The product of the bacteriophage lambda W gene: purification and...

The product of the bacteriophage lambda W gene:purification and properties

Helios Murialdo, Xuekun Xing, Dimitra Tzamtzis, Abraham Haddad,and Marvin Gold

Abstract: Gene W is one of the 10 genes that control the morphogenesis of the bacteriophage λ head. The morpho-genesis of the phage λ head proceeds through the synthesis of an intermediate assembly called the prohead. This is anempty shell into which the bacteriophage DNA is introduced — packaged — by the phage enzyme DNA terminase.The product of W (gpW) acts after DNA packaging, but before the addition of another phage product, gene productFII, and before the addition of tails. The role of gpW is unknown. The structure of N- and C-tagged gpW has beenpreviously determined by nuclear magnetic resonance (NMR) spectroscopy. Here we report some of the properties ofthe native protein. The purification of gpW to homogeneity, overproduced by a plasmid derivative, is described. To ob-tain large amounts of the protein, the ribosome-binding site had to be modified, showing that inefficient translation ofthe message is the main mechanism limiting W gene expression. The molecular weight of the protein is in close agree-ment to the value predicted from the DNA sequence of the gene, which suggests that it is not post-transcriptionallymodified. It behaves as a monomer in solution. Radioactively labeled gpW is incorporated into phage particles inin vitro complementation, showing that gpW is a structural protein. The stage at which gpW functions and other cir-cumstantial evidence support the idea that six molecules of gpW polymerize on the connector before the incorporationof six molecules of gpFII and before the tail attaches.

Key words: bacteriophage morphogenesis, lambda gene W, viral structure.

Résumé : Le gène W est un des dix gènes qui règlent la morphogenèse de la tête du bactériophage λ . La synthèse etl’assemblage de la procapside est une étape intermédiaire de la morphogenèse de la tête du phage λ . La procapside estune enveloppe vide dans laquelle l’ADN du bactériophage est introduit et empaqueté par une enzyme phagique, l’ADNterminase. Le produit du gène W (gpW) intervient après l’empaquetage de l’ADN, mais avant l’ajout d’un autre produitphagique, le produit du gène FII (gpFII), et la fixation de la queue. Le rôle de gpW est inconnu. La structure de laprotéine gpW marquée aux extrémités C et N a été déterminée antérieurement par spectroscopie de RMN. Dans cetarticle, nous rapportons certaines propriétés de la protéine native. La purification jusqu’à homogénéité de la protéinegpW, surproduite à l’aide d’un plasmide, est décrite. Afin d’obtenir une grande quantité de cette protéine, le site deliaison au ribosome a dû être modifié, ce qui indique que la traduction inefficace de l’ARN messager est le principalmécanisme limitant l’expression du gène W. La masse moléculaire de la protéine est près de la valeur prédite à partirde la séquence d’ADN du gène, ce qui suggère que la protéine ne subit pas de modification post-traductionnelle. Ensolution, la protéine se comporte comme un monomère. La protéine gpW radioactive est incorporée dans des particulesvirales au cours d’expériences de complémentation in vitro, ce qui indique que gpW est une protéine structurale.L’étape à laquelle gpW intervient ainsi que d’autres preuves circonstancielles appuient l’idée que six molécules de gpWpolymérisent sur le connecteur, avant l’incorporation de six molécules de gpFII et la fixation de la queue.

Mots clés : bactériophage lambda, morphogenèse, gène W, structure virale.

[Traduit par la Rédaction] Murialdo et al. 315

Introduction

The morphogenesis of bacteriophage λ involves the finalproducts of three distinct pathways: the replication of viralDNA, the assembly of proheads, and the polymerization of

tails. After the DNA has been matured and translocated intothe prohead, the portal vertex is closed off and tails areadded to complete the particle. The portal vertex containsthe connector, a ring composed of 12 gpB molecules with anaxial orifice (Kochan et al. 1984) through which the DNA is

Biochem. Cell Biol. 81: 307–315 (2003) doi: 10.1139/O03-059 © 2003 NRC Canada

307

Received 22 April 2003. Revision received 4 July 2003. Accepted 10 July 2003. Published on the NRC Research Press Web site athttp://bcb.nrc.ca on 22 September 2003.

Abbreviations: SDS-PAGE, sodium dedecylsulphate polyacrylamide gel electrophoresis; PFU, plaque forming unit; gp, geneproduct (i.e., gpB, product of gene B).

H. Murialdo,1 X. Xing, D. Tzamtzis, and A. Haddad. Fundación Ciencia para la Vida and Millennium Institute for Fundamentaland Applied Biology, Avenida Marathon 1943, Santiago, Chile.M. Gold. Department of Medical Genetics and Microbiology, University of Toronto, Toronto, ON M5S 1A8, Canada.

1Corresponding author (e-mail: [email protected]).

I:\bcb\bcb8104\O03-059.vpSeptember 11, 2003 11:04:08 AM

Color profile: DisabledComposite Default screen

thought to translocate into the head during DNA packagingand to exit during infection. The product of gene W func-tions late in head assembly, after DNA replication and pack-aging, but before the addition of gpFII to the connector ofthe head, which is the last step in head morphogenesis, cre-ating the site to which the tail attaches (Casjens 1971;Casjens et al. 1972; Perucchetti et al. 1988). Despite theknowledge of the morphopoietic stage at which gpW is re-quired, the function of gene W remains obscure (Casjens andHendrix 1988; Murialdo 1991). The W complementationgroup was discovered by Parkinson (1968). Cells infectedwith W mutants produce DNA-full phage heads; however,the DNA in these heads is unstable and it is easily released(Kaiser et al. 1975). Katsura and Tsugita (1977) showed thatactive gpW could be recovered from guanidinium-hydrochloride-disrupted, mature, infectious, phage particles,indicating that it might be a structural component.

GpW is a small protein, with a molecular weight of 7614(Sanger et al. 1982) and is highly basic, with a calculated pIof 10.8 (Smith and Feiss 1993). The mechanisms by which itstabilizes the DNA inside the phage head are unknown. Theright end of the λ DNA molecule is positioned very close tothe bottom of the connector in complete heads (Bode andGillin 1971; Chattoraj and Inman 1974). This is also proba-bly true in incomplete heads — heads lacking gpFII — and,therefore, the basic nature of gpW suggests that it binds tothe right end of the λ DNA molecule before the addition ofgpFII and tails (Casjens et al. 1972).

More recently, physical studies of gpW tagged with hexa-histidine at either the N or C terminus have shown that insolution, this protein is monomeric and 47% helical. Muta-genesis studies have demonstrated that the last three C-terminal residues of gpW are crucial for its activity in vitro(Maxwell et al. 2000). Also, the structure of gpW in solutionhas been determined using NMR spectroscopy and found topossess a novel fold consisting of two α helices and a singletwo-stranded β pleated sheet. It has been hypothesized that,although a monomer in solution, during head assembly gpWpolymerizes into a ring with six-fold rotational symmetrythrough which the DNA exits during infection (Maxwell etal. 2001). When gpW is modeled as a hexamer with six-foldrotational symmetry, it reveals a structural similarity to DNAsliding clamps (Maxwell et al. 2001).

To better understand of the function of gpW, we havestarted to study the properties of the protein. Previous stud-ies have concentrated on the activity and structure of N- andC-tagged gpW (Maxwell et al. 2000, 2001). Here, we fo-cused on the activity and properties of the native protein. Inthis report, we detail the complete purification of gpW fromoverproducing gene constructions, describe some of the pro-tein’s properties, and show that in vitro it can be added tofull heads and be incorporated into complete viable phage.

Materials and methods

Media and buffersLuria broth (LB) contained the following: 10 g Bacto

Tryptone (Difco, Detroit, Mich.); 10 g NaCl; 5 g yeast ex-tract/L (Difco), pH adjusted to 7.5 with 10 mol/L NaOH.

Nutrient broth (NB), used for phage dilutions, containedthe following: 8 g nutrient broth (Difco) and 5 g NaCl/L.

Tryptone agar (TA), used as the solid medium for bacte-rial and phage plating, contained the following: 10 gtryptone (Difco), 5 g NaCl, and 10 g agar (British DrughHousem, London, U.K.).

Top agar was 6 g agar/L of NB. Where needed, ampicillinwas added to liquid and solid media to 50 µg/mL.

Sucrose solutions for gradient sedimentation (15%–30%mass fractions) were prepared in 0.01 mol Tris–HCl/L (pH 8.0);1 mmol putrescine/L; 0.2 mmol EDTA/L.

M9 medium contains 3 g KH2PO4, 6 g Na2HPO4, 1 gNH4Cl, 0.49 g, MgSO4·7H2O, 0.5 mg FeSO4�7H2O, and 55 mgCaCl2 per litre.

RMM medium is 2.7 g KH2PO4, 6.3 g Na2HPO4, 0.9 gNH4Cl, 0.11 g MgSO4, 0,5 mg FeCl3, 28 mg CaCl2, 1.3 gKCl, 3.6 g D-maltose, 100 mL glycerol, 0.1 mL 2-mercaptoethanol, and 0.9 g putriscine per litre. λ -dil is10 mmol Tris–HCl/L (pH 7.4) and 10 mmol MgSO4/L.

Buffer A is 0.01 mol Tris–HCl/L (pH 8.0), 1 mmolEDTA/L, 0.10 volume fraction of glycerol, and 1 µM PMSF.

Buffer B is 50 mmol Tris–HCl/L (pH 8.0), 0.1 mol KCl/L,1 mmol Na2EDTA/L, and 5 mmol 2-mercaptoethanol/L.

TBE buffer is 0.45 mol Tris/L, 0.45 mol H3BO3/L, and12 mmol Na2EDTA/L. PSB (p-sample buffer) used forPAGE samples is 0.0625 weight fraction SDS, 0.1875 vol-ume fraction glycerol, 0.078 mol Tris–HCl/L (pH 6.8), and0.03125 volume fraction 2-mercaptoethanol (Schägger andvon Jagow 1987).

OligonucleotidesThe following three oligonucleotides were used for the

construction of plasmids overproducing gpW:Dsd: 5′-CTTGGTAAGGGATGTTTATGACGCGA-3′ (for-

ward); 3′-CATTCCCTACAAATACTGCGCTGTCCTT-5′ (re-verse).

Esd: 5′-CTTGGTTTTTTTACGGGATTTTTTTATGAC-GCGA-3′ (forward); 3′-CAAAAAAATGCCCTAAAAAAA-TACTGCGCTGTCCTT-5′ (reverse).

Wlink: 5′-CAGGAAGAACTTGCCGCTGCCCGTG-3′ (for-ward); 3′-CTTGAACGGCGACGGG-5′ (reverse).

Dsd contains (from 5′→3′ of the coding strand) a StyI end,the ribosome-binding site of the lambda D gene, the initia-tion codon, and a sticky end that is complementary to the 5′end of Wlink. Esd contains, in the same order, a StyI stickyend, the ribosome-binding site of lambda gene E, the initia-tion codon, and a sticky end complementary to the 5′ end ofWlink. Wlink contains a 5′ end complementary to the 3′ endsof both Dsd and Esd, and a BglI sticky 3′ end. The ligationof the oligonucleotides containing the ribosome-bindingsites to Wlink reconstitutes the DNA sequence of W up tothe BglI site normally present in the wild-type gene.

Bacteria and bacteriophage strainsAll the bacteria used are derivatives of Escherichia coli K12

OR1265 (Reyes et al. 1979), which is sup null (sup°) and car-ries a defective λ prophage that contains a thermosensitiverepressor and lacks all morphogenetic genes. Strain 594(Campbell 1961) is sup°, and QD5003 (Yanofsky and Ito 1966)is supF, which suppresses all the phage amber mutations usedin this study. The prophages in the 594(λcIts857Sam7),594(λWam403cIts857Sam7), and 594(λEam4cIts857Sam7) lyso-gens (Murialdo and Siminovitch 1972) have the following

© 2003 NRC Canada

308 Biochem. Cell Biol. Vol. 81, 2003



properties in common. The cIts mutation renders themthermoinducible (Sussman and Jacob 1962), and the Sammutation blocks cell lysis at the normal time (Goldberg andHowe 1969), allowing increased intracellular accumulationof phage particles or phage components. Upon induction andincubation, the first lysogen accumulates phage particles; theE– lysogen accumulates tails, gpW, and gpFII; and the W–

lysogen accumulates DNA-full heads, tails, and gpFII(Casjens 1971; Perucchetti et al. 1988). Strain BL21(λDE3)-pLysS (Novagen) is F– ompT, hsdSB (rBmB), gal, dem(DE3)pLysE(camR).

The procedures used for plaque formation and preparationof phage lysates by thermoinduction of lysogens have beendescribed in detail previously (Murialdo et al. 1981).

Recombinant DNA techniquesIn general, in vitro DNA manipulations followed the proto-

cols of Sambrook et al. (1989). Restriction enzymes, mungbean nuclease, calf intestine phosphatase, and bacteriophageT4 ligase were used as recommended by the manufacturers(New England Biolabs, Beverly, Mass., or Pharmacia Biotech,Piscataway, N.J.). The procedures and references for celltransformation, agarose gel electrophoresis, and plasmid prep-aration have been described before (Murialdo and Fife 1984).

Construction of plasmidsThe parent plasmid for the construction of a gpW over-

producer was pFM148 (Fig. 1) (Ziegelhoffer et al. 1992). Itcarries the gene coding for the λcI857 repressor and the λmorphogenetic region including a portion of gene A to a por-tion of gene E. The morphogenetic genes are transcribedfrom the λPR and λPL promoters juxtaposed in the sametranscriptional orientation, with both under the control of thethermosensitive λcI857 repressor. The first step in the con-struction of the gpW overproducer involved the eliminationof the λ genes downstream of W. For this purpose, pFM148was digested with DraIII, and the resultant single-strandedends were trimmed with mung bean nuclease and dephos-phorylated with calf intestinal phosphatase. The modifiedfragments were ligated to SalI linkers with T4 DNA ligase.This mixture was used to transform OR1265 bacteria, and li-gated constructs were selected by plating on ampicillinplates. Plasmids containing a SalI site were characterized byfurther restriction endonuclease analysis and also by measur-ing gpW production upon induction. One of these plasmids,pFM149 (Fig. 1), was selected for further modification ofthe W ribosome-binding site as follows. After linearizationwith StyI, the DNA was precipitated, washed, and resus-pended in BglI incubation buffer and partially digested withBglI. The fragment containing (most of) W and the StyI endwas isolated by electrophoresis in a 0.005 weight fractionagarose gel. The DNA was recovered from the excised gelband by electroelution into a dialysis membrane in 0.5×TBE; the solution was concentrated on an Elutip-d column(Schleicher and Schuell), ethanol precipitated, washed, andresuspended in ligation buffer. One aliquot of the fragmentsolution was mixed with oligonucleotides Dsd and Wlink;another aliquot was mixed with oligonucleotides Esd andWlink. Upon ligation, the mixtures were used to transformOR1265, and colonies were screened for the presence of aplasmid of the correct size by restriction with EcoRI. Cor-

rect candidates were then further screened by digestion withseveral restriction endonucleases, and putative constructswere assayed for the ability to express active gpW. The gpWoverproducing plasmid containing the D ribosome-bindingsite is pFM150; that containing the E ribosome-binding siteis pFM151 (Fig. 1).

For the isolation of labeled gpW, the 300-bp StyI–SalIfragment of pFM150 containing W was isolated, purified,and rendered blunt ended with Klenow-fragment DNA poly-merase I. This fragment was then ligated to the vectorpET15B (Novagen, Madison, Wis.), which had been di-gested with XbaI and blunt-ended as well. Transformantswere screened by DNA sequencing for the correct orienta-tion of the insert and for the ability to express gpW.

Preparation of cell extracts

Crude extracts of gpWFor small-scale preparation, the bacteria were grown in

250-mL flasks containing 25 mL LB with vigorous aeration

© 2003 NRC Canada

Murialdo et al. 309

Fig. 1. Genetic and physical maps of plasmids used in this work.Those regions of the plasmid containing λ DNA are shown asdoubled lines, whereas DNA from pBR322 is represented by asingle line. Known λ genes are shown as boxes containing thegene name. Truncated genes are designated by their names, followedby the prime symbol. Promoters used are as follows: pM,repressor maintenance; pL and pR, left- and right-ward λ earlypromoters, respectively. Other labels are as follows: nutL, thesite coding for gpN use; Bla, the B-lactamase gene. The smallarrows initiated by a dot designate the position and direction oftranscription from the above promoters. Some restrictionsendonuclease sites are indicated by lines intersecting the plasmiddrawing. A linearized representation of pFM148, starting at theunique EcoRI site, RI, is shown. The DNA contents of pFM149,pFM150, and pFM151, all of which were derived from pFM148as described in the text, are shown at the bottom. The insertionsites of the synthetic oligonucleotides used to construct pFM150and pFM151 are shown.



at 31 °C to a concentration of 2.7 × 108 cells/mL. For therm-oinduction, the flask was transferred into a 44 °C water bath,incubated with shaking for 15 min, transferred to a 37 °Cwater bath, and incubated with shaking for 1 h. For largerscale preparations, bacteria were cultured in 2-L flasks con-taining 500 mL LB with vigorous aeration at 31 °C to a con-centration of 2 × 108 cells/mL. The flasks were thentransferred to a 70 °C water bath and shaken until the cul-ture temperature reached 43 °C. The flasks were then trans-ferred to a 45 °C water bath, shaken for 15 min, partiallysubmerged, and swirled in ice water until the cultures’ tem-perature dropped to 39 °C, and were then incubated at 37 °Cfor 1 h.

For the preparation of [35S]-labeled gpW, BL21(λDE3)-pLysS[pET15B-W] was grown at 37 °C in 250 mL of M9and 0.02 weight fraction of glucose to A600 = 0.3. IPTG wasadded to 1 mmol/L and incubated for 1 h. Rifampicin wasadded to 100 µg/mL, and after 30 min, 5 mCi of [35S]-methionine (specific activity: 1000 Ci/mmol) was added andshaking continued for a further 3 h. The cells were centri-fuged, resuspended in 5 mL of buffer A, disrupted bysonication, and centrifuged at 12 000g for 10 min. Thesupernatant was loaded onto a trimethylammonium column(20 mL bed volume) and developed with a gradient of NaCl(0–1 mol/L, 150 mL total volume) in buffer A. Fractionscontaining gpW activity were pooled and concentrated withsolid ammonium sulfate (to 80% saturation). The pellet wasdissolved in 0.5 mL buffer A and desalted on a sepharoseG10 column (volume 30 mL) in buffer A. The final fractionhad a protein concentration of 1 µg/mL and contained 5 ×105 dpm/µg protein (60 dpm = 1 Bq).

Acceptor extractsFor routine gpW titration, these extracts were prepared

from 594(λ Wam403cIts857Sam7) by the small-scale protocoldescribed above. At the end of the incubation period, 20 mLof culture were centrifuged at 4 500 r/min for 10 min at10 °C, resuspended in 1 mL RMM (20× extract), transferredinto an Eppendorf tube containing 0.15 mL of chloroform,vortexed for 10 s, followed by shaking, three times each, andcentrifuged at 15 000 r/min in a Tomy microcentrifuge(18 700g) for 5 min at 2 °C. Good acceptor activity was ob-tained when a negligible interphase and cell debris pelletwere obtained. The top phase was separated, aliquoted, andstored at –80 °C. Such aliquots retained their activity forseveral months provided they were used soon after thawing;refreezing noticeably reduced activity. For the complementationreaction with radioactively labelled gpW, the acceptor ex-tract was prepared using the large-scale preparation protocol(see above) and the culture was concentrated 100 fold beforecell lysis (100× extract).

Head–tail joining activity of gpWAcceptor extract was thawed at room temperature and im-

mediately stored on ice. For the standard assay, 40 µL of ac-ceptor extract and 40 µL of a putative gpW activitycontaining extract or fraction (undiluted or diluted) weremixed, vortexed gently, incubated for 1 h at 37 °C, and kepton ice. Serial dilutions of the reaction mixture were thentitred for plaque formation on indicator lawns of QD5003.

Preparation of virionsWild-type bacteriophage λ were prepared from

594(λcIts857Sam7) (Murialdo and Siminovitch 1972), ac-cording to the procedure described previously (Dokland andMurialdo 1993).

Protein sequencing, mass spectroscopy, and gelfiltration

All protein analyses were performed on fraction MS41 ofpurified gpW (see bellow). The sequence of the first sevenresidues was determined in a Porton gas-phase micro-sequencer model 2090. The molecular weight of gpW wasdetermined by electrospray mass spectroscopy using aPerkin-Elmer–Sciex API-III triple quadruple instrument. Aswell, the protein was passed through a Sephadex G-100 gelfiltration column equilibrated in buffer B and calibrated withBlue Dextran 2000, bovine serum albumin, ovalbumin, ribo-nuclease A, and aprotinin. The concentration of the markersin the fractions was determined by SDS-PAGE (see below)and the concentration of gpW was measured by biologicalactivity with the complementation reaction.

SDS-PAGESDS-PAGE was carried out using the discontinuous

method described by Schägger and von Jagow (1987) withtricine as the trailing ion. The stacking gel was 4% T, 3% C;the spacer gel was 10% T, 3% C; and the separating gel was16.5% T, 3% C. One part sample was mixed with 1.8 partsPSB, and the mixtures incubated for 30 min at 40 °C or for3 min in a boiling water bath, and loaded into the wells withbromophenol blue. The slab gels, 1.5 mm thick, 15 cm wide,and 21 cm long (including the stacking and separating gels)were eletrophoresed for 3 h at 40 V, followed by 17 h at 120 V.Unless otherwise indicated, staining was with Coomassiebrilliant blue. Premixed molecular weight standards werefrom Bio-Rad (Hercules, Calif.). Protein concentrations weredetermined by the Bio-Rad assay.

Results

Production of gpWIt appears that W, like A, is poorly expressed in E. coli.

Earlier attempts at purifying gpW required very large vol-umes of bacterial cultures (Perucchetti et al. 1988). A com-parison of gpW in vitro biological activity from varioussources is shown in Table 1. Here, wild type is representedby an induced E-lysogen lysate. Its activity is compared withthat of extracts of cells carrying W on plasmids with alterna-tive promoters and ribosome-binding sites. Whereas a stronginducible promoter did indeed increase expression some 140fold (pFM149), the additional modification of a high-efficiency ribosome-binding site (pFM150 and pFM151) re-sulted in approximate 105-fold greater production than wildtype. It seems that the D- and E-ribosome-binding sites inpFM150 and pFM151, respectively, are equally efficient fortranslation of the W message, and that the low level of ex-pression in wild-type infections is indeed attributable to aweak W ribosome-binding site.

© 2003 NRC Canada




The gpW assayThe assay used in this study is based on the ability of

gpW-containing cell extracts to complement W-phage-infected bacterial extracts in vitro for the production ofPFUs (Casjens 1971). This assay is different from and sim-pler than the assay previously used in this laboratory(Perucchetti et al. 1988). However, as with all DNA-packaging systems, the gpW assay displayed nonlinear prop-erties. The kinetics of phage formation when crude extractswere mixed revealed that the reaction(s) were rapid (Fig. 2).Although a maximum yield of 4 × 109 PFU/mL was attainedwithin 20 min of incubation, an increase of four orders ofmagnitude is already evident after only 150 s. Yields ofphage were dependent on the particular acceptor (W–) ex-tract used. In several instances, levels of 5 × 109 to 6 × 109

PFU/mL were observed. Despite our efforts to carefully con-trol all the known parameters involved in acceptor extractpreparation, we have not been able to establish full consis-tency in the activity of the different acceptor extracts. Whenpresented as a log–log plot (Fig. 3), there was a nearly 3 to 4order of magnitude linear relationship between plaque for-mation and gpW concentration with fractions of various spe-cific activity and purity. Again, although the slopes (logyield/log[gpW]) depended on the pertinent acceptor extractused, and slopes as high as 5.8 were obtained in some mea-surements, the average slope of the straight lines was 3.6,which corresponds to a 12-fold increase in yield for everydoubling in pgW concentration. Since the system has an in-herent maximum (based on the levels of active filled headsand tails in the extracts), all assays were carried out at dif-ferent dilutions to ensure that they were in the linear range.In addition, it appeared that high concentrations of ammo-nium sulfate inhibited phage formation (see below, Fig. 4).

Purification of gpWFour liters of OR1265[pFM151]-induced bacterial culture

were centrifuged at 3400g for 10 min at 4 °C, and the cellsresuspended in 200 mL RMM. All subsequent steps werecarried out at 4 °C. The suspension was cleared bysonication (16 pulses of 20 s each, followed by 30 s of cool-ing on ice–salt) using the 9.5-mm probe of a VibraCell unitoperating at 300 W and centrifuged at 27 000g for 15 min toyield 180 mL of crude extract (CE fraction). The crude ex-

tract was mixed, while stirring, with an equal volume ofaqueous 0.005 weight fraction protamine sulfate (Perucchettiet al. 1988) and after 30 min the heavy precipitate was re-moved by centrifugation at 17 300g for 15 min to yield360 mL of the protamine sulfate supernatant fraction (PSfraction). This was concentrated by the addition of solid am-monium sulfate to 80% saturation, stirred for 1 h, and centri-fuged at 33 000 rpm (85 000g) for 50 min in a 45TiBeckman rotor. The pellet was dissolved in 10 mL of bufferB to give the ammonium sulfate (AS1) fraction, and then de-salted by filtration through a column (26 mm × 580 mm) ofSephadex G-100 (G100 fraction), and concentrated as abovewith ammonium sulfate to give 5.3 mL of the AS2 fraction.This was desalted by filtration through a column (16 mm ×260 mm) of Sephadex G50 fine in buffer B, and the activeeluates were pooled to give the G50 fraction, which wasthen passed through a Pharmacia HR5/5 Mono-Q column inbuffer B using a Pharmacia FPLC system running at1 mL/min and collecting 1.5-mL fractions. The gpW activityeluted completely in the first 16 fractions (24 mL), whichwere pooled as the Q fraction that was then chromatographedon a Biorad S2 column. The column was developed with alinear gradient of KCl (0.1–1.5 mol/L, 50 mL total volume)in buffer B at 1 mL/min, and 1-mL fractions were collected.All activity eluted close to the end of the gradient (1.45 molKCl/L) in two fractions, MS39, and MS40. An overall purifi-cation of approximately 200-fold with a yield of approxi-mately 30% was achieved. These fractions retained their

© 2003 NRC Canada

Murialdo et al. 311

GpW activity donor Activity (PFU)

None <1×102

594(λEam4cIts857Sam7) 1.3×104

OR1265(pTM149) 1.8×106

OR1265(pTM150) 9.3×108

OR1265(pTM151) 1.3×109

Note: The protein concentration of the extracts from thethree plasmid-containing cells was 2.0 mg/mL, that of thelysogen was 2.8 mg/mL, and that of the acceptor extractwas 2.4 mg/mL. The activity of the extracts was measuredusing three strengths, 1, 0.325, and 0.1, and the yields wereploted in a log–log plot against protein extract concentra-tion. The yields in the table correspond to the values inter-polated to a concentration of 1 mg/mL.

Table 1. Head–tail joining activity of crude extractsof gpW donor cultures.

Fig. 2. Kinetics of gpW activity. Equal volumes of acceptor extractand gpW donor source were preheated to 37 °C, mixed, andincubated at the same temperature. Ten seconds after mixing,and at designated times thereafter, aliquots were removed anddiluted 20 fold into either NB or suspensions of plating bacteria(QD5003) kept on ice. The background titre of the acceptoralone was 3 × 103 PFU/mL; the gpW donor’s titre was <10 PFU/mL.The graph in the inset corresponds to an expansion of the earlyincubation times in the main graph.



activity for several weeks when kept frozen at –20 °C. Thetitration of the fractions is shown in graphic form in Fig. 4.A summary of the purification is given in Table 2, and anSDS-PAGE analysis of various fractions is shown in Fig. 5.The final preparation appeared to be homogeneous as judgedby the Coomassie brillant blue stained gel.

Properties of gpWAmino acid analysis of the MS fractions showed that the

overall composition and the sequence of the first seven resi-dues — MTRQEEL — were identical to those predicted bythe gene sequence (Sanger et al. 1982). The molecularweight M from SDS-PAGE was estimated to be approxi-

mately 6600 Da, which is in rough agreement with the valuepredicted from the DNA sequence of the gene (7611.8 Da).In gel filtration under native conditions, a single peak of ap-proximately 7000 Da was obtained, indicating that gpW ex-isted as a monomer in solution. In four determinations, massspectroscopy gave an estimated mass of 7610.49 ± 0.92 Da.

Incorporation of gpW into phage particlesHead–tail joining reactions were carried out by mixing

150 µL of purified 35S-labeled gpW with 250 µL of 100acceptor extracts. After 1 h of incubation at 37 °C, 180 µLaliquots were layered on top of step CsCl gradients. The stepgradients were formed by gently layering in a SW60 rotor

© 2003 NRC Canada


Activity (PFU/mL;full strength)

Volume(mL)

Totalactivity

Protein concentration(mg/mL)

Specific activity(PFU/mg) Purification

Yield(%)

CE 2.7×109 200 5.4×1011 6.3 4.3×108 1 100PS 4.0×109 360 1.4×1012 2.38 1.7×109 4 259AS1 7.5×1010 10 7.5×1011 31.8 2.4×109 6 139G100 1.9×109 100 1.9×1011 0.21 3.0×109 21 35AS2 9.0×1010 4.5 4.0×1011 11 3.9×109 19 74G50 1.5×1010 20 3.0×1011 1.5 5.0×109 23 55Q 3.0×109 24 7.2×1010 0.4 3.0×109 17 13S39 1.0×1011 1 1.0×1011 0.43 2.32×1011 540 18.5S40 5.8×1010 1 5.8×1010 0.27 2.15×1011 500 10.7

Note: The specific activity of the fractions was obtained by dividing the activity at full strength by the protein concentration. When this was not possi-ble, owing to saturation of the acceptor capacity (fractions S39 and S40) or inhibition by (NH4)2SO4 concentration (fractions AS1 and AS2), the full-strengthactivity was obtained by extrapolation in the plots of log activity vs. log concentration (Fig. 4).

Table 2. Purification of gpW.

Fig. 3. gpW activity as function of concentration. Four typicalexperiments are shown. To avoid cluttering the graph, the ordi-nate axis for each experiment was displaced by a constant space.The symbols at the outside right bottom of the figure show the xaxis that corresponds to the curve with identical symbols. ThegpW concentration is expressed as the log of the microlitres ofextract added to the reaction tubes. Standard assay conditions asdescribed in Materials and methods were used.

Fig. 4. gpW activity of the different fractions. For each fraction ofthe purification procedure, a series of reactions were carried outwhere equal volumes of the same acceptor and various dilutions ofthe donor were mixed and incubated for 1 h at 37 °C, and phagetitres were determined as described in the text. CE, crude extract;PS, protamine sulfate supernatant; AS1, the ammonium sulfateconcentrate of PS; G100, Sephadex G100 filtrate of AS1; AS2,ammonium sulfate concentrate of G100; Q, Mono-Q eluate; S39

and S40, two consecutive fractions of the S2 column eluate.



tube 1.8 mL of CsCl (ρ = 1.55) and 1.8 mL of CsCl (ρ = 1.45),both in λ-dil. The step gradients were spun in a BeckmanSW60 rotor (not to equilibrium) for 3 h at 180 000g at 5 °C.Fractions from the gradients were assayed for both radioac-tivity and the presence of viable phage. As shown in Fig. 6,a single peak was obtained in each case where both the ra-dioactivity and the biological activity banded together at aposition characteristic of phage λ (ρ = 1.5). No such peakswere observed when control incubations lacking acceptorwere centrifuged (data not shown). These results indicatethat gpW is not only acting to promote head-to-tail joining,but is also incorporated into the finished virion.

Discussion

The results presented here substantially support previousfindings (Casjens et al. 1972; Perucchetti et al. 1988) thatgpW in vitro promotes the joining of tails to heads to pro-duce viable phage particles. The purification from the con-structed overproducers described in this report is fairlysimple. Since all of the morphogenetic genes are transcribedas a long polycistronic message from the late promoter(Gariglio and Green 1973; Chowdhury and Gupta 1973), thelarge variation in the numbers of the various proteins hasbeen ascribed to translational control (Ray and Pearson1975). Therefore, we substituted the ribosome-binding siteof W with that of either D or E. The results, in Table 1, showthat a large degree of overproduction was thus achieved.

The assay used in these studies is based on that of Casjenset al. (1972). It is convenient and differs from the one describedearlier, in which all the components were partially purified

and consisted of terminase, phage DNA (either mature orconcatameric), proheads, gpD, gpFII, and tails (Perucchettiet al. 1988). We have confirmed that gpW is a structuralcomponent of the bacteriophage virion (Fig. 6). Calculationsbased on the specific radioactivity of purified gpW and thenumber of phage produced in the complementation reaction,and recovered from the CsCl density gradient, results in 86 gpWmolecules per biologically active (able to form PFU) phage.There is circumstantial evidence to indicate that this figureis an artifact. Firstly, this amount of gpW would correspondto about 2% of the phage proteins. However, we have failedto detect a band of a molecular weight corresponding togpW in electropherograms of disrupted phage using thetricine electrophoresis system described in the Materials andmethods section. In these experiments, the gels were stainedwith Sypro orange, and the fluorescence was revealed in aphosphoimager. Other viral proteins corresponding to 2.9%(gpJ) and 2.3% (gpH) of the viral particle mass showed asthick bands. In fact, this procedure would have detected aband corresponding to no more than 0.2% of the phage pro-teins (about 20 ng/band). Secondly, the initial velocity of thecomplementation reaction is proportional to the PFU yieldand, as we have seen, the log yield – log[gpW] dose–responsecurves show slopes of 3 to 6, suggesting that the number ofgpW molecules per phage is 6. Thus, it appears as if a substan-tial amount of radioactivity is incorporated into biologically in-active particles. The assembly of biologically inactive virionsin the in vitro complementation reaction would explain the

© 2003 NRC Canada

Murialdo et al. 313

Fig. 5. Electropherogram of gpW fractions. The fractions obtainedduring the purification of gpW described in the text were analysedby SDS-PAGE and stained with Coomassie brilliant blue R, asdescribed in Materials and methods. The content of the lane isshown on top. For nomenclature of the fractions see the legendto Fig. 4. MWSt, molecular weight standards.

Fig. 6. Incorporation of gpW into phage particles. Reaction mix-tures for head–tail joining were set up with 35S-labeled gpW andincubated as described in the text. The reactions were then lay-ered on top of a step gradient, as described in Materials andmethods, and the step gradients were centrifuged at 180 000g at5 °C. Fractions of 0.2 mL were collected from the bottom of thetubes and assayed for radioactivity and viable phage. The bottomof the tube is at the left of the figure. In reactions omitting ac-ceptor, all of the radioactive gpW remained at the top of thetubes (not shown). The peak of purified phage in an identicalcontrol tube was found in fraction 10.



rather small maximum yield obtained (3 × 109 PFU/mL);about 5% of that expected assuming 100% assembly of theextract components.

It is known that gpW acts once the DNA has been pack-aged into the head, but before the addition of the six gpFIImolecules, a step that completes head morphogenesis andrenders it able to join the tail (Casjens et al. 1972; Casjensand Hendrix 1974). It is therefore likely that gpW assembleson the connector, which is composed of gpB and its cleavedproduct gpB*, 12 molecules in total (Kochan et al. 1984). Itis also noteworthy that gene W maps beside gene B and thatgenes whose products interact have a strong tendency tomap contiguously in lambdoid phages (Casjens and Hendrix1974, 1988). The kinetics and strong dependency on [gpW]of the complementation reaction strongly suggests that, al-though monomeric in solution, gpW polymerizes as it as-sembles in the substrate surface of the connector. Since theconnector has 12-fold rotational symmetry and there are 6molecules of gpFII (Casjens and Hendrix 1974), it is likelythat the number of pgW molecules in the virion is either 12or 6. The solution structure of gpW has recently beensolved. This structure can be modelled into a hexameric ringwith structural similarity to the DNA polymerase slidingclamp. The overall diameter and orifice diameter of the gpWhexamer are consistent with the dimensions of the connectorand DNA, respectively (Maxwell et al. 2001). Therefore, itis posible that six gpW molecules bind to alternate subunitsof the connector and that by interacting with the terminus ofthe DNA, they stabilize it inside the head. It is known thatupon gpFII and tail attachment, the DNA moves about 100 bpdownstream, occupying about one third of the tail shaft(Thomas 1974; Chattoraj and Inman 1974).

Acknowledgements

The authors would like to thank Andrew Becker for hisconsiderable contribution to this research. This work waspartially supported by funds from The Medical ResearchCouncil of Canada, Fondecyt 1980209 (Chile), and from theP. Valenzuela and B. Méndez Foundation.

References

Bode, V.C., and Gillin, F.D. 1971. The arrangement of DNA inlambda phage heads. I. Biological consequences of micrococcalnuclease attack on a portion of the chromosome exposed intailess heads. J. Mol. Biol. 62: 493–502.

Campbell, A. 1961. Sensitive mutants of bacteriophage λ . Virology,14: 22–32.

Casjens, S. 1971. The morphogenesis of the phage lambda head:the step controlled by gene F. In The bacteriophage lambda.Edited by A.D. Herskey. Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. pp. 725–732.

Casjens, S., and Hendrix, R. 1974. Comments on the arragement ofthe morphogenetic genes of bacteriophages lambda. J. Mol. Biol.131: 20–23.

Casjens, S., and Hendrix, R. 1988. Control mechanisms in dsDNAbacteriophage assembly. In The bacteriophages. Vol. 1. Editedby R. Calendar. Plenum Publishing Corp, New York. pp. 15–91.

Casjens, S., Hohn, T., and Kaiser, A.D. 1972. Head assembly stepscontrolled by genes F and W in bacteriophage λ . J. Mol. Biol.64: 551–563.

Chattoraj, D.K., and Inman, R.B. 1974. Location of DNA ends inP2, 186, P4 and lambda bacteriophage heads. J. Mol. Biol. 87:11–22.

Chowdhury, D.M., and Gupta, A. 1973. Characterization of themRNA transcribed from the head and tail genes of phagelambda chromosome. Nature New Biol. 241: 196–198.

Dokland, T., and Murialdo, H. 1993. Structural transitions duringmaturation of bacteriophage lambda capsids. J. Mol. Biol. 233:682–694.

Goldberg, A.R., and Howe, M. 1969. New mutations in the Scistron of bacteriophage lambda affecting host cell lysis. Virology,38: 200–202.

Kaiser, D., Syvanen, M., and Masuda, T. 1975. DNA packagingsteps in bacteriophage lambda head assembly. J. Mol. Biol. 91:175–186.

Katsura, I., and Tsugita, A. 1977. Purification and characterizationof the major protein and the terminator protein of the bacterio-phage λ tail. Virology, 76: 129–145.

Maxwell, K.L., Davidson, A.R., Murialdo, H., and Gold, M. 2000.Thermodynamic and functional characterization of protein Wfrom bacteriophage λ . J. Biol. Chem. 275: 18 879 – 18 886.

Maxwell, K.L., Yee, A.A., Boothe, V., Arrowsmith, C.H.,Gold, M., and Davidson, A.R. 2001. The solution structure ofthe bacteriophage λ head-tail joining protein, a small morpho-genetic protein processing a novel fold. J. Mol. Biol. 308: 9–14.

Murialdo, H. 1991. Bacteriophage lambda DNA maturation andpackaging. Annu. Rev. Biochem. 60: 125–153.

Murialdo, H., and Fife, W.L. 1984. The maturation of the coliphagelambda DNA in the absence of its packaging. Gene, 30: 183–194.

Murialdo, H., and Siminovitch, L. 1972. The morphogenesis ofbacteriophage lambda. IV. Identification of gene products andcontrol of the expression of the morphogenetic information.Virology, 48: 785–823.

Murialdo, H., Fife, W.L., Becker, A., Feiss, M., and Jochem, J.1981. Bacteriophage λ DNA maturation. The functional rela-tionships among the products of genes Nu1, A and Fl. J. Mol.Biol. 145: 375–404.

Parkinson, J.S. 1968. Genetics of the left arm of the chromosomeof bacteriophage lambda. Genetics, 59: 311–325.

Perucchetti, R., Parris, W., Becker, A., and Gold, M. 1988. Latestages in bacteriophage λ head morphogenesis: in vitro studieson the action of the bacteriophage λ D-gene and W-gene prod-ucts. Virology, 165: 103–114.

Ray, P.N., and Pearson, M.L. 1975. Functional inactivation ofbacteriophage lambda morphogenetic gene mRNA. Nature (Lon-don), 253: 647–650.

Reyes, O., Gottesman, M., and Adhya, S. 1979. Formation oflambda lysogens by IS2 recombinantion: gal operon – lambdaPR promoter fusions. Virology, 94: 400–408.

Sambrook, J., Frisch, E.F., and Maniatis, T. 1989. Molecular clon-ing: a laboratory manual. 2nd ed. Cold Spring Harbor Labora-tory Press, Cold Spring Harbor, N.Y.

Sanger, F., Coulson, A.R., Hong, G.F., Hill, D.F., and Petersen,G.B. 1982. Nucleotide sequence of bacteriophage λ DNA. J.Mol. Biol. 162: 729–773.

Schägger, H., and von Jagow, G. 1987. Tricine-sodium didecylsulfate-polycrylamide electrophoresis for the separation of pro-teins in the range of 1 to 100 kDa. Ann. Biochem. 166: 368–379.

© 2003 NRC Canada




Smith, M.P., and Feiss, M. 1993. Sequence analysis of the phage21 genes for prohead assembly and head completion. Gene, 126:1–7.

Sussman, R., and Jacob, F. 1962. Sur un systeme de repressionthermosensible chez le bacyeriophage λ d’Escherichia coli. C.R. Acad. Sci. (D) Paris, 254: 1517–1519.

Yanofsky, C., and Ito, J. 1966. Nonsense codons and polarity in thetryptophan operon. J. Mol. Biol. 21: 313–334.

Ziegelhoffer, T., Yau, P., Chandrasekhar, G.N., Kochan, J., Georgo-poulos, C., and Murialdo, H. 1992. The purification and proper-ties of the scaffolding protein of bacteriophage λ . J. Biol. Chem.267: 455–461.

© 2003 NRC Canada

Murialdo et al. 315



The product of the bacteriophage lambda W gene: purification and...

Documents

Transcript of The product of the bacteriophage lambda W gene: purification and...