of the wild-type chromosome size. In contrast, if pD stabilizes rather than enlarges the head, lack of pD will not change the normal headful size. As this size is far larger than 82% (Sternberg & Weisberg, 19’75), the excess DNA might destabilize the pD-deficient head which would therefore be incapable of packaging headfuls. This is in fact what we observe. Phage h eficiently packages headfuls of DNA after induction of an excisiondefective DC prophage (Little & Gottesman, 1971; Sternberg & Weisberg, 1975; also see Fig. 1 of Sternberg & Weisberg, accompanying paper). Packaging initiates at thth prophage cos and proceeds rightwards into the bacterial DNA until the head is full. The left end of the packaged DNA is the normal h left end formed by cos cutting. The right end of the DNA is formed in vitro by DNase treatment. Such treatment digests unpackaged DNA that protrudes from the head and blocks tail attachment. The resulting particles, which contain a headful of DNA, are matured by the addition of

746

N.

STERNBERG

AND

R.

TABLE

WEISBERG

4

The density change associated with pD addition Dam16 deletion mutants

- Asup + k cm-7

-f

hb22limmh hb538imm434nin6 /\b221imm21

0.223t

0.0271t

0.255t 0.272t

0.0308 0.0332

- Asup (g cme3)

APD (g cme3)

Copies of pD

0.0098 0.0125 0.0161

0.0173 0.0183 0.0171

426 449 424

t Values taken or calculated from Table 2 of Davidson & Szybalski (1971) or Fiandt et al. (1971). The Dam16 deletion mutants were grown in strain 594 (sup-) or strain YMC (sup+) and centrifuged to equilibrium in a C&l density gradient. The gradients were fractionated and the fractions assayed for viable phage. The following phages were centrifuged in the same tubes aa density markers: hb2cIts857Sam7; p = 1.491; and hb221cIts867Sam7; p = 1.481.fis the fractionalchange in molecular weight of the mutant DNA relative to that of wild-type h. Asup+ and Asupare the changes in density relative to that of wild-type X after growth on t,he sup+ and sup- hosts, respectively. ApD, the density change associated with pD addition, is the difference between Asupand Asup + . The number of copies of pD per virion was calculated using a suitable modification of equation (5) of Davidson & Szybalski (1971) to relate ApD to the fractional change in mass and assuming the monomer molecular weight of pD is 11,000 (Murialdo & Siminovitch, 1971). We used the values of Davidson & Szybalski (1971) and Fiandt et al. (1971) for the buoyant densities of DNA, protein, and wild-type h.

pFI1 and phage tails. They are called MocL (defective, one cohesive end, left; Little & Gottesman, 1971). To see if hdocL particles are produced in the absence of pD, we induced a single lysogen of hDam15b2. The b2 mutation prevents normal prophage excision (Gottesman & Yarmolinsky, 1968). We looked for hdocL particles in the lysate in two ways. (1) We measured the number of bio transducing particles. The bio operon, which is carried by D+ XdocL, is located 0.5 h lengths to the right of the b2 prophage cos (Hradecna & Szybalski, 1970). Therefore D- hdocl, even if their DNA size were reduced to 82%, should carry it. In fact the yield of bio transducing phage in the Dlysate was only 5 x 10m6 that of a control D+ lysate (Table 5). (2) We labeled the cells TABLE

5

hdocL and h generalizedtransducing phage production from D + and D excisior&efective lysogens Prophage allele

D

pD added in vitro

+

No

am15 am15

No Yes

Relative hdocL (bio transducers) 100 5x 10-k 1 x 10-z

yield of: XaroA generalized transducers 100

<10-a 5x10-3

Phage lysates were prepared 90 min after induction of either strain NS490 or strain NS432 as described by Sternberg & Weisberg (1975). pD in vitro complementation of the NS432 lysate was performed as described in Materials and Methods, section (1). AroA generalized transducing particles were assayed on strain NS604 and hdocL was assayed by biotin transduction of strain R876. These transductions were performed as described by Sternberg & Weisberg (1976).

THE

ROLE

OF

pD

IN

THE

PACKAGING

OF

h DNA

747

wit,h [3H]thymine before and during induction and measured the amount of radioactivity in virions as follows. After lysis, cold carrier phage were added, the lysate was purified and centrifuged to equilibrium in a CsCl density gradient, and the gradient was fractionated and counted (Fig. 7). The number of 3H counts at the densit) predicted for D- hdocL (see legend to Fig. 7) was less than 2% that found in a comparably treated D + control lysate. These results show that DNL4 packaging by a headful mechanism requires PD. The simplest interpretation is that uptake of an excessive amount of DNA destabilizes the pD-deficient head. This implies the exist,ence of a mechanism that prevents the uptake of excess DNA during packaging of concatemeric DNA of D- deletion mutants. We suggest that this is accomplished by recognition and binding to the head of the next adjacent cos site to the right of t,he one at which packaging initiated. We shall consider this point further in the Discussion. Three other classes of particles in addition to MocL are formed in low yield after induction of an excision-defective prophage, and we asked if production of these particles requires PD. (1) Generalized transducing particles are formed by headful packaging of bacterial DNA (Sternberg & Weisberg, 1976). Like hclocl, t’hey are not formed after induction of a D- prophage (Ta,ble 5). (2) About lo-” plaque-forming

FIG. 7. CsCl equdibrium density gradient analysis of XdocL phage from strain NS432 and NS490. MocL was labeled with [3H]thymidine, purified by centrifugation to equilibrium in a CsCl density gradient, and assayed as described by Sternberg & Weisberg (accompanying paper). The Figure is a composite of 2 gradients, 1 containing labeled hdocL from strain NS490 (----O-e-) and the other any presumpt,ive labeled XtZocL from strain N8432 ( ---i ; (,) ~- ). The gradients were aligned by the 2 marker phage (hdoeL and /\wt) I:wcwmt in each gradient (open arrows). The positions represented by the closed arrows are those expected for pD-deficient hdocL particles containing either 0.82 X length DNA (fraction 16) or I.05 h length DNA (frection 7).

N. STERNBERG

748

AND

R. WEISBERG

phage per cell are produced after induction of a hb2 lysogen (Gottesman & Yarmolinsky, 1968). When the prophage was also D-, the yield decreased loo-fold. More than 50% of these phage were int- and/or xis- and were able to form plaques on an sup- host. Presumably their DNA content has been reduced by deletion so that their growth is pD-independent. (3) Lambda docR particles (defective, one cohesive end, right) contain prophage and bacterial DNA from the left of cos(seeFig. 1, Sternberg $ Weisberg, accompanying paper). The right end of MocR DNA is the wild-type right cohesiveend formed by coscutting (Gottesman & Yarmolinsky, 1968; Little & Gottesman, 1971); the left end appearsto be formed by an uncharacterized nucleaseof little or no site specificity. The chromosomesof a hdocR population therefore have a broad distribution of sizesranging from lessthan 75% to greater than 106% wild-type size (Little & Gottesman, 1971; seeFig. 8). About 20% of the chromosomesare lessthan 82% wild-type size. If pD stabilizes particles against disruption by overfilling with DNA, we expect that the production of XdocR with chromosomesizesexceeding 82% will require pD, and the production of those with smaller chromosomesizeswill not. The following results are consistant with this prediction. We can determine the total number of MocR without regard to chromosome size by measuring the number of particles in the lysate capable of giving plaque-forming recombinants in a crosswith a repressedheteroimmune prophage (Little 6 Gottesman, 1971). This assay, which requires only that the hdocR contain the region from costo the left prophage terminus, has an efficiency of at least 50% (Sternberg & Weisberg, 1975). We found that mutation in gene D reduces the production of infective XdocR to about 1% of the D+ value (Table 6, column 3). However, there are many noninfective particles in this lysate: the XdocR titer increased to ZOoA,of the DC value upon addition of a pD-containing extract (Table 6, column 3). The mechanism of this increase in infectivity is not understood, but it presumably involves addition of pD to the non-infective particles. The value of 20“/o is what we expect if only XdocR particles with chromosomeslessthan 82% wild-type size are stable in the absence of PD. Do the hdocR produced by induction of a D- prophage in fact contain shorter chromosomes?We answered this question in two ways. TABLE

XdocR production Prophagge allele

D

pD added in vitro

6

from D + and D- prophzges

Total XdocR

galP transduction

100 1-2 16-20

20-23 0.1-0.13 1.4-1.6

g&T transduction

gaZK transduction -

+ am16 am16

No No Yes

2P31 0~010-0~013 0.18-0.20

9-15 0.001 0.02

Lambda docR lysates were prepared 90 min after induction of either strain NS490 or strain NS432 as described by Sternberg t Weisberg (1976). pD in vitro complement&ion of the NS432 lysate was performed as described in Materials and Methods, section (1). Total XdocR phage were assayed as plaque-formers on strain NSlO60 (column 3; see text). AdocR was also measured by galactose transduction of strains carrying either a gal promoter mutation (NS443) (column 4), a galTern mutation (NS443) (column 6) or gaZKT missense mutations (694) (column 6). These transductions were performed as described by Sternberg & Weisberg (1976). The total AdocR phage titer in the NS490 lysate (1.2 x lOma phagelinduced cell) was arbitrarily given the value 100 and all other AdocR titers are presented as a percentage of this value. The 2 values for each of the titers represent the range of values obtained from 3 different experiments.

THE

ROLE

OF

pD

IN

THE

Xb2imm434

PACKAGING Xb538nnm434

OF

X DNA

749

105

104

%

103

2 6 'ij ; f

102

IO Froctlon

no

Fm. 8. CsCl equilibrium density gradient analysis of hdocR phage. XdocR lysates were prepared from strrtin NS490 and NS432 8s described by Sternberg & Weisberg (1975). The in vitro addition of pD to hdocR phage lacking this protein (D -XdocR) ~8s performed 8s described in Materials and Methods, section (1). The Figure is 8 composite of 3 parallel gradients prepared as described in Msteri8ls and Methods, section (g) end aligned by the 3 merker phages (himm434utt2, hb2imm434 and hb638imm434c) added to each gredient. The peak positions of the marker phages are indicated by the arrows. Each gradient contains one of the following AdocR phage : D + AdocR (- A-- &> - . 3 X IO5 phage); D- hdocR (-e-o-, IO4 phage); D- AdoeR + in vitro added pD (-O--O-, lo5 phage). hdocR ~8s assayed by plaque formation on strain NS1060 and the himm434 marker phage on strain 594. To distinguish between Ab2imm434 and hbb38imm434c plaque assays were carried out at 32°C. At this temperature the former phage makes 8 turbid plaque and the letter 8 cleer plaque.

(1) We determined the DNA content of D+ MocR, D- MocR and D- MocR + pD by centrifugation in CsCl density gradients. The results (Fig. 8) confirm that the D- MocR to which pD was added in vitro correspond essentially to that subclass of D+ hdocR with a DNA content ranging from 0.73 to 0.82 h wild-type length. DAdocR, with or without in vitro added pD, have the same DNA content. The difference in their densities can be completely accounted for by the difference in their pD content (see section (e), above). (2) The gal promoter (gulp), which is at the right end of the gal operon, is 0.69 h lengths to the left of cos (Ahmed $ Johansen, 1975). We therefore expect that the left ends of AdocR chromosomes that are packaged in the absence of pD should lie within 0.13 h lengths (=0*82-0.69) or about 6000 base-pairs to the left of gaZP. This prediction can be tested by measuring the efficiency with which D- and DC hdocR transduce different gal markers : we expect that transduction frequency should increase with increasing distance of the wild-type allele of a marker from the DNA 48

N. STERNBERG

760

AND

R. WEISBERG

ends. (This assumesthat crossovers do not occur preferentially at the left end of AdocR DNA.) We used three mutants: the galP marker is closest to the prophage, the gaETmarker is next, and galK is most distant (seeFig. 1 of Sternberg & Weisberg, accompanying paper). We found (Table 6) that the proportion of D+ hdocR capable of transducing galK was several hundred fold more than the corresponding proportion of D- AdocR. In contrast, the D +ID- ratio for the proportion of particles transducing gulp was lessthan 3. Transduction of galT, which lies between the first two, gave an intermediate ratio. The sameratios were obtained after addition of a pD-containing extract to the D- lysate. We conclude that the chromosomesof the hdocR formed in the absence of pD are, on the average, shorter than those of the D+ hdocR. The particles whosepresencewas revealed by addition of a pD-containing extract resemble the pD-deficient AdocR in the distribution of their chromosomelengths. (g) The head size of phage with and without pD Lambda phage with and without pD were visualized in the electron microscope and their gross appearance is the same (Fig. 9). The area of pX and of phage heads was measured as described in Materials and Methods and the data for hDam15b221~supand hDam15b221-sup+ plotted in Figure 10. The measurementsfor all of the phage heads relative to those for ph are presented in Table 7. No significant difference (t > 2) in head size was seen for any of the phages. In particular, the relative head size and standard error of the mean for an artificial mixture of h wt and hDam156221imm21-sup- was not significantly different from those of either of the two phages alone. TABLE 7 Head size measurementsof phage with and without pD

Phage

hwt ADam15b221~sup+ XDamlSb22l.qwADam16b221imm21~sup+ hDam15b221imm21-suphwt + ADam15b221imm21*sup-

Number of heads measured

Mean

Standard error of the mean

60 120 93 69 37 34

1.68 1.71 1.71 1.72 1.73 1.72

0.017 0.011 0.033 0.014 0.016 0.019

The area of pX and phage heads was measured as described in Materials and Methods, section (n) and in the legend to Fig. 10. The value for any mean head size is that relative to the mean size of the ph particles present on the same grid field. In the mixture containing X wt and XDam15b221imm21 .supthe concentrat,ion of each phage was approximately the same.

These results show that the role of pD cannot simply be to enlarge the head. They are consistent with the hypothesis that pD stabilizes the head against disruption by overfilling with DNA. The data presented in the next section also support this view. (h) Phage lacking pD are extremely sensitive to EDTA The rate of inactivation of h by chelating agents such as EDTA increases with increasing chromosomessize (Parkinson $ Huskey, 1971), and inactivation occurs by rupture of the phage head (Yamagishi et al., 1973). Apparently excess DNA

THE

ROLE

OF

pD

IN

THE

PACKAGING

OF

h DKA

1'3~. 9. Electron micrographs of pD-deficient and pD-containing phage. These micrographs at-(’ of 4srtificial mixtures of phage and p/\, and were prepared aq described in Materials and Methods. ret :tion (n). (A) /\wt + hDaml5b22limm21-sup+ pX. (B) hDam15b221imm21~s~cp~ $- ph. + pX. (D) /\Dam166221~sup+ ph. (E) hDamlSb221.sup+ / II/\. ((3 XDam15b221imm21~sup+ arrow in micrograph (B) points to a pl head and the rightmost arrow (F”) h wt + ph. The leftmost to a phage.

762

N.

STERNBERG

AND

R.

WEISBERG

‘0

h B

35

j

30

30

2

25

25

(b) p I

20

Head

size bxea,

82)

Flu. 10. The head size distributions of hDam166221c126.supw and hDamlSb221cI26~sup+ phage. PX and phage head sizes were measured as described in Materials and Methods, section (n). The cross-hetched bars are measurements of ph particles and the empty bars are measurements of either XDamlSb22lcI26*supheads (a) or UkmlSb221cI26*sup+ heads (b).

disrupts the head in the absence of divalent cations. If pD acts during DNA packaging to stabilize an overfull DNA-prohead complex, a phage particle lacking pD might have an increased sensitivity to EDTA. The inactivation curves shown in Figure 11 show that this is so: hDam15b221 *sup- is inactivated much faster than wild-type h and XDam15b221 *supF. We shall show in a subsequent article (Sternberg 6 Weisberg, manuscript in preparation) that resistance to EDTA is a sensitive measure of the number of phage that have a full complement of pD : particles with a partial complement remain quite sensitive. (i) D-independent

virions that lack pD cm accept this protein both in vivo and in vitro

Measurements of labeled pD, of phage buoyant density, and of EDTA resistance show that D-independent Dam phage grown in a sup+ host or D-independent Dam+ phage grown in a sup- host contain pD (see sections (a), (e) and (h), above). Hohn & Hohn (1973) have reported that addition of an extract containing pD to a lysate of XDaml5 increases infectivity from low7 to low5 plaque-forming units per induced cell; thus such lysates contain D-deficient non-infective particles that can interact ila vitro with PD. We find that hDsm15b221‘sup- particles can add pD from a pD-containing extract to such an extent that they are indistinguishable from hDam15b221*wpF or Adam+ b221 in buoyant density and resistance to EDTA (Fig. 6 and 11). Exposure of the phege to an extract lacking pD had no effect (data not

THE

ROLE

OF

pD

IN

THE

5 Time

PACKAGING

IO after

exposure

15

OF

h DNA

753

20

to EDTA hnl

Fra. 11. The kinetics of EDTA inactivation of h wild type and hDem15b221cI26 phage. The inactivation reactions were performed at 32°C as described in Materials and Methods, section (f). hDam156221cI26.sup+ ; hDam15b221cI26.sup-; -A-A--, -A---G--. -0-O-9 ADam166221cI26..sup+ in vitro added pD; -a-@-, X wt. At 4°C no significant EDTA inactivation of h wt was detectable throughout the 20 min incubation period. hDam15b221cI26. sup- was inactivated with the same kinetics at 4°C as at 32°C.

shown). To confirm this finding and to see if any proteins other than pD add to pD-deficient particles, we incubated lysates containing XDam15b221 -sup- and )rDam+b221 with [35S]methionine-labeled extracts made from induced hDam and hDam+ lysogens. After incubation, unlabeled carrier phage was added, the mixture purified, and a portion analyzed by gel electrophoresis. The gel (Fig. 12, channel 2) containing hDam15b221 ‘sup- phage that had been exposed to the pD+ extract contained two radioactive bands. The major band had the mobility of pD and the faint band the mobility of pV (the major tail protein). As expected no labeled pD band was present in the channel (channel 4) containing XDam156221 *SUP- phage that were exposed to the extract lacking PD. However, a faint band with the mobility of pE, the other major h head protein, is seen in this channel. The pE and pV bands cannot reflect a substantial addition of these proteins because the autoradiographs indicate they are present in greatly reduced amounts relative to pD, and because the density of the /Warn156221 -sup- phage incubated with the extract lacking pD did not change (data not shown). More likely, it reflects contamination of the purified phage w
N.

764

STERNBERG

AND

R.

WEISBERG

PJ-

PM*-

+*-

PEPV-.

I

2

3

4

5

6

Fro 12. The in vitro addition of labeled pD to hDamlbb221*su~phage. The procedures used in preparing the [s%]methionine-labeled pEand pDextracts and in performing the in vitro addition reaction are described in Materials and Methods, sections (k) and (1). After the in vitro pD addition reaction, X wt was added in lo-fold excess, the phage mixture pursed, subjected to aorylamide gel eleotrophoresis, and the gels stained and autoradiographed (see Materials and Methods, sections (i) and (m)). Slots 1, 3 and 6 are the Coomassie blue-stained protein bands of the h wt phage added subsequent to the pD addition reactions. Slots 2, 4, and 6 are the labeled proteins, respectively, from addition reactions XDamlSb221cIts857~am7-sup+ pEextract, XDamlSb22lcIts867Sam7~su~+ pDextract and hD+b221cIts867Sam7 + pEextract. The small amount of labeled pV and pE protein present, respectively, in slots 2 and 4 is probably due to residual contamina tion of the pursed phage by these major extract proteins.

THE

ROLE

OF pD

IN THE

PACKAGING

OF A DNA

755

(j) The DNA of particles lucking pD appears to be cut at cos Wake et al. (1972) have shown that pD is required for production of the normal cohesive ends in intracellular concatemeric X DNA where the monomer unit is a wild-type chromosome. The ends are presumably formed by cutting the concatemers at cos (see Yarmolinsky, 1971). The ability of D-deficient deletion phage to form plaques shows that DNA cutting must occur during assembly of the virion: uncut and unpackaged DNA protruding from the phage head is expected to block tail attachment thereby rendering the particle non-infectious (Sternberg t Weisberg, 1975). The agreement between the change in buoyant density and the change in pD content (section (e), above) supports this conclusion: any appreciable excess DNA associated with the particles should have increased their buoyant density. Was this DNA cut at cos? We attempted to answer this question by electron microscopic examination of heteroduplexes formed by annealing denatured DNA from hDam15b221‘sup- and hDam15b221imm21-sup- with denatured DNA from Xb1451imm21b3000.The b1451 and b3000 mutations delete DNA from the left and right, respectively, of the immunity region and thus serve as convenient markers for electron microscopy (Enquist & Weisberg, 1977). Measurement of duplex lengths in these heteroduplexes (Table 8) shows that the ends of at least some of the DNA moleculesextracted from the particles lacking pD were within a few hundred nucleot’ide pairs of their normal locations. No circular heteroduplexes were observed (see legend to Table 8). Circles are expected if any of the chromosomescontain an uncut

TABLE

8

Mapping of DNA from pD-dejicient Phagpe

Mean I

length II

of duplex III

phage

regions IV

(%A)

Total

duplex

V -

,Wam15b221.sup-

40.150.3

1.7*0.2

5.040.1

5.2hO.l

13.250.2

65.0&

~am156221.sup+

39.4*0.7

2.1+0*2

S.l&O.l

5.2fO.l

13.3*0*1

65.0_+ 1.3

Expected?

40.6

5.2

5.5

13.3

I XDam15b221imm21~supExpectedt

409f0.5 40.6

2.0 II 2.OhO.2 2.0

VI 14.2hO.2 14.1

1.0

66.6

V 13.3hO.l

70.2:to.g

13.3

70.3

t Expected values for these duplex lengths are taken from the compilations and observations of Davidson & Szybalski (1971), Szybalski & Szybalski (1974), and Enquist & Weisberg (1977). Lambda Dam15b221cI26 and ~aml5b22limm2lcIts were grown in either strain 594 (sup-) or strain YMC (sup+). DNA was extracted, denatured, annealed with DNA from Abl451imm. 2lcIts b3000Sam7, and mounted for electron microscopy. Molecules with 3 single-stranded deletion loops and, for ADamlSb221cI26, a substitution bubble, were selected for photography and measurements made. Circular PM2 DNA molecules measured in the same photographs were used as an external length standard. At least 5 heteroduplexes of each type were measured. It was necessary to reject many molecules because the disposition of single-stranded DNA in the large b221 deletion loop made the location of the b221 branch point uncertain. None of the heteroduplexes were circular, as might have been expected if one of the polynucleotide chains in the heteroduplex had an uncut coa. (The X cohesive ends do not join stably in these conditions (data not shown).) Column I, left end to b221; column II, b221 to b1451; column III, b1451 to imm21; column IV, 1:mm21 to b3000; column V, b3000 to right end; column VI, b1451 to b3000.

766

N.

STERNBERG

AND

R.

WEISBERG

ws and ends that are displaced from ws by more than a few hundred base-pairs. These observations strongly suggestthat oligomeric DNA is cut at cosduring assembly of virions lacking PD.

4. Discussion We have shown that deletion mutants lacking about 18% or more of the wild-type chromosomecan grow in the absenceof the capsid protein PD. The resulting particles differ from normal virions in extreme sensitivity to EDTA, increased density and ability to accept PD. We have offered evidence supporting the hypothesis that pD acts to stabilize a prohead-DNA packaging intermediate against disruption by overfilling with DNA. Does other evidence support this view? Kaiser et al. (1975) showed that concatemeric XDNA became partially DNase-resistant when exposed to extracts made from cells infected with h D-. They proposed that partial DNase resistance in the absence of pD is a consequenceof entrance of the DNA into the prohead, an event required by our hypothesis. Murialdo & Siminovitch (1972) examined lysates of h D--infected cells with the electron microscope and found abnormal empty heads of approximately normal size. Such heads could be the products of abortive DNA packaging. This interpretation is consistent with the more recent results of Lickfeld et al. (1976), who examined sections of h D--infected cells with the electron microscope. They found heads of normal size that were partially filled with DNA. Such heads could be either precursors or by-products of DNA packa,gingin the absenceof PD. In any event, these observations show that pD is not directly required for the head volume increase of approximately twofold that accompanies DNA packaging. Our own results also support this conclusion: the heads of pD-deficient virions containing deleted DNA are indistinguishable in size from those of wild-type h. We do not know how severe the DNA-induced disruption of pD-deficient heads is. Clearly some or all of the packaged DNA must be releasedfrom the damaged head. The results cited in the previous paragraph also suggest that at least some of the damaged structures are recognizable as phage heads in the electron microscope. Perhaps studies of in vitro packaging in the absenceof pD will answer this question. We find that pD-deficient heads resemble normal heads in shape as well as size. In the model of X head structure proposed by Wurtz et al. (1976) these heads should lack the trimer subunit clusters, while in the model proposed by Williams & Richards (1974) and Howatson & Kemp (1975) the pentamer and hexamer clusters should be missing. A more detailed examination of the head surface of pD-deficient phage should reveal which model is correct. We have shown that the DNA of deletion mutants is cut, almost certainly at cos, in the absenceof PD. In contrast, the DNA of undeleted A is not, and concatemers accumulate after infection (Wake et al., 1972). Cos cutting of wild-type length DNA is also known to require proheads, pA, and several other h proteins (Dove, 1966, Ma&inlay t Kaiser, 1969; Wake et al., 1972; Skalka et al., 1972; McClure el al., 1973). These multiple requirements suggestthat Ter, the nucleasethat cuts ws, acts only on DNA that is bound to or within the capsid. Indeed, Emmons (1974) has shown that the h cohesive endsare not formed by a free nucleaseacting on unpackaged DNA. The results reported in this article show that pD is not Ter and imply that a DNA segment as long as 82% of the wild-type chromosomecan enter the head in the absence of PD. Why, then, is pD required for the formation of cohesive ends in

THE

ROLE

OF

pD

IN

THE

PACKAGING

OF

A DNA

751

concatemers of wild-type length chromosomes? We propose two models which differ in the assumed timing of cos cutting. In both, we postulate that the entrance of concatemeric DNA into the head initiates at ws and proceeds sequentially rightwards (Emmons, 1974; Feiss & Bublitz, 1975; Sternberg & Weisberg, 1975). In the first model, ws can be cut to form cohesive ends as soon as it enters or is bound to the prohead. However, when the chromosome length exceeds S2% of the wild-type, pD is needed to prevent the release of packaged DNA. If such DNA release rapidly follows cos cutting, and if free intracellular ends rapidly cohere to their complements, the lifetime of the cohesive ends will be short and their steady-state level will consequently be low. In the second model we assume that Ter acts late after the initiation of packaging. Perhaps Ter must be activated by entrance of some minimum amount of DNA into the head. If so, the time interval between Ter activation and head disruption by overfilling with DNA of wild-type length may be too short for efficient cos cutting in the absence of PD. Alternatively, or in addition, DNA might not be susceptible to Ter action until entrance into the head has ceased. Our evidence suggests that DNA uptake stops when the next adjacent cos site on the concatemer has bound to the head (Results, section (f)). If the distance between adjacent ws sites is greater than 82% of a wild-type chromosome, the pD-deficient head will be disrupted, and Ter will be inactivated. For model (1) to be correct, end joining inside the cell would have to be much more rapid than it is in vitro. This appearsto be true for cyclization of X chromosomesafter infection (Wang & Davidson, 1968), but accelerated joining of the ends of different moleculeshas not been demonstrated. It is noteworthy, however, that the number of cohesive ends found after infection with hD-, although low, is measurably higher than that found after infection with XE- or AA- (Wake et al., 1972). (pE is a major capsid protein that is required for prohead formation (Casjenset al.. 1970), and pA is believed to be Ter (A. Becker, personal communication).) As for model (2), the studies of Emmons, Feiss, and their collaborators on the packaging of the DNA of h mutants t,hat contain a tandem duplication of cos do indeed suggest that ws cutting occurs after DNA entrance has begun, and that Ter action is regulated by the amount of DNA in the head (Emmons, 1974; Feiss & Campbell, 1974; Feiss t Bublitz, 1975; Feiss et al., 1977). We summarize the relevant conclusions as follows. The DNA between two adjacent cos sites that are separated by lessthan 0.2 h lengths is not packaged as such and is probably not cut from a concatemer. In contrast, the DNA hetween two adjacent cossites that are separated by 0.75 to 1.05 h lengths usually is cut as such. However, the right-hand ws site of such a pair can occasionally he packaged uncut with a probability that increases as the distance between the two decreases.It is thus plausible that Ter requires some minimum length of packaged DNA in order to be active, and that its activity increasesgradually as the amount of packaged DNA exceeds this minimum. Although both models account for the results reported in this article, model (l), without additional assumptions, fails to explain the packaging of cos duplication chromosomes.Therefore we favor model (2). We assumethat the DNA of deletion mutants doesnot destabilize the head in the absenceof pD hecauseDNA entrance pausesat cos. Pausing may be a consequence of cos--PA-head binding (Hohn, 1975). We note that DNA entrance can resume after pausing provided that coscutting hasnot occurred. This is implied by the observation, cited above, that uncut copiesof ws can also be packaged. In order to explain headful

768

N. STERNBERG

AND

R. WEISBERG

packaging of prophage DNA, which occurs only in the presenceof pD, we assumethat DNA entrance stops when the head is filled to capacity. The D-independence of phage particles with short chromosomesgives us a powerful method for selecting new deletion mutants : derivatives of Adam that can form plaques on a non-permissive host have usually lost a segment of DNA equal to at least 18% of the wild-type chromosome. If we start with a Adam stock that carries a deletion of less than lSo/o, the minimum DNA loss needed for D-independence is reduced accordingly. In this way we have isolated deletion mutants in which less than 4% of the X chromosome has been deleted (Sternberg, Enquist & Weisberg, manuscript in preparation). We have also used D-independence to select and screen addition mutants that were constructed by in vitro recombination (Sternberg et al., 1977). We note here a remarkable similarity between the D-independence of h deletion mutants and the ability of phage T4 to produce a normal yield of plaque-forming phage in the absenceof two proteins (hoc and sot) that are normally components of the viral capsid (Ishii & Yanagida, 1975). pD resemblesthe T4 sot protein in three ways. First, the molecular weight of both proteins is about 11,000. Second, just as pD will add in vitro to D - phage, so the sot protein will add in vitro to T4 soc- phage. Finally, the number of moleculesof the dispensiblemajor capsid protein (either pD or sot), when present on the viral capsid, is equal to the number of molecules of the indispensible major capsid protein (either pE or p23*). Because of this similarity we asked if T4 sot protein would add to pD-deficient Xphage in vitro. The results of these experiments were negative. Our proposed role for pD is especially interesting in view of the relation between packaging efficiency and chromosomesize that was discovered by Feiss et al. (1977) for xD+. They found that packaging efficiency was maximal and independent of chromosomesize in the range 80 to 105% of wild type and suggestedthat this favored free genetic exchange between X and other lambdoid phages with different chromosome sizes. Clearly pD facilitates such genetic exchange by enlarging the range of DNA lengths that h can package, and this may be its function in nature. We would like to acknowledge the following people for their helpful suggestions and criticisms during the courseof this work: Lynn Enquist, Howard Nash, Michael Feissand Andrew Becker. We are also indebted to K. Kunkle for her careful preparation of this manuscript.

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