Role of the Host Cell in Bacteriophage T4 Development I. Characterization of Host Mutants That Block T4 Head Assembly

Transcription

1 JOURNAL OF VIROLOGY, Jn. 1980, p X/80/ /11$02.00/0 Vol. 33, No. 1 Role of the Host Cell in Bcteriophge T4 Development I. Chrcteriztion of Host Mutnts Tht Block T4 Hed Assembly HELEN R. REVEL,t BARBARA L. STITT4, ILGA LIELAUSIS, AND WILLIAM B. WOOD * Division of Biology, Cliforni Institute of Technology, Psden, Cliforni To study the role of the host cell in bcteriophge T4 infection, we selected more thn 600 mutnt host-defective bcteri tht dsorbed nd were killed by phge T4+ but were unble to support its growth. The mutnts were grouped into seven clsses by the growth ptterns of T4 phges crrying compensting muttions (go mutnts [,grows on]), selected on four prototype host-defective strins. Lysis nd DNA synthesis experiments indicted tht clsses A, AD, D, nd B (the mjority of the host-defective mutnts) block T4+ development t n ssembly step, clss C mutnts ffect n erly stge in phge development, nd clss F mutnts pper to ct t more thn one stge. Anlysis of T4+ infection in the ssembly-defective mutnts by in vitro complementtion, electron microscopy, nd sodium dodecyl sulfte-polycrylmide gel electrophoresis showed tht the host-defective muttions interfere with T4+ cpsid formtion t the level of phge gene 31 function, before ssembly of ny recognizble cpsid structure. The muttions mp ner pura, but t two or possibly three different sites. The go mutnt phges ble to overcome the host defect crry muttions in either gene 31, s found by others for similr defective hosts, or in the gene for the mjor cpsid protein (gene 23). The gene 23 go muttions do not bypss the requirement for gene 31 function. These results suggest tht t lest three components must interct to initite T4 hed ssembly: gp3l, gp23, nd one or more host fctors. The compenstory effects of muttionl ltertions in these components re highly llele specific, consistent with the view tht phge nd host components interct directly in protein complexes. The morphogenesis of bcteriophge T4 is known to proceed in stepwise fshion by the subssembly of heds, tils, nd til fibers, which re subsequently joined in sequence to form ctive phge prticles (40). Although most of the ssembly steps cn proceed in infected-cell extrcts (5), the erliest steps in these subssembly pthwys hve not yet been demonstrted in vitro (14, 22, 28). Consequently, we wondered whether the host cell might ply n obligtory role in inititing some ssembly processes. Viruses utilize much of the existing host cell synthetic mchinery, specificlly redirecting it to mke virus prticles. Perhps the host lso supplies scffolding, templtes, or other components essentil for inititing virl ssembly. Electron microscopic evidence suggests tht the first steps in the ssembly of T4 heds, nd perhps tht t Present ddress: Deprtment of Microbiology nd Moleculr Biology, Tufts Medicl School, Boston, MA t Present ddress: Public Helth Reserch Institute of the City of New York, Inc., New York, NY Present ddress: Deprtment of Moleculr, Cellulr nd Developmentl Biology, University of Colordo, Boulder, CO of bsepltes, proceed on the host membrne (29, 30). We sought to investigte these possibilities by the study of host mutnts tht specificlly block T4 ssembly. When this work ws initited, two host mutnts tht interfere with T4 hed ssembly hd been described. Georgopoulos et l. (9) hd reported tht groea44, K-12 mutnt selected for its inbility to support phge X growth nd found to block X hed ssembly, lso blocked T4 morphogenesis erly in cpsid formtion, t the step controlled by gene 31. A single mutnt derivtive of Escherichi coli B clled mop (37) gve similr phenotype fter T4 infection. Furthermore, Pulitzer nd Yngid (20) hd reported tht mutnt of W3350 ws deficient in the ssembly of functionl til fibers, nd we hd inferred from genetic experiments tht host involvement in T4 til fiber ssembly occurs t the level of the gene 57-controlled step in polymeriztion of the til fiber components (22). These results encourged us to serch for more ssembly-defective mutnt hosts. Accordingly, we selected host-defective (HD) bcteri unble to propgte T4 nd grouped 366

2 VOL. 33, 1980 them into clsses bsed on their bility to propgte mutnt T4 go (grows on) phges selected for growth on vrious HD strins. We report here genetic nd physiologicl studies on four clsses of HD mutnts tht block T4 ssembly nd on the T4 go phge mutnts tht overcome these blocks. All of these HD mutnt hosts pper to interfere with T4 cpsid ssembly t the step controlled by gene 31, in greement with results found for similr mutnts by other investigtors (3, 9, 36, 37). Genetic nlysis shows tht the HD muttions define t lest two nd perhps three host genes nd tht compensting phge go muttions cn occur in either of two phge genes. The ptterns of compenstion mong these mutnts re discussed with reference to the detiled model of Tkhshi et l. (36) nd the possible nture of phge-host interctions in the initition of T4 cpsid ssembly. HD mutnts of fifth clss ffect T4 ssembly but lso exhibit dditionl pleiotropic effects on T4 development. We shll describe the chrcteriztion of these mutnts in subsequent pper (B. L. Stitt, H. R. Revel, I. Lielusis, nd W. B. Wood, J. Virol., submitted for publiction). We lso found mutnts of sixth clss, which ffect n erly step in T4 growth; we hve not further investigted these mutnts. (A preliminry report of some of the work described here hs been published [39]. These studies re included in the doctorl disserttion of B.L.S., submitted in prtil fulfillment of the requirements for the Ph.D. degree, Cliforni Institute of Technology, Psden, 1978.) MATERIALS AND METHODS Medi. H broth for phge nd bcteril growth nd EHA top nd bottom grs for plting were prepred s described previously (33). LB broth nd top nd bottom grs (21) were supplemented with 2.5 x 10-3 M CCl2 for growth of P1 or 0.3% mltose for growth of phge A. M9, synthetic, phosphte-buffered medium (13), ws used when phge-infected cells were lbeled with '4C-mino cids. M9 top nd bottom grs contined 6.5 nd 10.0 g of gr (Difco Lbortories) per liter, respectively. M9 ws supplemented s required with mino cids, purines, or pyrimidines t 20 to 50,ug/ml nd vitmins t 0.1,ug/ml for the growth of uxotrophic bcteril strins in P1 trnsduction nd F' trnsfer experiments. Buffers used were the dilution medium (DM) described by King (15); Tris-mlete (TM) buffer, ph 6.0 (1); nd Tris-mgnesium buffer (TMg), ph 6.8 or ph 7.4, which ws 0.05 M Trishydrochloride-0.02 M MgSO4. Chemicls nd enzymes. Crystlline DNse I nd RNse A nd 2'-deoxydenosine were obtined from Sigm Chemicl Co. Acrylmide nd N,N'-methylenebiscrylmide were from Estmn Orgnic Chemicls. TEMED (N,N,N',N'-tetrmethylethylenedimine) ws from Mtheson, Colemn & Bell. Nitrosogunidine (N-methyl-N'-nitro-N-nitrosogunidine) ws HOST MUTANTS THAT BLOCK T4 HEAD ASSEMBLY 367 from Aldrich Chemicl Co. Liquifluor ws from New Englnd Nucler Corp. Csmino Acids were from Difco Lbortories. All other chemicls were regent grde. Rdioctive compounds. '4C-mino cids were reconstituted protein hydrolyste no from Schwrz/Mnn; [2-'4C]thymidine (61 mci/mmol) ws from Amershm Corp. Bcteri nd bcteriophges. E. coli K-12 CR63, permissive for mber (m) mutnts, ws the host for growth of phge stocks. E. coli B S/6/5, nonpermissive for m mutnts, ws used for selective plting of m + phge. SKB178, n E. coli K-12 strin tht is F- gle nd nonpermissive for m mutnts, is the prent of the HD strins to be described (9). WH-1 (F' [F196 supd32] his-45 trp-37phoa4 recal stra144 "vr-116" [12; CGSC 4612]) ws used to mke HD strins supd. C600 (thr-1 leu-6 thi-i supe44 lcy) tona2 A-) ws the donor in P1 trnsductions of supe. F' strins KLF17/KL132 (F' [F117] pyrb31 thi-1 thya25 his-i pro-27 leu-6 thr-1 recal xyl-7 mlau r-13 gl-6 lcyl tona2 str-9 rel-i Ar A- [CGSC 4255]) nd KLF18/KL132 (F' [F118] pyrb31 thi-1 thya25 his-i pro-27 leu-6 thr-1 recal xyl-7 mial r-13 gl-6 lcyl tona2 str-9 rel-1 Ar A- [CGSC 4259]) (18) were used for mpping muttions in HD strins. K-12 strin T832 (rgh his pro thi-1 pura mpa ml tsx-9 Strr A' A-) ws obtined from T. Tkno (37) nd used s recipient in trnsduction experiments. Our isolte of this strin crries n unidentified m suppressor. groea44 nd groeb515 were obtined from C. Georgopoulos (9). T4D+ nd T4 (m) nd temperture-sensitive (ts) mutnts were from the Cliforni Institute of Technology (Cltech) collection (now mintined t the University of Colordo, Boulder). m mutnts used were mb8 (gene 20), me209 (gene 22), mb270 (gene 22), mhll (gene 23), mb17 (gene 23), mb272 (gene 23), me506 (gene 23), mn67 (gene 25), mn120 (gene 27), mn54 (gene 31), mng71 (gene 31), mn52 (gene 37), mn122 (gene 42), nd X4E [mb25 (gene 34):mA455 (gene 34):mN52 (gene 37):mB262 (gene 38):mB252 (gene 35)]. ts mutnts used were tsa70 (gene 31) nd tsa56 (gene 31). r67 in gene riii ws included s n outside mrker in crosses between gene 31 mutnts. T2L, T3, T5, T6, nd T7 were from the Cltech collection. T4e ( T4 mutnt tht grows on groea44) AcIb2, nd AEA30 were received from C. Georgopoulos (9). P1 (Plkc) ws used for trnsduction experiments; fl phge is mle specific nd ws used to identify F'-crrying strins. HD mutnt bcteri. SKB178 cells grown in H broth, wshed three times, nd suspended t 4 x 108/ ml in TM buffer were treted with nitrosogunidine (100 Ag/mi) for 15 min t 37 C by the procedure of Adelberg et l. (1). The survivl rte ws 40%. Nitrosogunidine ws removed by wshing three times with cold TM buffer, nd 26 subcultures (numbered 0 through 25) were mde by inoculting 5 ml of H broth with 0.1 ml of mutgenized cells nd growing overnight t 370C. To select HD mutnts, subcultures were diluted 1: 25, grown to log phse t 37 C, nd 0.05-ml smples contining bout 106 cells were spred on reltively

3 368 REVEL ET AL. dry EHA pltes. The pltes were incubted for 60 to 90 min t the desired selection temperture nd then spryed with 108 phge in 1 /A, using n erosol spryer nd rotting the plte to ensure uniform coverge. A mixture of T4 nd T6, relted phges with different host rnges, ws used to reduce the recovery of phgeresistnt cells. In SKB178, which is r6- (permissive for T6 with nonglucosylted DNA [21,24]), this procedure lso prevented the selection of glu-defective host mutnts which produce nonglucosylted T4 nd T6 progeny. Such mutnts comprised bout 25% of the recovered colonies when selection ws with T4 lone. After overnight incubtion, there were 50 to 100 surviving colonies per plte. Cells from these colonies were purified by streking on EHA pltes. No mutnts were found when the selection procedure ws pplied to nonmutgenized cells. Three seprte selections were crried out (see below). Mutnts from the three selections were clled HDXO.1-HDXO.29 nd HDX1.0-HDX1.9 through HDX25.0-HDX25.9 (selection I), HDX3.01-HDX3.26 (selection II), nd HDX26.01-HDX26.30, HDX HDX27.30, nd so on through HDX35.01-HDX35.30 (selection III). HD is phenotype designtion; "X" is clssifiction letter (see below). The digits preceding the deciml point indicte the subculture of origin. In the genetic nlysis of the HD strins nd in the Discussion, we use the genotypic designtion hdh for the HD strins blocked in T4 hed ssembly. A prticulr muttion is designted by the number of the HD strin; e.g., HDA17.5 crries the muttion hdh- 17.5, the hyphen to be replced by letter when the identity of the host genes is better defined. go mutnt phge. The go phges were selected by plting 107 to 108 T4+ prticles per plte on ech of the first 29 HD strins isolted in selection I (HDX0.1- HDXO.29). Mutnt phge were purified by stbbing nd replting from lrge plques tht ppered with frequencies of 106 to An nlysis of plting ptterns by spot testing with purified phge on the 29 HD host strins resulted in the identifiction of four different clsses of go mutnt phge, designted goa, goc, god, nd gof (see below). The following representtives of ech of the four clsses were used s the stndrd go phge for typing of HD strins: goa 1 (selected on HDADO.1), gocl (selected on HDCO.13), godl (selected on HDDO.18), nd gofl (selected on HDF0.26). Additionl go mutnts were purified similrly from the smller plques tht ppered t frequencies of 10' to 105 when T4+ ws plted on some HD hosts (see below). m:go double mutnts were constructed by pproprite crosses nd screened for their bility to grow on HD hosts tht crried supd or supe nd for their inbility to grow on the corresponding nonsuppressing HD hosts. Strek tests. About 10 IlI of two T4 phge stocks contining 108 nd 10" prticles per ml, respectively, ws streked cross EHA pltes nd llowed to dry. Bcteril cultures (-4 x 10' cells per ml) were crossstreked seprtely over ech phge strek, nd the pltes were incubted overnight. Cell lysis or its bsence in the strek overlp res differentites sensitive, resistnt, nd HD cells: sensitive cells re lysed t both phge concentrtions, resistnt cells re not lysed t either concentrtion, nd HD mutnts re J. VIROL. lysed t the high phge concentrtion but not t the low concentrtion. Spot tests. Smll drops (-5 1l) of phge solutions t concentrtions of 108, 106, nd 104 prticles per ml were spotted on gr pltes seeded with 108 cells of the bcteril strin to be tested, llowed to dry, nd incubted overnight t the pproprite temperture. Mpping of phge muttions. Phge crosses were performed s described by Wilson nd Kells (38). Specil conditions used to distinguish recombinnt progeny re described in the pproprite figure legends. Burst size mesurements. Burst sizes were determined s described previously (22). Mesurement of infected-cell lysis. Bcteril cells were grown in 20 ml of H broth to 2 x 108 to 3 x 108/ml (opticl density t 660 nm = 0.50 to 0.60) nd infected with phge t multiplicity of infection of 6 to 7. Smples (1 ml ech) were withdrwn t intervls, nd the opticl density ws mesured t 660 nm. Surviving bcteri mesured t 7 min were less thn 1%. A few drops of CHCl3 were dded to the HDC nd HDF cultures t 80 min fter infection, nd the opticl density ws mesured once more t 90 min. The time of lysis ws lso determined visully for mny phge-infected HD strins. Lbeling phge proteins with "C-mino cids. Cells were grown in M9 t 370C to 5 x 107/ml nd concentrted to 4 x 108/ml; 2.0-ml cultures were infected with phge t multiplicity of infection of 5.0 t t = 0 nd were superinfected with the sme mount of phge t t = 7 min. '4C-mino cids were dded to finl concentrtion of 1 to 2 jlci/ml to infected cultures t the desired time. After the lbeling period, cultures were usully chsed for t lest 1 min by the ddition of 0.5 ml of 10% Csmino Acids. Chilled cells were pelleted, suspended in 0.1 ml of TMg, ph 6.8, contining 1,ig of DNse, nd frozen in solid CO2- ethnol bth. Upon thwing, the preprtion ws mde 2% in sodium dodecyl sulfte nd /3-mercptoethnol nd ws boiled for 1 to 3 min. Incorported rdioctivity ws determined by trichlorocetic cid precipittion of 2-/A portion, nd smples contining equl numbers of counts (nd equl volumes when identicl lbeling conditions were used) were loded onto gels. Sodium dodecyl sulfte-polycrylmide gel electrophoresis. Smples prepred s described bove were electrophoresed in the discontinuous sodium dodecyl sulfte buffer system described by Lemmli (16), s modified by Dickson (4), nd dpted for use with slb gels by the method of Studier (35). Gels were run t constnt current of 10 ma until the bromophenol blue mrker dye entered the seprting gel nd then t 20 ma until the mrker dye reched the bottom of the gel. Gels were fixed nd stined for 1 to 2 h in n queous solution of 50% (wt/vol) trichlorocetic cid nd 0.2% Coomssie brillint blue (Schwrz/Mnn). Destining ws for 10 to 20 h in 10% methnol-10% cetic cid. The gels were dried onto Whtmn 3MM chromtogrphy pper under vcuum nd exposed to Kodk No-Screen X-ry film. In vitro complementtion tests. In vitro complementtion ssys were performed by the procedure of Edgr nd Wood (5). For the preprtion of defective

4 VOL. 33, 1980 extrcts, nonpermissive cells were grown to 4 x 108/ ml in H broth with vigorous ertion, infected t t = 0 with n pproprite m mutnt t multiplicity of infection of 7, superinfected t 7 min t the sme multiplicity of infection, chilled t t = 35 min, nd pelleted t 45 min. The pellets from 250-ml culture were suspended in 0.5 to 1.0 ml of TMg, ph 7.4, contining 10,tg of DNse, frozen t -70 C, nd thwed once. Smples (20 p1 ech) were mixed t 4 C with either 20 p1 of TMg or 20 pl of different extrct, incubted t 30 C for minimum of 2 h, nd ssyed for phge. Electron microscopy. Smples of infected-cell extrcts prepred s described bove were plced on crbon-coted grids nd negtively stined with 1% urnyl cette. Grids were exmined in Philips 201 electron microscope t 60 kv. P1 trnsductions. Trnsductions using phge Plkc were ccording to Miller (19) or Rothmn (25). F' trnsfer experiments. Cells grown in H broth were mted t rtio of 10 donor cells to 1 recipient cell for 60 min t 37 C without ertion. The mixture ws diluted nd plted on M9 gr pltes to select ginst the multiply uxotrophic donors. F-ductnts were purified by streking on M9 pltes nd tested by the strek test for their bility to support fl nd T4 growth. RESULTS Selection nd clssifiction of HD bcteril mutnts. HD mutnts, isolted s colonies unble to propgte T4 or T6, were grouped into t lest six clsses ccording to the plting properties of four stndrd go phge mutnts tht were selected for growth on four prototype HD mutnt hosts (Tble 1). Lysis mesurements, shown by Epstein et l. (6) to differentite between T4 erly nd lte nonsense mutnts, were used to distinguish those bcteri tht cused n erly rrest of T4 development from those tht blocked morphogenesis. Mutnts of clsses A, AD, D, nd B showed norml lysis, suggesting n ssembly defect. Lysis did not occur in T4- infected clss C mutnts nd ws delyed in HOST MUTANTS THAT BLOCK T4 HEAD ASSEMBLY 369 TABLE 1. clss F mutnts. T4 DNA synthesis mesurements supported these tenttive ssignments of phenotype: DNA synthesis ws norml in the puttive ssembly mutnts of clsses A, AD, D, nd B but delyed nd depressed in mutnts of clsses C nd F, which exhibited defective lysis (dt not shown). When physiologicl nd genetic studies reveled tht mutnts from ll four ssembly-defective clsses blocked T4 development t the sme stge, two further mutnt isoltions were done, using either goa 1 (selection II) or godl: gofl (selection III) in combintion with T6 s selecting gents to fvor the detection of rrer clsses of ltered bcteri tht might ffect other morphogenetic pthwys. After these selections, the mjority AD clss ws eliminted; other defined clsses were enriched, nd mny mutnts (17 to 64%) ppered in the "other" ctegory (dt not shown). These mutnts, however, did not represent blocks in different ssembly pthwys. Mesurements of lysis, in vitro complementtion nlysis, nd genetic studies reveled the new muttions to be vritions of the sme defect (see HD3.10, representtive of the "other" clss, below). Growth nd plting properties of ssembly-defective HD mutnts. The bility to propgte T4 phges nd the growth properties of the ssembly-defective mutnts described in this pper re shown in Tble 2. Although most HD strins grew normlly, some clss D nd ll clss B mutnts showed temperture-dependent growth ptterns. Phge propgtion in these two clsses lso ppered to be influenced by temperture: some strins were defective t high tempertures but supported phge growth t 300C (HDDO.18, HDD25.9, nd HDD4.3), wheres others showed the converse behvior (HDD3.6, HDD7.1, nd HDB4.5). Severl observtions indicted tht the four Clssifiction of HD mutnts Growth of phge' HD clss Lysisb Frequency (%)c T4+ goal godi gocl gofl HD A D AD B 0 (+) (+) C F D 1 Other or D 5 The four stndrd go phge mutnts were isolted s lrge-plque formers on the pproprite HD host strins s described in the text. Phge growth ws determined by spot tests. Symbols: 0, no phge growth; +, phge growth; (+), phge growth is better thn T4+ by fctor of bout 1,000. b Lysis mesurements re described in the text. Symbols: +, lysis; 0, no lysis; D, delyed lysis. "A totl of 279 mutnts derived from 26 subcultures were clssified to yield 75 independent HD strins.

5 370 REVEL ET AL. J. VIROL. Strin' Bcteri Growth TABLE 2. Properties of HD strins tht block T4 ssembly Growth of phge EOPb Burst sizec T4+ goal godl T4+ goal godl HD HDA17.5 < < HDADiJ < HDD3.6 lo-4-lo-6 < HDDUYI ts 10-4_10-6 < HDD7.1 cs 10-4_10-6 < HDDO.18d 10-4_10-6 < < HDD25.9d 10-4_10-6 < HDB4.5 cs 10-4 < <0.1 NTe NT HDB8.4 cs+ 1.0 < HDB17.3 ts < < NT NT HD3.10 Cs 10-4 < NT NT groea44 ts <10-6 < NT 251 groeb NT 0.3 The course of T4 infection ws chrcterized in detil in the underlined strins. b EOP, Efficiency of plting; mesurements were t 37 C unless otherwise specified. ' Phge dsorption nd killing were norml. d Efficiency of plting nd burst size mesurements t 420C. e NT, Not tested. ssembly-defective clsses of HD mutnts re relted. (i) Both goa nd god phges grew on HDAD hosts. However, goa phges showed lower efficiency of plting thn did T4+ on HDD hosts. This property ws most obvious t permissive tempertures in host strins with temperture-dependent defectiveness; for exmple, t 370C on HDDO.18 nd HDD25.9, T4+ grew well, but goal showed n efficiency of plting of i0-' (dt not shown). (ii) Both goal nd godl showed efficiencies of plting bout 103 times higher thn tht of T4+ on HDB hosts. However, cold-sensitive (cs+) derivtive of HDB8.4 tht hd regined the bility to propgte T4+ still inhibited the growth of both go mutnts. (iii) After selection with godl:gofl in combintion with T6 t 300C, mny of the survivors tht were HD for both T4 nd T6 under the selection conditions propgted T4+ t 370C but were specificlly defective for goal, godl, nd god3. These observtions suggest tht the four HD mutnt clsses impose common block to T4 development. Additionl evidence for this suggestion is presented below. The bility of other phges to grow on the ssembly-defective HD mutnts ws investigted. T2 nd T6 behved like T4. T3, T5, T7, nd P1 grew on ll strins with one exception: T5 filed to grow on HDB17.3. HD strins HDB4.5, HDB8.4cs+, HDB17.3, nd HDD4.3 inhibited the growth of XcIb2, but llowed the growth of its derivtive XeA30, phge tht compenstes for mny groe host muttions (8). Course of T4 infection in ssembly-defective HD mutnts. The course of T4 infection ws studied in mutnt host of ech clss. First, in vitro complementtion tests were crried out to determine whether ctive structurl intermedites in ssembly ccumulte in T4+-infected HD strins. Extrcts of T4+-infected HD cells were mixed with either prticles lcking til fibers or with n extrct (prepred by using the pproprite mutnt phge [see bove]) tht provided heds nd til fibers (til defective) or tils nd til fibers (hed defective). The production of infectious phge prticles in these mixtures ws ssyed. As shown in Tble 3, T4+ infection of representtives of the four clsses of ssembly-defective HD mutnts produced ctive til fibers nd tils comprble in quntity to those in the hed-defective control, but yielded no ctive heds. These results show tht in the HD hosts, ssembly of til bsepltes nd fibers is norml, wheres ssembly of heds is defective. The extrcts of T4+-infected HD cells used for in vitro complementtion lso were exmined by electron microscopy using negtive-stining techniques. In greement with the in vitro complementtion dt, the HD extrcts contined norml numbers of tils but no heds or hedrelted structures. As controls, the hed-defective nd til-defective extrcts used in the complementtion tests showed norml numbers of tils nd heds, respectively (dt not shown). The phge protein gene products (gp) lbeled during the ltter hlf of the ltent period fter T4+ infection of HD hosts were nlyzed by sodium dodecyl sulfte-polycrylmide gel elec-

6 VOL. 33, 1980 TABLE 3. In vitro complementtion with extrcts of T4+-infected HD strins Complementing N Til fiber Til de- Hed deprepn one defective fective fective T4+ + HDA T4+ + HDAD T4+ + HDB T4+ + HDD T4+ + HD b Hed defective Til defective Til fiber 0.3 defective Two extrcts (50 ttl of ech) were mixed, incubted for 300 min t 30 C nd then ssyed for plque-forming phge. Results re expressed s the titer (ctive phge per milliliter) x 109 in the rection mixture. Extrcts of phge-infected cells were prepred s described in the text. The til fiber-defective preprtion (hed nd til donor) ws prticles lcking til fibers purified from X4E-infected cells; 5 x 10" prticles per ml were used in the complementtion rections. The tildefective preprtion (hed nd til fiber donor) ws n extrct of SKB178 infected with mn120 (gene 27). The heddefective preprtion (til nd til fiber donor) ws n extrct of SKB178 infected with mb17 (gene 23). b The mximum possible vlue in this experiment ws 50 becuse only 5 x 1010 prticles were dded to the rection mixture. trophoresis (Fig. 1). In ll infections, the mjor cpsid protein (gp23 plus its clevge product gp23*) ws produced t pproximtely the sme level. In the permissive infections (T4+ infection of SKB178 nd HDB8.4cs+ nd goal infection of HDA17.5), gp23 ws cleved to gp23*. In the nonpermissive infections [mn54 (gene 31) infection of SKB178, goal infection of HDB8.4cs+, nd T4+ infection of HDA17.5 nd HD3.10], ll of the gp23 remined uncleved. Similr results lso were found in T4+-infected HDD3.6 nd HDAD1.1. These findings support the conclusion tht ll of the HD strins presumed to ffect phge morphogenesis block T4+ development t n erly stge in hed ssembly t the level of gene 31 function (17). The dt lso suggest tht the muttion in HDB8.4cs+ which prevents the growth of goal probbly cts t the sme level. Genetic nlysis of go mutnts. The goal nd godl muttions mp in gene 31 t n internl site ner tsa70 nd give wild-type recombinnts in crosses of goal by godl (Fig. 2). Complementtion tests with gene 31 m mutnts confirned the ssignment to gene 31 (dt not shown). Further crosses showed tht godl nd number of spontneous, independently isolted go phge mutnts with identicl plting ptterns, selected on clss A, AD, or B mutnt hosts, filed to recombine with T4e. This go mutnt, selected previously ongroea44 (9), hs godl growth chrcteristics t 37 C but differs by its inbility to grow t 42 C. Similrly, HOST MUTANTS THAT BLOCK T4 HEAD ASSEMBLY 371 number of independently isolted mutnts with goal plting properties showed the sme frequency of recombintion with tsa70 s did the prototype phge goal. Thus, goal-like nd godl-like muttions pper to recur t sites identicl or closely linked to those of the originl muttions. However, not ll go mutnts selected on HDA strins were similr to goa1. Four such mutnts, isolted in selection III, filed to plte on HDADO.1 or HDA17.5. One of these novel muttions mpped in gene 31 but gve wild-type recombinnts with both goal nd godl (percent recombintion = 0.16 nd 0.17, respectively). These results re consistent with the view tht the different plting chrcteristics of go mu- _ - cc cc < < co <0: C = I I I + cc c * < + < A z m B2 _11P1 -,0 v,i - cc Co s3ii _2- - _. 4.sw-. * _. _ 4d 2 3 5% FIG. 1. Sodium dodecyl sulfte-polycrylmidegel electrophoresis of extrcts of phge-infected cells. Cells were infected with phge, lbeled with 14Cmino cids from 13 to 24 min fter infection t 37 C, nd prepred for sodium dodecyl sulfte-polycrylmide gel electrophoresis s described in the text. Smples contining bout 40,000 cpm were electrophoresed on 10% polycrylmide slb gels nd processed s described in the text. Phge nd bcteri used in the preprtion of extrcts re listed bove ech trck: "+" mens T4+; "31" refers to phge crrying mn54 (gene 31), nd "23:63:rII" refers to phge crrying mb17, mm69, nd reddf4l; "B178" refers to SKB nd 23 * designte, respectively, the product of gene 23 nd the cleved product of gene 23. (A) nd (B) re gels from two different experiments. 3 _

7 372 REVEL ET AL. J. VIROL. gene 31 pset rm 30 4) 4- I _ FIG. 2. Genetic loction of goal nd godi. The mp of the gene 31 region of the T4 genome is from the dt of Revel nd Lielusis (23) nd shows the positions of mng71 nd mn54 in gene 31 reltive to outside mrkers pset (to the left) nd riii nd gene 30 (to the right), s well s recombintion percentges between mng71 nd mn54, mn54 nd r67, nd mng71 nd r67. The top line indictes the reltive order of the genes. The second line is the genetic mp showing the positions of phge muttions. Ech number in the figure is the observed percent recombintion in cross between mutnts t the ends of the corresponding rrows. Two sets of three-fctor crosses [(i) r67(riii):mn54 with T4e, godl, mng71, nd tsa70; nd (ii) r67(rhii):tsa70 (gene 31) with T4e, god1, mn54 nd mng71] nd two-fctor cross between T4e nd god) were nlyzed by plting totlprogeny on CR63 nd wild-type recombinnts on groeb515 t 42 C, conditions under which godl, T4e, tsa70 nd m mutnts cnnot grow. In three-fctor cross between r67(riii):tsa70 nd goal, wild-type recombinnts were ssyed on HDD4.3 t 30 C, where goal nd tsa70 fil to plte. In cross between goal nd godl, wild-type recombinnts were ssyed on HDB8.4cs+ t 37 C. The rtio of wild-type progeny with the r phenotype to totl wild-type progeny permitted ordering of the mutnt lleles with respect to the outside mrker r67 in gene riii. Recombintion percentges re clculted s follows: (wild-type recombinnts/totlprogeny) x 200. No wild-type recombinnts were found in the cross between T4E nd godl. tnts reflect different muttionl sites. When T4+ ws plted on HDD host strins, two distinct go mutnt plque types were observed. Lrge plques similr to those of godl ppered t frequency of -10-6, nd smll plques ppered t frequency of _10-4 (Tble 2). Plting ptterns of three such smll-plque isoltes, god2, god3, nd god4, re shown in Tble 4. Two-fctor crosses showed tht these new go muttions were not in gene 31 but were closely linked to mb17 in gene 23. More definitive mpping of one of these mutnts, god3 (selected on HDD3.6), plced this muttion within gene 23 ner mb272 (Fig. 3). Thus, go phge mutnts with ltertions ffecting the gene for the mjor cpsid protein lso cn overcome the ssembly block in some HDD host strins. Two experiments showed tht god3 does not simply bypss the requirement for gp3l function: (i) god3:mn54 (gene 31) double mutnts grew on HDD3.6 only when gp3l ws supplied by complementing phge, nd (ii) in mixed infection of HDD3.6 with god3:mn54 nd god3 in vrying rtios, the phge yield ws function of the level of wild-type gp3l present (dt not shown). Inbility of T4 go mutnts to grow on some HD strins. As noted in Tble 2, some go phge mutnts selected on specific HD strins fil to grow on certin other temperturedependent HD strins t low tempertures t TABLE 4. Plting properties of vrious god phge mutnts Bcteril Growth of phge strin T4+ godl god2 god3b god4 HD groea HDDO.18c HDD HDD HDB Phge growth ws determined by spot test t 37 C s described in the text. Symbols: +, phge growth; 0, no phge growth. b The burst size of god3 on SKB178 (HD+) ws the sme s tht of T4+ (-150). On HDD3.6, the burst size of god3 ws reduced to -30. c Spot test t 42 C. which wild-type T4 cn grow. Similrly, we found tht tsa70 (gene 31) did not plte on HDDO.18, HDD25.9, nd HDD4.3 t 30 C. The growth of tsa56 (gene 31) ws inhibited only on the lst host. Genetic nlysis of hdh strins. P1 trnsduction studies showed tht most of the host muttions cotrnsduced with pura with frequencies rnging from 8.5 to 17.3% for different muttions (Tble 5 nd Fig. 4). Two strins, HDB4.5 nd HDD7.1, were clerly different from the rest: there ws less thn 1% cotrnsfer of the HD muttion with the pura+ mrker.

8 VOL. 33, HOST MUTANTS THAT BLOCK T4 HEAD ASSEMBLY Gene I C 1., I I I I I I t g0od-3 I -- Mutnt I_ 4.55 _ 2.95 _ _ _ 3.31 _ 1.28 _ ~ ~ ~ L b _ I ( N67(25) FIG. 3. Genetic mp of the gene 23 region of the T4 genome. The top line gives the reltive order nd sizes of genes 21 to 23 (41). The second line is the genetic mp showing the position of m muttions. () Recombintion percentges between muttions in genes 20, 22, 23, nd 25. Genetic crosses were done in CR63 under stndrd conditions described in the text. Totl progeny ws determined by plting on CR63. m' recombinnts were ssyed on S/6/5. Recombintion percentges re clculted s follows: (m+ recombinnts/totl progeny) x 200 nd re verges obtined in two or more crosses. (b) Recombintion percentges from the crosses between god3:mx double mutnts with wild-type phge. god3:mx double mutnts were constructed s described in the text. The god3:mx double mutnts were bckcrossed to wild-type T4+ in CR63 under stndrd conditions. Totlprogeny were determined byplting on CR63. god3:m+ recombinnts were ssyed on HDD3.6 t 37 C, where wild-type nd m phge cnnot grow. Recombintion percentges re clculted s follows: (god3:m+ recombinnts/totl progeny) x 200. Trnsfer of either of the F' fctors, F'117 or F'118, into these strins or into HDD3.6 rendered the hosts ble to support T4+ growth. This result indictes tht the wild-type lleles re probbly dominnt nd tht the HD muttions in ll three of these strins must lie between the ends of the F'117 fctor. P1 trnsduction experiments with mela nd pyrb (Fig. 4) mrkers to locte these muttions more precisely hve not been done. DISCUSSION Bcteril hdh muttions block n erly step in hed ssembly. Our combined physiologicl nd genetic dt show tht the HD muttions described here block T4 morphogenesis t the level of gene 31 ction. After infection of these strins by T4+, neither heds nor hedrelted structures re mde, nd hed proteins re not cleved s in norml infection. This phenotype is distinct from tht of n ssembly core defect (27), but is the sme s observed fter infection of wild-type (nonsuppressing) hosts by gene 31 m mutnts (17). It is lso the sme s the phenotype reported for T4+ infection of the previously described mutnt host strins mop (37), groea44 (9), tbb (3, 36), nd hdb3 (31). As ws reported for these strins, we find tht muttions in T4 gene 31 cn compenste for the host defect, nd tht certin gene 31 ts mutnts (k mutnts [3, 36]) interct negtively with certin temperture-dependent HD mutnt hosts, in tht the k mutnt phge fil to propgte under conditions where T4+ phge cn do so. Therefore, the HD mutnts, like those described previously, pper to interfere with the norml function of T4 gp3l in hed ssembly. However, we hve shown, in ddition, tht muttion in gene 23, god3, lso cn compenste for the host defect in certin HD strins such s HDD3.6. Gene 23 codes for the mjor T4 cpsid protein, which presumbly must interct with gp3l in order to ssemble correctly (17). In possibly nlogous mutnts of phge X, muttions

9 374 REVEL ET AL. in gene E (mjor cpsid protein) restore phge growth on certin HD groe bcteril mutnts pprently by lowering the mount of gpe produced, thereby restoring required blnce between the levels of this protein nd the host groe function (8, 34). However, it ppers unlikely tht the god3 muttion in T4 cts by reducing the mount of functionl gp23 produced. The god3 muttion lies fr from the gene 23 promoter (Fig. 3), nd god3 phge give norml burst size on wild-type host strins (Tble 4). Furthermore, severl gene 23 m mutnts were found not to grow in HDD3.6 supe host (dt not shown), in which gp23 should be un- TABLE 5. Frequency of Pl cotrnsduction of the HD phenotype of HD strins with pura+ HD strin HD trnsductnts/ Cotrnsduction (donor) PurA + trnsductnts tested (% HDD3.24b 16/ HDA / HDD4.3 8/ HDAD1.1 22/ HDB / HDB8.4cs+c 29/ HDDO.18d 51/ HDD3.6 52/ HDD7.1 0/90 <1.0 HDB4.5 0/199 <0.5 groea44 22/ Plkc ws grown on the HD strins nd used to trnsduce T832 pura s described in the text. pura trnsductnts were selected on supplemented miniml medium in the bsence of purines, purified, nd tested by strek test t 370C for the bility to grow T4+ (except HDB8.4cs+ [see footnote c]). Frequencies of pura+ trnsductnts rnged from 5 x 10-7 to 100 x 10-7 in different b experiments. The test for host defectiveness ws t 300C. CHDB8.4cs+ is sensitive to T4+ but defective for goa 1; therefore, goa 1 ws used to screen the pura + trnsductnts for host defectiveness. d The test for host defectiveness ws t 420C. J. VIROL. derproduced becuse of the low efficiency of the supe suppressor. We hve lso presented evidence tht the god3 muttion does not simply bypss the norml requirement for gp3l function. Therefore, the most likely interprettion is tht the HDD3.6 host defect cn be overcome by specific chnge in either gp23 or gp3l of T4, implying interction between these proteins nd one or more host functions in the first step of T4 hed ssembly. This view is supported by the findings tht k muttions, s well s compensting muttions, cn occur in gene 23 nd tht the corresponding k mutnts cn be used to select specificlly for tbb-defective host mutnts (36). hdh muttions define t lest two host functions. Most of the hdh muttions tested cn be cotrnsduced with the pura mrker, s hs been reported for mop (37), groe (7), nd tbb (36). In our experiments, the cotrnsduction frequencies of vrious hdh mutnts rnge from 8.5 to 17.3%, but show two pprent clusters (HDD3.24, HDA17.5, HDD4.3, HDAD1.1, HDB17.3, nd groea44 nd HDB8.4, HDDO.18, nd HDD3.6) with mens of bout 10 nd 16%, respectively. Dt in the literture for mop, groe, nd tbb mutnts show similr rnges nd tendencies to cluster round 10 nd 21%. The finding of two pprent frequencies could be due to n effect of some hdh muttions on the recovery ofpura+hdh trnsductnts to give lower pprent cotrnsduction frequency. Altemtively, the two frequencies could represent two different sites t which hdh muttions occur. From the difference in trnsduction frequency, the two sites would be bout 10i nucleotide pirs prt nd, therefore, probbly in different genes. This hypothesis could be tested by genetic complementtion nlysis. Of interest in this connection is recent report tht A trnsducing phge crrying 8,000 bse pirs of bcteril DNA, including mutnt groe gene cnnot grow on some groe hosts but cn grow on others,, I ( chromosome F118 F Iq R,,,,,,, E co/i 4 F117 FIG. 4. Loction ofhdh muttions on the E. coli chromosome. The upper line shows the min segment of the E. coli chromosome with relevnt mrkers (2). The hevier lines below the chromosome show the extents of F' fctors F'117 nd F'118 bsed on the dt of Low (18) nd Tkno nd Kkefud (37), s djusted to correspond to the most recent mp ofthe E. coli chromosome (2). The brcket bove the chromosome indictes the loction ofmost of the muttions in our HD strins s obtined by P1 cotrnsduction ofhost defectiveness with the pura + mrker (Tble 5). Cotrnsduction frequencies hve been converted to minutes on the E. coli chromosome by the eqution of Wu (42) s described in reference 2.

10 VOL. 33, 1980 suggesting tht two complementing groe genes my be present (10). In ddition to the pura-linked muttions, we hve found two hdh muttions tht re not cotrnsduced with pura, lthough F' trnsfer experiments show tht they re in the sme region of the bcteril chromosome. The defects in strins HDB4.5 nd HDD7.1 cn be overcome by the phge muttions god3 nd god1, respectively. Therefore, hdh muttions my occur in three or more host genes. To complicte the picture still further, two other HD bcteril mutnts tht block T4 hed ssembly nd cn be overcome by muttions in T4 gene 31 hve been reported. These mutnts differ from our chrcterized hdh mutnts nd from mop, groe, nd tbb mutnts in tht one of them mps ner pro on the E. coli chromosome (31) nd the other shows dissimilr T4- defective phenotype (32). We hve selected go mutnts on the ltter strin, hd590, kindly furnished to us by L. D. Simon. Most of the corresponding muttions do not mp ner gene 23 or gene 31. A few mp in gene 31, but these mutnts fil to grow on ny of our HDA, HDAD, HDB, or HDD strins (Stitt, unpublished observtions). A comprehensive model for the interction of gp3l with host functions must ccount for these HD mutnts s well s the other clsses described here. go nd hdh muttions probbly define specific phge-host protein interctions. Two kinds of models cn be proposed to explin the interctive systems of host nd T4 phge muttions described here nd by others (3, 9, 31, 36, 37). The interction could be indirect; for exmple, host muttions might lter membrne trnsport properties so s to cuse chnge in the intrcellulr ionic environment tht could prevent the gp3l-medited step in T4 hed ssembly. Alterntively, the interction could be direct; hdh muttions might block hed ssembly by ltering host protein so s to prevent required specific ssocition of gp3l nd gp23 with host protein complex. In either model, compensting muttions in phge gene 23 or 31 could overcome the block. From the evidence vilble so fr, we cnnot rule out either model. Indirect interction seems HOST MUTANTS THAT BLOCK T4 HEAD ASSEMBLY 375 to be supported by our findings tht: (i) single phge muttion in gene 31 cn overcome severl host defects tht my represent muttions in three different host genes, nd (ii) certin host defects cn be overcome by muttions in either of two phge genes. Either model would be consistent with the findings of both compenstory (go) nd killing (k) muttions in both genes 23 nd 31 of T4: in some cses, phge muttion cn compenste directly or indirectly for detrimentl host muttion, wheres in other cses, phge muttion nd host muttion, both innocuous lone, cn interct either directly or indirectly to give lethl phenotype. However, one spect of the dt supports the type of direct-interction model proposed by Tkhshi (36), involving specific ctive complex of phge nd host proteins required for phge hed ssembly. Host mutnts tht pper relted on the bsis of similr cotrnsduction frequencies with pura+ show different ptterns of compenstion by different clsses of go mutnt phge. Conversely, different go muttions or k muttions in prticulr gene show different plting ptterns on the HD strins. In other words, the properties of hdh, go, nd k muttions pper to be llele specific rther thn clss specific or gene specific. This feture would be less likely if the HD muttions cuse ltertions in physiologicl prmeter for which the go muttions cn compenste. Allele specificity would be more likely if these muttions re cusing compensting conformtionl chnges in n intercting complex of proteins. From the observtions reported here, the functionl complex might consist of t lest three host proteins nd the virl proteins gp3l nd gp23. The pprent bility of compensting muttions to occur in ny phge-host pir of these components would seem puzzling. However, possibly nlogous observtion hs been mde with the multimeric regultory enzyme sprtte trnscrbmylse: muttionl chnges in the regultory subunit ffect the specificity of the ctlytic subunit ctive site, solely by interction between the heterologous subunits (26). The recent identifiction of host protein defined by groe muttion (10, 11) should led to more direct tests for the existence of functionl phge-host protein complex in hed ssembly. ACKNOWLEDGMENTS These studies were supported by Public Helth Service grnt AI from the Ntionl Institutes of Helth nd by specil grnt no. 573 from the Americn Cncer Society, Cliforni Division, to W.B.W. B.L.S. ws supported by Public Helth Service trining grnt GM from the Ntionl Institute of Helth. The technicl ssistnce of Mrie Bell is grtefully cknowledged. LITERATURE CITED 1. Adelberg, E. A., M. Mndel, nd G. C. C. Chen Optiml conditions for mutgenesis by N-methyl-N'- nitro-n-nitrosogunidine in Escherichi coli K12. Biochem. Biophys. Res. 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