Shape and DNA packaging activity of bacteriophage SPP1 procapsid: protein components and interactions during assembly1

Article No. jmbi.1999.3450 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 296, 117±132

Shape and DNA Packaging Activity of Bacteriophage SPP1 Procapsid: Protein Components and Interactions during Assembly Anja DroÈge1*, MaÂrio A. Santos2,3, Asita C. Stiege1, Juan C. Alonso4 Rudi Lurz1, Thomas A. Trautner1 and Paulo Tavares1 1

Max-Planck-Institut fuÈr Molekulare Genetik, Ihnestraûe 73, D-14195, Berlin, Germany 2 Instituto de Tecnologia QuõÂmica e BioloÂgica, Apart.127 2781, Oeiras, Portugal 3

Universidade de Lisboa Faculdade de CieÃncias Campo Grande, Bloco C2, Departamento de Biologia Vegetal, 1750, Lisboa, Portugal 4 Centro Nacional de BiotecnologõÂa, CSIC, Campus Universidad AutoÂnoma de Madrid, Cantoblanco 28049, Madrid, Spain

The procapsid of the Bacillus subtilis bacteriophage SPP1 is formed by the major capsid protein gp13, the scaffolding protein gp11, the portal protein gp6, and the accessory protein gp7. The protein stoichiometry suggests a T ˆ 7 symmetry for the SPP1 procapsid. Overexpression of SPP1 procapsid proteins in Escherichia coli leads to formation of biologically active procapsids, procapsid-like, and aberrant structures. Co-production of gp11, gp13 and gp6 is essential for assembly of procapsids competent for DNA packaging in vitro. Presence of gp7 in the procapsid increases the yield of viable phages assembled during the reaction in vitro ®ve- to tenfold. Formation of closed procapsid-like structures requires uniquely the presence of the major head protein and the scaffolding protein. The two proteins interact only when co-produced but not when mixed in vitro after separate synthesis. Gp11 controls the polymerization of gp13 into normal (T ˆ 7) and small sized (T ˆ 4?) procapsids. Predominant formation of T ˆ 7 procapsids requires presence of the portal protein. This implies that the portal protein has to be integrated at an initial stage of the capsid assembly process. Its presence, however, does not have a detectable effect on the rate of procapsid assembly during SPP1 infection. A stable interaction between gp6 and the two major procapsid proteins was only detected when the three proteins are co-produced. Ef®cient incorporation of a single portal protein in the procapsid appears to require a structural context created by gp11 and gp13 early during assembly, rather than strong interactions with any of those proteins. Gp7, which binds directly to gp6 both in vivo and in vitro, is not necessary for incorporation of the portal protein in the procapsid structure. # 2000 Academic Press

*Corresponding author

Keywords: Bacillus subtilis phages; virus assembly; in vitro DNA packaging; protein-protein interaction; portal protein

Introduction A common theme in head morphogenesis of all icosahedral double-stranded DNA (dsDNA) bacteriophages is the formation of a precursor capsid, called procapsid or prohead, that is subsequently Present address: Paulo Tavares, is at Unite de PathogeÂnie Microbienne MoleÂculaire, Institut Pasteur, Rue du Dr. Roux 28, F-75724 Paris Cedex 15, France. Abbreviations used: cfu, colony forming units; dsDNA, double-stranded DNA; EM, electron microscopy; gpX, the product of gene X; i.m., input multiplicity; pfu, plaque forming units; wt, wild-type. E-mail address of the corresponding author: [email protected] 0022-2836/00/60117±16 $35.00/0

®lled with DNA. The shells of these procapsids are in most cases formed by several hundred copies of a single protein which are precisely arranged in an icosahedrally shaped lattice. Caspar & Klug (1962) predicted that multiples of 60 subunits, de®ned by the T numbers (1, 3, 4, 7, 9, 12, 13...), are positioned in T different quasi-equivalent environments of such lattices. For T > 1 lattices the coat subunits are organized in pentamers at the vertices of the icosahedron while hexamers form the faces of the structure. Structural data on phages and other viruses supported but for a few exceptions the CasparKlug theory and showed that the structural basis for quasi-equivalence is the ability of the capsid proteins to switch to different conformations # 2000 Academic Press

118 depending on the environment they are placed in (Rossmann, 1984; Rossmann & Johnson, 1989; Johnson, 1996; Thuman-Commike et al., 1996). This structural ¯exibility is essential for capsid formation but implies that at each position within the lattice the capsid subunit must adopt the single ``correct'' conformation out of several ``incorrect'' ones. Failure to do so leads to formation of aberrant lattice structures. The vast majority of dsDNA phages requires a scaffolding protein to guarantee correct polymerization of the coat protein subunits (Casjens & Hendrix, 1988; see Duda et al., 1995a,b for the exception HK97). Cellular chaperonins are also involved in the correct folding of coat proteins from several bacteriophages (e.g. T4, P22, HK97; Georgopoulos et al., 1972; Gordon et al., 1994; Xie & Hendrix, 1995). One vertex of the dsDNA phage proheads is distinct from the remaining 11 vertices by the presence of a cyclical hollow oligomer, the portal protein. Portal proteins play a role in procapsid assembly of several phages, are essential for DNA packaging, and provide an interface for tail attachment or assembly (reviewed by Valpuesta & Carrascosa, 1994). The construction of an icosahedron with a single portal complex presents an intriguing assembly problem. It requires an active mechanism to incorporate an oligomer positioned asymmetrically within a highly symmetric structure and to prevent incorporation of additional portal proteins at the other capsid vertices. Portal complexes, which may either have 12- or 13-fold symmetry, are inserted in a structural environment of 5-fold symmetry, replacing the coat pentamer at the portal vertex (Tao et al., 1998). The symmetry mismatch precludes strong speci®c interactions between portal and coat proteins leading to the proposal that the portal complex is attached to the head shell-like ball bearings in the hub of a wheel (Hendrix, 1978). It seems thus unlikely that targeting of the portal protein to the procapsid is promoted by speci®c bonding to the coat protein. The scaffolding protein was shown to play a role in recruitment of the portal protein to the procapsid structure (Bazinet & King, 1988; Greene & King, 1997; Trauls et al., 1984; Kahn et al., 1987; Lee & Guo, 1995) but its interaction with the portal protein in absence of coat protein was only detectable in case of bacteriophage f29 (Lee & Guo, 1995). An intricate interplay between the three proteins appears necessary to ensure the ef®cient assembly of procapsids carrying a single portal protein but the underlying mechanism remains unknown. We are investigating head assembly of the lytic Bacillus subtilis dsDNA bacteriophage SPP1. Assembly intermediates, which can mature into infective virions, and the phage particles are visualized in Figure 1(a). The gene clusters encompassing the phage cistrons required for head assembly were identi®ed and the general outline of its morphogenetic pathway has been de®ned (Becker et al., 1997; Figure 1(b)). The SPP1 procapsid is composed of the major capsid protein (gp13), the scaf-

Assembly of Bacteriophage SPP1 Procapsid

folding protein (gp11), the portal protein (gp6) and the accessory protein gp7 (Becker et al., 1997; Figure 1(b)). Here we identify the minimal requirements for procapsid shape determination and assembly of biologically active procapsids, and de®ne the network of protein-protein interactions necessary for the assembly reaction. SPP1 procapsid assembly requires the interplay between the major capsid protein, the scaffolding protein and the portal protein, a common feature to tailed icosahedral bacteriophages. gp7, which binds gp6, enhances the biological activity of procapsids but is not an essential component of the structure. We found that the SPP1 portal protein plays a role in capsid size determination not previously described for any other phage system. Polymerization of gp13 into closed icosahedral lattices requires uniquely gp11, and the assembly kinetics is apparently not in¯uenced by presence of the portal protein, a situation analogous to bacteriophage P22 (Bazinet & King, 1988). However, in contrast to the P22 system, presence of the SPP1 portal protein is important to direct the procapsid assembly reaction towards the T ˆ 7 normal structure by preventing formation of small-sized procapsids (T ˆ 4?). The capacity of the portal protein to affect polymerization of gp13 suggests an interaction of gp6 with gp11 and/or gp13 at an initiation step that in¯uences the procapsid build-up. This feature was proposed for other isometric phages based on a different phenotype associated with portal protein absence: assembly of aberrant structures, more frequently polyheads, in addition to normally shaped procapsids (l, T7, T3; reviewed by Murialdo & Becker, 1978; Bazinet & King, 1985). Presence of the portal protein in the prolate procapsid of bacteriophage f29 is necessary to de®ne the elongation axis of the icosahedral structure, a shape determining role that implies again an early participation of that protein in procapsid assembly (Hagen et al., 1976; Guo et al., 1991).

Results Morphology and T number of the SPP1 prohead and head Figure 1(a) illustrates the morphology of the SPP1 head structure at different morphogenetic steps. A major structural change occurs upon the transition of the spherically shaped procapsid, Ê , to the with an outer diameter (do) of about 550 A Ê full head (do ˆ 660 A) which displays a hexagonal outline (Becker et al., 1997). As a consequence of this 20 % expansion in size the thickness of the shell is reduced. The full head and the phage head are morphologically similar. We carried out a quantitative analysis of the protein composition of proheads and phages to determine the T number de®ning the symmetry of the SPP1 capsid shell. Stoichiometry calculations were based on densitometry of protein bands from autoradiograms of 35S-labeled virions and from Coo-

119

Assembly of Bacteriophage SPP1 Procapsid

Figure 1. (a) Bacteriophage SPP1 head assembly intermediates and (b) morphogenetic pathway. (a) Stable morphogenetic intermediates were negatively stained with uranyl acetate. SPP1 procapsids (pc), full heads (fh) and phages (ph) were puri®ed from SPP1sus70, sus9, and wild-type infections, respectively. Different type of structures are indicated by arrows and identi®ed by the abbreviations used on top of the structures cartoon in (b). The bar represents 100 nm. (b) The morphogenetic pathway adapted from Becker et al. (1997) shows the gene products involved in procapsid formation and their position in the structure. The terminase-DNA complex (gp1-gp2-DNA; Chai et al., 1992, 1995) proposed to dock in the procapsid portal vertex is also represented. The subsequent morphogenetic events are also depicted but to simplify the scheme the proteins involved are not indicated.

massie blue stained SDS-PAGE of procapsids and virions (Table 1). Assuming the presence of a single tredecameric portal protein (Dube et al., 1993; Orlova et al., 1999) per capsid the calculated T number is approximately 6 both for proheads and phage heads. This value suggests that the SPP1 capsid displays most probably a T ˆ 7 symmetry. Underestimation of the number of coat protein subunits in capsids might be due to the presence of truncated versions of gp13 with heterogeneous Mr that were detected in anti-gp13 Western blots (DroÈge, 1998). Quanti®cation of the gp11 signal showed that the procapsid is ®lled with approximately 100-180 copies of the scaffolding protein. This is an average number that might vary among individual structures, as suggested from the different amount of scaffolding protein observed inside

procapsids on our preparation (Figure 1(a)). Both proheads and phages contain gp7, which is present in one to two copies per virion. Minimal requirements for the formation of biologically active procapsids Overexpression of the SPP1 prohead gene clusters (construct pBT368; Figure 2) in Escherichia coli BL21(DE3)pLysS leads to formation of procapsidlike structures, a designation used here to describe any closed shell lattices ®lled with scaffolding protein, that are competent for SPP1 DNA packaging in vitro (DroÈge & Tavares, 2000). This ®nding validated the use of the E. coli heterologous system to overproduce SPP1 proteins and to test their activity in assembly. To determine the minimal

120

Assembly of Bacteriophage SPP1 Procapsid

Table 1. Mass and stoichiometry of procapsid proteins (gp6, gp7, gp11 and gp13) on viral particles

Gene product

Estimated Mr Percentage of total (theoretical Mr) (kDa) virion proteinsb

Number of copies per virionc Coomassie

35

S

gp6

67(2) (57.3)

4.9  1.4

13

13

gp7 gp11 gp13 T number

34(2) (35.1) 22(2) (23.5) 37(1) (35.4)

<1 68  12

n.d. 137  39d 356(35) 345(161)d 5.9(0.4) 5.7(2.4)d

1.6  0.4 n. d. 342  60 5.7  1

Function Portal protein Accessory head protein Scaffolding protein Major head protein

Protein samples prepared from proheads, phages and 35S-labeled phages were resolved by SDS-PAGE. Quanti®cation of the different proteins was based on the density of their corresponding bands on Coomassie blue stained gels and on autoradiograms, performed as described in Materials and Methods. a The molecular mass of the different proteins was estimated based on their electrophoretic mobility in comparison to a protein standard size marker. The Mr values predicted from the nucleotide sequence of their coding genes are given within parentheses. b To determine the percentage of the total virion mass associated to individual proteins, the relative mass per virion of each protein, estimated densitometrically from Coomassie blue stained gels, was divided by the sum of all proteins relative mass and multiplied by 100. c The number of subunits from each protein type per phage was calculated under the assumption that 13 copies of gp6 are present per virion (Dube et al., 1993; Orlova et al., 1999). d The number of subunits from each protein type per phage was calculated under the assumption that 13 copies of gp6 are present per procapsid (Dube et al., 1993; Orlova et al., 1999).

requirements for procapsid formation and the role of the individual components expressed from pBT368 on procapsid assembly, we inactivated individual or different combinations of the genes present on this construct (Figure 2(a)). The biological activity of structures produced in E. coli BL21(DE3)pLysS bearing the various constructs depicted in Figure 2(a) was measured using the in vitro assembly system. The system yields viable phages upon combination of the prohead donor extract under analysis with an extract of B. subtilis cells infected with the procapsid defective mutant SPP1sus115sus7sus31, which is de®cient in genes 6, 11, and 13 (DroÈge & Tavares, 2000). E. coli BL21(DE3)pLysS/pBT368 extracts led to the production of between 106 and 107 pfu/ml in vitro (Table 2; DroÈge & Tavares, 2000). Inactivation of the scaffolding or coat protein encoding genes from pBT368 abolished this activity almost completely (Table 2) con®rming that both proteins are required for formation of the procapsid structure (Becker et al., 1997). Deletion of gene 6 from the original pBT368 construct caused a ca. 300-fold reduced in vitro activity (Table 2). For cloning convenience genes 4 and 5 were also deleted from construct pBT369 used to assay the role of gene 6. Deletion of genes 4 and 5 from other constructs (e.g. pBT373; Table 2) or from the SPP1 phage genome (unpublished results) demonstrated that the reduction in biological activity was associated speci®cally to the absence of gene 6 in plasmid pBT369. The lack of gp6 during prohead assembly was not compensated by addition of puri®ed portal protein to the in vitro reaction (concentrations between 0.01 and 1 mg/ml were tested; data not shown) most likely because it cannot be incorporated on the procapsid structure after formation of the icosahedral lattice. Substitution of SPP1sus115sus7sus31 by SPP1sus7sus31 for preparation of the prohead de®cient extract, which in this case con-

tains free gp6 encoded by the infective phage, did also not increase phage yield in vitro (data not shown). This yield was, however, reproducibly tenfold higher than the background or the titer obtained when constructs lacking the scaffold or the major capsid genes were tested (Table 2). This might re¯ect that a basal level of procapsid assembly is occurring in vitro when extracts are mixed. The small amount of gp6 required for this reaction, presumably not the limiting factor, would be contributed by revertants of the mutation in gene 6 (sus115). Procapsids produced in the absence of gp7 (pBT370, PBT373) were ®ve- to tenfold less active in the in vitro assembly reaction than pBT368 derived structures (Table 2). This reduction is considerably lower than those caused by absence of gp6, gp11 or gp13. Our characterization was limited to SPP1 proteins found in procapsid structures. Five other polypeptides are coded by pBT368 (Figure 2(a)). gp15 was implicated in stabilizing the capsid structure after DNA packaging (Becker et al., 1997). Deletion of genes 4, 5 and 12 of the phage genome does not affect phage growth (DroÈge et al., unpublished results; see above). The function of gp14 remains unknown. Gp11 and gp13 are required for formation of procapsid-like structures Coomassie blue stained SDS-PAGE (Figure 2(b)) and Western blot analysis (data not shown) con®rmed that the various constructs depicted in Figure 2(a) led to production of the expected SPP1 proteins. The exception is gp11 found reproducibly in lower amounts when gp13 is not co-produced (Figure 2(b); data not shown). To investigate the effect of inactivating individual genes on procapsid formation, extracts of the overproducing strains were treated with 10 %

Assembly of Bacteriophage SPP1 Procapsid

121

Figure 2. Expression of the SPP1 procapsid gene clusters from a recombinant plasmid in E. coli. (a) Physical map of the different constructs used for co-production of different combinations of procapsid proteins. The translation products of the DNA fragment are represented on top. The construct pBT368 carries all identi®ed genes coding for SPP1 procapsid proteins (DroÈge & Tavares, 2000). Please note that due to cloning the order of the genes 4 to 7 and 11 to 15 in pBT368 is opposite to the one found in the SPP1 genome (Becker et al., 1997). The inserts cloned in the constructs represented below pBT368 are aligned relative to the pBT368 insert. The full lines indicate SPP1 DNA and the dotted line represents vector DNA. Restriction sites relevant for the cloning procedures are indicated. Sites that were eliminated as a consequence of cloning are crossed out. The BamHI site shown within brackets was generated by PCR. Abbreviations: A, Asp718; B, BamHI; Bc, BclI; Bs, BsiWI; Bsm, BsmBI; E, EcoRI; H, HpaI (only the site relevant for cloning is indicated); N, NruI; S, SalI; Sm, SmaI; X, XhoI. (b) Synthesis of proteins in E. coli BL21(DE3)pLysS carrying the plasmids indicated above each individual gel lane. The position of SPP1 proteins shown in the left is identi®ed by comparison to the pattern of puri®ed SPP1 virions (lane SPP1wt) and procapsids (lane SPP1sus70). gp7 is not detectable because of its low abundance and partial co-migration with gp13. Protein extracts of E. coli induced cells were separated via SDS-15 % PAGE and stained with Coomassie blue. The migration of Mr standard proteins is shown on the right.

(v/v) polyethylene glycol (PEG) 6000 to precipitate the major bulk of aberrant structures formed under overexpressing conditions (DroÈge & Tavares, 2000). Procapsids concentrated by ultracentrifugation were sedimented through 10 %-30 % glycerol gradients. Biologically active procapsids formed in B. subtilis cells infected with the terminase de®cient

mutant SPP1sus70 (gp1ÿ) (Becker et al., 1997) and in the overproducing strain bearing pBT368 sediment in the middle of the gradient under the running conditions used (fractions 6 to 8 of 13 fractions collected in total; DroÈge & Tavares, 2000). Figure 3 shows a comparison of the material found in the procapsid peak fraction 6 of gradients separ-

122

Assembly of Bacteriophage SPP1 Procapsid

Figure 3. Analysis of the structures produced in E. coli BL21(DE3)pLysS bearing the plasmids coding for different combinations of procapsid genes (Figure 2(a)). Protein extracts prepared from the transformed strains were cleared from the bulk of aberrant major head material by PEG precipitation and sedimented through 10 %-30 % glycerol gradients as described (DroÈge & Tavares, 2000). (a) Coomassie blue stained SDS-PAGE of the enriched procapsid fraction (fraction 6) of each gradient. Standards and symbology is as in Figure 2(b). (b) Agarose gel electrophoresis (1.75 %) of the same fractions stained with Coomassie blue. Discrete bands correspond to procapsid structures (Figure 4): pc, procapsids with normal size; spc, small procapsids.

ating structures derived from different plasmid constructs. The presence of procapsids is indicated by co-sedimentation of the procapsid proteins (Figure 3(a)) and by the presence of a de®ned band upon migration through 1.75 % agarose gels (Figure 3(b); see below). No prohead-related structures are produced in absence of gp13 (electron microscopy (EM), observations not shown; Figure 3), con®rming its role as the coat protein. Deletion of gene 11 leads to the formation of a variety of large gp13 aggregates often with a spiral-like appearance (data not shown) as observed in gene 11ÿ infections (Becker

et al., 1997). gp13 is spread all over the gradient explaining the low concentration of this protein in fraction 6 (Figure 3(a)). In all cases that gp11 and gp13 are co-produced (constructs pBT368;
4-6;
7;
4-7;
4,5,7) the two proteins co-sediment in fractions characterized by the enrichment of procapsid-like structures (data not shown). A signi®cant amount of the material present in these fractions co-migrates with puri®ed SPP1sus70 procapsids in agarose gel electrophoresis (Figure 3(b)). The SPP1 scaffolding protein thus appears to be suf®cient to direct the ef®cient polymerization of gp13 into closed icosahedral lattices.

Table 2. Biological activity of procapsids produced in E. coli BL21(DE3)pLysS bearing plasmid pBT368 and its derivatives Construct pBT368 pBT369 (
4-6) pBT370 (
7) pBT371 (
11) pBT372 (
13) pBT373 (
4, 5, 7) pBT374 (
4-7) pBluescript SK‡

Experimentsa 7 6 7 3 2 3 4 3

(5) (3) (5) (2) (2) (2) (2) (2)

Titer (pfu/ml)b

Titer (%)c

3.1  106  (3.2  106) 8.1  103  (8.7  103) 4.6  105  (3.1  105) 8.4  102  (3.5  102) 3.5  102  (1.5  103) 7.2  105  (3.1  105) 7.0  103  (6.1  103) <100

100 0.3 14.6 0.03 0.01 23.1 0.2 <0.003

(
), gene(s) inactivated in the construct Figure 2(a)). Protein extracts from the corresponding strains were prepared and tested in the in vitro assembly system as described (DroÈge & Tavares, 2000). The background, that is associated to the terminase donor extract, was between 5  102 and 103 pfu/ml. a Number of in vitro tests carried out. The value within parentheses indicates the number of extracts used that were prepared independently. b The mean of the titers obtained in the in vitro assays and their standard deviation are indicated. c Titers expressed as percentage were calculated based on the mean titers observed for the different constructs relative to the one obtained for construct pBT368 (100 %).

123

Assembly of Bacteriophage SPP1 Procapsid

gp6 is necessary to ensure correct procapsid size gp6 is the third component of the SPP1 procapsid, in addition to gp11 and gp13, necessary for formation of biologically active structures (Table 2). The in¯uence of the portal protein in procapsid assembly varies among phage species: it can be part of an initiator complex (l), de®ne the axis of elongation of prolate capsids (e.g. f29), or have no apparent effect (P22) in formation of the icosahedral structure (reviewed by Valpuesta & Carrascosa, 1994). The gp6 has a distinct effect in procapsid assembly. When SPP1 procapsid enriched fractions were separated in agarose gels one predominant band (pc-procapsid) and a faint one (spc-small procapsid; see below), corresponding to a faster migrating species, can be observed (Figure 3(b)). The spc band was signi®cant only in procapsid preparations missing the portal protein (
4-6 and
4-7; Figure 3(b)). Procapsids derived from constructs lacking gene 4 and 5 but not gene 6 did not exhibit this feature (
4, 5, 7). The spc band corresponded to material sedimenting slower than the material associated to the most abundant pc band which co-sedimented with biologically active procapsids (data not shown). The SPP1 major head and scaffolding proteins can thus coassemble into two types of structures. Presence of the portal protein directs the assembly reaction towards the particle with procapsid properties. In order to characterize the pc and spc structures we used procapsid material formed in B. subtilis cells infected with the portal-de®cient mutant SPP1sus115 (gp6ÿ). Enrichment of the spc band was also found in this case (Figure 4(a)). When the SPP1sus115 procapsids were analyzed by EM, procapsid-like structures of two different sizes were observed (Figure 4(b), lower panel). In contrast, a homogenous population of procapsids was found in case of SPP1sus70 (gp6‡). To test whether the large and small particles are the origin of slower and faster migrating bands on agarose gels, the two bands observed after electrophoresis of SPP1sus115-procapsids were puri®ed by electroelution (Figure 4(c)). EM analysis of the puri®ed material con®rmed that the small procapsids are enriched in the spc band (Figure 4(d)). These small structures have an outer diameter of approximately Ê while the larger procapsid species has the 410 A Ê also found for SPP1sus70 prodiameter of 550 A capsids (Figure 1). Kinetics of procapsid assembly are not influenced by the portal protein gp11 and gp13 co-assemble forming icosahedral structures with two sizes. In presence of gp6, the latter component is incorporated at a single vertex of the procapsid icosahedron ensuring a homogeneous overall shape of the structure. A possible mechanism to account for both features would be an initial interaction between gp6, gp11 and gp13

that favors the kinetics of procapsid assembly and directs the reaction towards formation of normalsized procapsids. The kinetics of procapsid assembly were analyzed during infection of non-permissive B. subtilis strain YB886 with the procapsid producing mutants SPP1sus70 (gp6‡) and SPP1sus115 (gp6ÿ). Production of procapsid particles during infection, monitored by agarose gel electrophoresis, was detectable at ten minutes postinfection and increased until the onset of host cell lysis at around 30 minutes (data not shown). Procapsids with normal size assembled at a similar rate during SPP1sus70 and SPP1sus115 infections leading to accumulation of an equivalent amount of structures (data not shown). The rate of SPP1 prohead assembly is thus apparently not affected by presence of the portal protein suggesting that a complex interaction between the procapsid proteins or a yet unknown factor, rather than a kinetic mechanism, ensures ef®cient incorporation of gp6 in the procapsid. The same feature was observed for bacteriophage P22 (Bazinet & King, 1988). Protein-protein interactions during procapsid assembly The interplay between the major capsid protein, the scaffolding protein and the portal protein is essential for formation of correctly shaped, biologically active procapsids. Incorporation of gp7 enhances signi®cantly the procapsid activity in the in vitro assembly system (Table 2). The molecular basis of the assembly process relies on the network of interactions carried out between these four proteins. We performed co-immunoprecipitation to identify their interaction partners in SPP1 infected cells (Figure 5) and con®rmed afterwards that these are direct interactions using extracts of E. coli producing selected SPP1 proteins (Figure 6). Immunoprecipitations from lysates of B. subtilis infected cells were carried out with speci®c antibodies directed against the four procapsid components (gp6, gp7, gp11, gp13). 35S- or 14Cradiolabeled lysates were prepared from infections with SPP1 wild type and with mutants that accumulate different intermediates of the SPP1 morphogenetic pathway in the host cytoplasm (Table 3). Immunoprecipitates were separated by SDS-PAGE and autoradiograms of these gels were analyzed to identify proteins co-precipitating with the antigens (Figure 5; Table 3). Phage proteins were identi®ed by comparison with the protein pattern of puri®ed SPP1 phages labeled with [35S]methionine residues and by their absence in extracts of SPP1sus mutants defective in production of individual capsid proteins. Co-immunoprecipitation requires strong interactions and that the antigen is accessible to the antibody, either at the surface of supramolecular complexes or free in the extract. Procapsid proteins co-immunoprecipitated selectively with one or more of the other prohead components among all the proteins present in cell extracts (Figure 5) con-

124

Assembly of Bacteriophage SPP1 Procapsid

Figure 4. Role of gp6 in procapsid shape. (a) Procapsids produced in cells infected with SPP1sus115 (gp6ÿ) and with SPP1sus70 (gp6‡), and procapsids assembled in E. coli BL21(DE3)pLysS bearing plasmids pBT368 and pBT369 (
4-6) were separated on a 1.75 % agarose gel and visualized by staining with Coomassie blue. (b) Negatively stained procapsids puri®ed from SPP1sus70 and SPP1sus115 infected cells. (c), (d) Separation of the species corresponding to the two bands found in agarose gels of SPP1sus115 procapsid preparations (a). Individual bands were electroeluted from agarose gels and re-electrophoresed. The 1.75 % agarose gel in (c) con®rms that the species separated exhibit the same electrophoretic mobility as the original material and that a good separation was achieved. Negative staining of the two samples (d) shows that these correspond to small (spc) and normal (pc) sized procapsids. The apparent loss of the scaffolding protein during the electro-elution procedure (d) did not affect the electrophoretic mobility of the structures (c). The bars in (b) and (d) represent 100 nm.

®rming the speci®city of the interactions detected. Anti-gp13 antibodies immunoprecipitated also some SPP1 full heads and virions (Figure 5(d); data not shown). The results showed two clear proteinprotein interactions detectable on all infections where both interaction partners were present: gp6gp7 and gp11-gp13. The gp6-gp7 interaction occurs in absence of other procapsid components (SPP1sus7sus31 infected cells; Figure 5(a) and (b)) and when the two proteins are produced in E. coli (Figure 6(a)). gp6 and gp7 co-immunoprecipitate also after mixing of extracts containing each individual protein (Figure 6(a)) suggesting a direct protein-protein interaction. gp11 and gp13 interact in infected cells (Figure 5(c); Table 3) and when co-produced in

E. coli (Figure 6(b)). (gp11 is not detected in the autoradiogram of Figure 5(d) because it contains a single methionine residue that can be labeled with 35 S but its co-immunoprecipitation with gp13 is evident when 14C-labeling is used, Table 3). The two proteins, however, do not interact when synthesized independently and the extracts of the overproducing strains are combined together (Figure 6(b); Becker et al., 1997). Immunoprecipitation with anti-gp6 antibodies showed some co-precipitation of gp6 and gp13 in absence of the scaffolding protein but this interaction was not con®rmed by the reciprocal experiment with anti-gp13 antibodies (Table 3). No detectable interaction was observed between gp6 and gp11. When gp6, gp11 and gp13 are co-produced we can detect a clear co-immunoprecipita-

Assembly of Bacteriophage SPP1 Procapsid

125

Figure 5. Immunoprecipitations from extracts of SPP1 infected cells with polyclonal sera raised against the procapsid proteins ((a) gp6, (b) gp7, (c) gp11 and (d) gp13). 35Slabeled extracts of B. subtilis Prot1 and of the strain infected with the SPP1 phages indicated on top of the lanes were used for immunoprecipitation as described in Materials and Methods. SPP1 wild type is identi®ed by wt and the other phages are SPP1 suppressor sensitive mutants whose phenotype is described in Table 3. Immunoprecipitates were separated via SDS-15 % PAGE and labeled proteins were visualized by autoradiography. The position of SPP1 proteins shown in the left is identi®ed by comparison to the pattern of puri®ed SPP1 virions labeled with 35S. This assignment is further con®rmed by the absence of those proteins in extracts of cells infected with SPP1 mutants de®cient on their synthesis.

tion of all three proteins with antibodies raised against any of the three (Figure 5, Table 3; data not shown). This suggests that a stable interaction with gp6 requires presence of the two other proteins. Interestingly, when an amino terminus fragment of gp6 produced during infection with SPP1sus115 is immunoprecipitated a small amount of gp13 is coprecipitated (Figure 5(a)), hinting that the gp6 amino terminus participates in the interactions with the procapsid proteins.

Discussion The icosahedral head shell of bacteriophage SPP1 is formed by a single protein, the major capsid protein gp13, as with most isometric bacteriophages and viruses. Protein stoichiometry calculations assuming a 13mer portal protein (Dube et al., 1993) result in a T number of about 6. This suggests a T ˆ 7 symmetry, implying that 415 coat protein subunits form the head shell lattice if the SPP1 capsid is constructed under the principles of the quasi-equivalence theory (Caspar & Klug, 1962). The SPP1 capsid has an outer diameter Ê ) comparable to the one found for other (660 A Ê ) and the T ˆ 7 phages (e.g. l and P22 with 630 A molecular mass of the coat subunits of these phages is within the same range (SPP1: 35.4 kDa; P22: 47 kDa; l: 38.2 kDa; Becker et al., 1997; Prasad et al., 1993; Dokland & Murialdo, 1993). The mass of DNA packaged within these capsids, normally found to be approximately correlated with the capsid inner volume, is also very similar among the three viruses. Like the majority of icosahedral tailed phages, SPP1 requires three different proteins for assembly of biologically active procapsids: the capsid protein, a scaffolding protein, and a portal oligomer located at the unique DNA translocating capsid vertex (reviewed by Casjens & Hendrix, 1988). In addition, SPP1 procapsids contain one to two copies of the accessory protein gp7 localized very probably in the portal vertex because it binds strongly to gp6 (Figures 5(a) and (b) and 6(a)). gp7

presence increases the biological activity of SPP1 procapsids ®ve- to tenfold by an unknown mechanism (Table 2). Detailed analysis of gp7 function during viral infection was mainly hampered by the lack of SPP1 mutants defective on its synthesis (Becker et al., 1997). This limitation was recently overcome by engineering of a gp7ÿ mutant (unpublished results) whose characterization is presently being carried out. Phenotypes associated with the absence of any of the procapsid proteins and study of their interactions (Figure 7) revealed an overall similarity between SPP1 and other phage systems but highlighted also particular features of SPP1. The most relevant of these features is the ®nding that the SPP1 portal protein drives the assembly reaction towards formation of T ˆ 7 structures while, in analogy to phage P22, the rate of assembly or correctness of major head protein polymerization into a closed lattice is not affected by its absence. Lack of the portal protein during morphogenesis of the prolate B. subtilis phage f29 leads to formation of procapsids with different size and shape (Hagen et al., 1976; Guo et al., 1991), a more drastic phenotype than in SPP1. Another particularity of SPP1 is the presence of the accessory protein gp7 which apparently has no functional analogue in other well-characterized phages. Assembly of the procapsid icosahedral lattice: the gp11-gp13 interaction The gp13 has the inherent capacity to polymerize into curvilinear structures. Since gp13 does not form detectable amounts of pentamers or hexamers in solution (Becker et al., 1997), it is probable that the SPP1 procapsid is assembled by a polymerization reaction of major capsid protein monomers, as described for bacteriophage P22 (Prevelige et al., 1988, 1993), rather than by joining of preformed pentamers (and hexamers in some cases) as proposed for phage HK97 and eukaryotic viruses (Xie & Hendrix, 1995, and references therein). Ef®cient polymerization of the coat protein into closed

126

Figure 6. Co-immunoprecipitation of (a) gp6-gp7 and (b) gp11-gp13. Immunoprecipitations were carried out as described in Materials and Methods. Lysates were prepared from E. coli BL21(DE3)pLysS bearing plasmids that lead to overproduction of the procapsid proteins indicated on top of each individual lane: gp6 (pBT376); gp7 (pBT377); gp6 and gp7 (pBT375); gp11 (pBT386), gp13 (pBT385), gp11 and gp13 (pBT374), gp6, gp7, gp11, gp13 (pBT368). In the cases indicated, extracts of cells producing individual proteins were mixed in equal amounts and used for immunoprecipitations as described. ``pBluescript SK ‡ `` is the strain bearing the plasmid without any phage DNA insert (negative control). Proteins were identi®ed (right side) by immunodetection with polyclonal antibodies. The strong band below gp6 in (a) corresponds to IgG heavy chains of the antibody used for immunoprecipitation. The precipitating antibodies are identi®ed below the Western blots.

lattices requires co-assembly with the scaffolding protein. Closed lattices are the very predominant gp13 polymerization products found when gp11 and

Assembly of Bacteriophage SPP1 Procapsid

gp13 interact in the cytoplasm of B. subtilis cells infected with a SPP1 mutant de®cient in the portal protein. Co-expression of genes 11 and 13 outside the infected cell by a recombinant plasmid in E. coli yielded similar closed icosahedral lattices but also numerous spiral-like and curvilinear structures (Becker et al., 1997; DroÈge & Tavares, 2000) that resemble gp13 polymerization products formed in absence of gp11 (Becker et al., 1997; Figure 7(a)). This effect is probably due to an increase of the gp13:gp11 ratio in E. coli. A stoichiometric requirement for gp11 to regulate gp13 polymerization seems conceivable. An optimal gp13:gp11 intracellular ratio would thus be important for correct procapsid assembly if the SPP1 scaffolding protein is not recycled (participation of the phage P22 scaffolding protein in several rounds of procapsid assembly was demonstrated by King & Casjens (1974)). The scaffolding protein is thought to interact with the capsid protein subunits to keep their spatial orientation and to direct the growing structure curvature as the polymerization reaction proceeds towards a closed lattice (Prevelige et al., 1988, 1993 and references therein). Failure to achieve correct curvature yields spiral structures (Berger et al., 1994). The interaction of gp11 with gp13, on the other side, stabilizes or protects the scaffolding protein as signi®cant higher amounts of gp11 are found when it is co-produced with gp13 (Figure 2(b); data not shown). The requirement that gp11 and gp13 must be coproduced to permit their interaction (Figure 6(b)) suggests that the gp13 binding site(s) for gp11 is created by a transient conformation or becomes buried during gp13 uncontrolled polymerization. Greene & King (1994) showed that the P22 scaffolding protein binds selectively to the P22 major capsid protein in the procapsid lattice but not to aberrant polymerization products or expanded capsid shells. The interaction between the scaffold and major capsid proteins thus occurs ef®ciently only during productive co-assembly of the two proteins into closed lattices that retain exposed the binding sites for the scaffolding protein forming a metastable structure. Procapsid expansion would eliminate or render those sites inaccessible with concomitant release of the scaffolding protein preventing that it competes with DNA being packaged for the capsid inner space. This is one of the steps that ensures irreversibility of the morphogenetic pathway. The asymmetric portal vertex: gp6 incorporation and role in procapsid assembly Co-production of gp11 and gp13 leads to formation of closed icosahedral lattices with two different sizes, one population morphologically identical to SPP1 procapsids and a second one of smaller structures (Figures 3(b), 4 and 7(a)). The Ê) small procapsids size (approximately 410 A suggests a T ˆ 4 or T ˆ 3 geometry. The ability to form T ˆ 4 or T ˆ 7 procapsids depending on the

Table 3. Co-immunoprecipitation of procapsid proteins from cell extracts of B. subtilis YB886 cells infected with different SPP1 mutant phages Lysate

anti-gp6

Phage

Relevant genotype

Structures accumulated

SPP1sus7sus31

11ÿ, 13ÿ

Tails gp13-spirals**, tails Tails Procapsids, tails Portal-less procapsids, tails Full heads Mature virions

SPP1sus7 SPP1sus31 SPP1sus70

11ÿ 13ÿ 1ÿ

SPP1sus115 SPP1sus222 SPP1wt

6ÿ ?* -

Label

anti-gp11

anti-gp13

gp6

gp13

gp7

gp11

gp6

gp7

gp6

gp11

gp13

gp6

gp11

gp13

35

S

‡

ÿ

‡

n.d.

‡

‡

ÿ

ÿ

ÿ

ÿ

n.d.

(‡)****

14

C C 35 S

‡ ‡ ‡

(‡) ÿ ‡

‡ ‡ ‡

ÿ ÿ n.d.

n.d. n.d. ‡

n.d. n.d. ‡

n.d. ÿ ‡

n.d. ‡ (‡)

n.d. ÿ ‡

ÿ ÿ ‡

ÿ ÿ n.d.

‡ (‡)**** ‡

(‡)*** ‡ ‡

(‡) ‡ ‡

ÿ ‡ ‡

ÿ n.d. n.d.

ÿ (‡) ‡

‡ ‡ ‡

ÿ ‡ ‡

(‡) (‡) ‡

‡ ‡ ‡

ÿ ‡ ‡

‡ n.d. ÿ

‡ ‡ ‡

14

35

S,

14

35

35

anti-gp7

S,

S

C

14

C

The mutants used for infection, their relevant genotype and the main type of structures accumulated in the non-permissive host are shown. Infected cells were radioactively labeled with [35S]methionine/[35S]cysteine or with 14C-labeled amino acids as indicated. Please note that due to its amino acid composition (only one methionine residue, no cysteine residue) gp11 is only weakly labeled by [35S]methionine/[35S]cysteine. Small amounts of gp11 (as expected for co-precipitates) can therefore not be visualized with this label. The antibodies used for immunoprecipitation are shown in the top row. The second row lists the procapsid proteins analyzed for each antibody immunoprecipitation. ‡, Precipitation or co-precipitation of a protein; (‡), weak signals, but clearly above background; ÿ, the lack of signal; *, SPP1sus222 (Behrens et al., 1979) is a mutant de®cient in a not identi®ed gene required for phage tail morphogenesis (our unpublished results); **, structures described by Becker et al. (1997); ***, a truncated version of gp6 is detected (Figure 5(a)); ****, immunoprecipitation of gp13 minor amounts in extracts of cells infected with SPP1 carrying the sus31 allele, which is de®cient in gene 13, is due to basal reversion of that marker leading to some synthesis of gp13.

128

Assembly of Bacteriophage SPP1 Procapsid

Figure 7. (a) Assembly defects and (b) network of protein-protein interactions during SPP1 procapsid morphogenesis. (a) Structures formed by the procapsid proteins when all components are present in the cell cytoplasm or when one of those proteins is omitted (indicated on the left) are drawn schematically based on EM data (Becker et al. 1997; this paper). These structures were found in B. subtilis cells infected with different SPP1 mutants and in E. coli producing different combinations of procapsid proteins. The exception are particles assembled in absence of gp7 which were produced only in the E. coli system. The expected position of proteins in the procapsid structure is indicated. The capacity of those structures to yield infective virions on our in vitro assembly system (Table 2) is shown on the right. (b) Our current understanding of the protein-protein interactions during SPP1 procapsid assembly. Thick black bars represent strong interactions detected both in vivo and in vitro. Thick gray bars represent interactions observed only when the proteins are co-produced, and the dotted line indicates a potential interaction between a gp6 amino terminus fragment and gp13 in infected cells. Arrows indicate proteins participating in formation of the stable complexes represented inside the boxes. These complexes were puri®ed by sedimentation (Figure 3) and/or by immunoprecipitation (Figures 6 and 7).

presence or absence of additional factors, has been shown for the coat proteins of l (Katsura, 1983; Katsura & Kobayashi, 1990), P2 (Lindqvist et al., 1993; Marvik et al., 1994) and P22 (ThumanCommike et al., 1998). Thuman-Commike et al. (1998) proposed that the coat proteins of all T ˆ 7 phages are intrinsically capable of forming capsids with both T numbers. The scaffolding (P2, P22) and the coat protein (l) were previously shown to play a role in directing assembly towards T ˆ 7. We now report that in case of SPP1 a third procapsid component, the portal protein, has a determinant effect on the gp11-gp13 co-polymerization reaction to form normal sized procapsids (Figures 3, 4 and 7(a)). An in¯uence in procapsid shape was so far only demonstrated for the portal protein of the prolate phage f29, where isometric instead of elongated procapsids are formed in absence of the portal protein (Hagen et al., 1976; Guo et al., 1991). The SPP1 portal complex located at a single vertex of the procapsid icosahedron in¯uences the overall shape of the head shell. Its integration must thus occur at an early stage during procapsid assembly since the decision to form a T ˆ 7 or a smaller capsid is made after the assembly of the ®rst round of hexamers around the portal vertex (Thuman-Commike et al., 1998). This feature supports models in which the portal protein is part of an initiator complex that directs the subsequent copolymerization reaction of coat and scaffolding protein irradiating from the portal vertex (Bazinet & King, 1985; Valpuesta & Carrascosa, 1994). Presence of the SPP1 portal protein in the organizer site appears necessary to ensure that the coat pro-

tein assumes the local conformations in the lattice required to form a T ˆ 7 procapsid. The structural in¯uence of the portal protein is thus propagated throughout the complete icosahedral lattice during its formation. This mode of assembly could avoid restoring structural conditions (i.e. creation of new binding sites for gp6) that would allow for incorporation of additional portal proteins in other vertices of the growing polymer. The structural organization of the initiation complex in assembly of procapsids with T ˆ 7 geometry is unknown. The scaffolding and coat protein are essential components while the portal protein, when present, is incorporated at high ef®ciency in the icosahedral lattice, a requirement necessary to produce procapsids competent for DNA packaging. The mechanism underlying ef®cient recruitment of the portal protein to the initiation complex is not understood. In case of P22 and of SPP1 the portal protein is not required to initiate procapsid assembly and it does not affect the assembly kinetics (Bazinet & King, 1988; this paper). It is possible that incorporation is achieved by the excessive production of the portal protein in case of SPP1 infections (Tavares et al., 1995). The scaffolding protein was proposed to be the organizer of the procapsid initiation complex (Bazinet & King, 1985) based on experimental evidence that it interacts with the portal protein (P22: Bazinet & King, 1988; Greene & King, 1997; T4: Traub et al., 1984; Kuhn et al., 1987; f29: Lee & Guo, 1995) and with the coat protein (Murialdo & Becker, 1978). We could only detect an interaction of the SPP1 portal protein with gp11 and gp13 when all three proteins were co-produced in vivo

129

Assembly of Bacteriophage SPP1 Procapsid

(Figures 5, 7(b)). This does not exclude that the proteins bind sequentially (e.g. several bi-molecular reactions) but shows that formation of a gp6gp13-gp11 complex is necessary to observe a stable interaction and that it occurs before extensive copolymerization of gp11 with gp13 (see above). Knowledge of the organization of this complex and of the dynamic interactions between its components will be invaluable to understand the mechanisms of procapsid assembly, expansion, and release of its scaffold. Presence of the portal protein in the initiation complex appears to add ®delity to the subsequent gp11-gp13 polymerization reaction. It also confers asymmetry to the initiation complex within the icosahedral lattice that provides the procapsid with a specialized vertex serving as a target of choice for interactions that trigger overall effects in the procapsid structure, to support DNA packaging, to measure the level of DNA head®lling, and for attachment of the phage tail (Tavares et al., 1995).

Materials and Methods Bacterial strains, bacteriophages and plasmids Phage and bacterial strains were as described by (Behrens et al., 1979; Chai et al., 1992; Tavares et al., 1992; Becker et al., 1997; DroÈge & Tavares, 2000). The prototrophic strain B. subtilis Prot1 was constructed by transformation of B. subtilis YB886 with chromosomal DNA from B. subtilis 168T‡. In a ®rst step the transformed strain was selected for methionine prototrophy in minimal medium plates. The met‡ strain was transformed with B. subtilis CU1965 chromosomal DNA and now selected for tryptophan prototrophy. The isogenicity of Prot1 to YB886, including the absence of the defective phage PBSX and of the SPb prophage, was con®rmed as described (DroÈge & Tavares, 2000). The plasmids used were pBluescript SK ‡ /ÿ (Stratagene, Heidelberg) pLysS (Studier et al., 1990), pBT376 (gene 6 cloned in pBluescript SK; Jekow et al., 1999), pBT368, pBT369 (
4-6), pBT374 (
4-7), and pBT382 (DroÈge, 1998; DroÈge & Tavares, 2000). Microbiological, genetic procedures and DNA manipulations All microbiological, genetic and cloning procedures were performed as previously described (Chai et al., 1992; Tavares et al., 1992). Correctness of recombinant plasmids generated by PCR cloning was con®rmed by DNA sequencing. Construction of new plasmids Plasmid pBT370 (
7) was generated by sub-cloning a fragment XhoI-BsmBI from pBT368, which carries genes 11 to 15, 4, 5 and most of gene 6 (Figure 2(a)), in a pBluescript SK‡ derivative carrying gene 6 (Jekow et al., 1999) that was cleaved with XhoI and BsmBI. The construct is isogenic to pBT368 except for deletion of most of gene 7 (Figure 2(a)). Plasmid pBT371 (
11), which lacks the 50 region of gene 11 (Figure 2(a)), was constructed by cleavage of

pBT368 with XhoI and partial digestion with SalI followed by re-ligation of the resulting 8.4 kb fragment. Gene 13 was inactivated by cleavage of pBT368 with BsiWI followed by ®lling in with Klenow polymerase (Sambrook et al., 1989), and religation. The insertion of four nucleotides shifts gene 13 out of frame shortly after its 50 end. The resulting construct is pBT372 (
13). Plasmid pBT373 (
4, 5, 7) (Figure 2(a)) was constructed by sub-cloning a PstI-XbaI-fragment carrying gene 6 from plasmid pBT376 (Jekow et al., 1999) into the same restriction sites from plasmid pBT374. Plasmid pBT375 (
11-15) was constructed by subcloning a 3.1 kb EcoRI (partial digest)-PstI fragment from plasmid pBT382 (DroÈge & Tavares, 2000), carrying genes 4 to 7, into pBluescript SK-cleaved with the same enzymes. Plasmid pBT377, expressing gene 7, was constructed by sub-cloning an Asp718-BamHI fragment from plasmid pBT368 in pBluescript SK- cleaved with the same endonucleases. Plasmid pBT385, expressing gene 13, was constructed by sub-cloning a 1.1 kb SmaI-EarI fragment from plasmid pBT374 in pBluescript SK- cleaved with HincII and EcoRV. The correct orientation of the insert was con®rmed by restriction analysis. Plasmid pBT386, expressing gene 11, was constructed by cleavage of pBT374 with SmaI and re-ligation of the plasmid. Purification of SPP1 particles and head-related structures SPP1 procapsids, full heads and phages were produced and puri®ed as described by Becker et al. (1997). For the preparation of 35S-labeled phages an overnight culture of Prot1 was diluted 1:50 in modi®ed MIII medium (composition adapted from Esche et al. (1975): 10 mM (NH4)2SO4, 8 mM K2HPO4, 4.1 mM KH2PO4, 0.15 mM MnSO4, 31 mM MgSO4, 0.15 mM FeCl3, 10 mM sodium citrate, 10 mM asparagine, 0.7 % (w/v) glucose) and grown to an optical density equivalent to 108 cfu/ ml. After supplementation with 10 mM CaCl2 the culture was infected with SPP1 wild-type (input multiplicity of ten). Ten minutes after infection, 1 mCi 35S-Translabeling Mix (ICN, Eschwege) was added to the culture. After two hours the cells were lysed by addition of 1 mg/ml lysozyme and 2 % (v/v) chloroform. The labeled phages were precipitated with PEG/NaCl and puri®ed by centrifugation through a caesium chloride gradient as described for bacteriophage l (Sambrook et al., 1989). Production of procapsid-like structures in E. coli and subsequent puri®cation was carried out as described (DroÈge & Tavares, 2000). Quantification of SPP1 procapsid proteins Highly puri®ed SPP1 phages and 35S-labeled phages were incubated for 30 minutes at 55  C in presence of 50 mM EDTA to disrupt virions (Tavares et al., 1996). After addition of 100 mM MgCl2, DNA was digested overnight at 37  C with ten units of benzonase (Merck, Darmstadt). Protein samples of puri®ed procapsids and disrupted phages were boiled for three minutes in presence of SDS-PAGE-loading buffer and separated on long 15 % or 7.5 %-20 % gradient SDS-PAGE. Proteins visualized by Coomassie blue staining and autoradiography, in case of labeled proteins, were quanti®ed by densitometry on a Personal Densitometer (Molecular Dynamics). Data analysis was performed using the ImageQuant software

130 (Molecular Dynamics). Bands were encircled using the rectangle tool and their intensity quanti®ed by volume integration. Quanti®cation was carried out twice with at least two different gels. Characterization of head-related structures The morphology of procapsids and procapsid-related structures was investigated by EM, SDS-PAGE and agarose gel electrophoresis. For EM analysis, aliquots of the fractions collected from glycerol gradients were stained with 1 % (w/v) uranyl acetate as described by Steven et al. (1988). SDS-PAGE was performed usually with 20 ml aliquots of the gradient fractions separated on 15 % (w/v) polyacrylamide gels. Protein bands were visualized with Coomassie brilliant blue staining. Procapsid-related structures (20 ml aliquots of the gradient fractions) were resolved by gel electrophoresis on 1.75 % (w/v) agarose gels run at approximately 5 V/cm in TAE buffer (Sambrook et al., 1989). Electrophoresis was stopped after the bromophenol blue tracking dye had moved 6 cm in the gel. Gels were rocked gently at room temperature for ten minutes in 10 % (v/v) acetic acid and ten minutes in absolute ethanol. Gels were dried under vacuum without heat for at least three hours. Proteins were then stained for ten to 20 minutes in 0.4 % (w/v) Coomassie blue, 25 % (v/v) 2-propanol, 10 % acetic acid and destained in 25 % 2-propanol, 10 % acetic acid for 30 minutes to overnight. Subsequently gels were kept for several hours in 10 % acetic acid (Duda et al., 1995b). To compare the kinetics of procapsid formation in SPP1sus70 and SPP1sus115 infected cells, a 26 ml culture of YB886 was grown to an optical density equivalent to 108 cfu/ml. After addition of CaCl2 to 10 mM, the culture was split into two cultures of 13 ml that were infected with SPP1sus70 and SPP1sus115. Samples of 1 ml were taken at different time points post-infection, mixed with 0.5 ml of frozen TBT (0.005 % NaN3, 30 % glycerol) and frozen immediately in liquid nitrogen (Chai et al., 1992). After collection of the last sample, all samples were thawed and span down for ®ve minutes at 18,000 g. The pellets were resuspended in 100 ml of lysis buffer (500 mM NaCl, 1 % (v/v) NP-40, 50 mM Tris, 5 mM MgCl2, 1 mg/ml pepstatin, 1 mg/ml leupeptin, 100 mg/ml PMSF) and lysed by three cycles of freeze and thaw. Samples were treated with DNAase (Benzonase; Merck, Darmstadt) for ten minutes at 37  C. Cell debris was removed by spinning for two minutes at 18,000 g. Synthesis of procapsid proteins and formation of procapsid structures were monitored by SDS-PAGE (15 ml of sample) and agarose gel electrophoresis (20 ml of sample), respectively. Quanti®cation of procapsid proteins and procapsid structures was performed by densitometry of the corresponding bands in dried gels as described above for SPP1 procapsid proteins. Immunoprecipitations Immunoprecipitations were carried out in protein extracts prepared from B. subtilis cells infected with SPP1 and from E. coli expressing different combinations of SPP1 procapsid proteins. For preparation of protein extracts from infected B. subtilis cells, the non-permissive strain Prot1 was grown at 37  C in 10 ml of modi®ed MIII medium. When the culture reached an optical density equivalent to 108 cfu/ml, it was supplemented with 10 mM CaCl2

Assembly of Bacteriophage SPP1 Procapsid and infected with SPP1 wild-type or SPP1sus mutant phages (input multiplicity of 30). Twelve minutes after infection, 80 mCi of 35S-Translabeling Mix (ICN, Eschwege) or of 14C-labeled algal protein hydrolysate (ICN, Eschwege) were added. Chase of the radioactive label with an excess of Casamino acids or no chase yielded identical results in the experiments presented here. At 25 minutes after infection the cultures were gently mixed with 5 ml of ice-cold TBT (10 mM NaN3, 20 % glycerol). Cells were collected by centrifugation (ten minutes, 4  C, 8000 g) washed with ice-cold TBT, and resuspended in 300 ml resuspension buffer (50 mM glucose, 1 mM EDTA, 25 mM Tris (pH 8.0)). After incubation for ®ve minutes at room temperature, 0.5 ml of lysis buffer (500 mM NaCl, 1 % NP-40, 50 mM Tris, 5 mM MgCl2, 1 mg/ml pepstatin, 1 mg/ml leupeptin, 100 mg/ml PMSF) were added and the samples were kept on ice for 30 minutes with occasional mixing. Cell debris was removed by centrifugation (30 minutes, 4  C, 14,000 g). The supernatant was used either immediately for immunoprecipitation or was kept at ÿ20  C. Extracts of SPP1 infected cells (200 ml) were rocked for one hour at room temperature with 30 ml of protein ASepharose CL-4B (Pharmacia, Uppsala) (150 mg/ml in 100 mM Tris-HCl (pH 7.5)) to eliminate material binding non-speci®cally to the resin. Protein A-Sepharose beads were pelleted (20 seconds, room temperature, 10,000 g) and the supernatant was mixed with 10 ml of antiserum used for immunoprecipitation. After incubation for one hour, 30 ml of protein A-Sepharose CL-4B were added and the samples were rocked for one hour at room temperature. Protein A-Sepharose was pelleted (20 seconds, room temperature, 10,000 g) and the supernatant was removed. Pellets were washed three times with washing solution I (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.2 % Triton X-100, 5 mM MgCl2), twice with washing solution II (10 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.2 % Triton X-100, 5 mM MgCl2) and once with washing solution III (10 mM Tris-HCl (pH 7.5), 5 mM MgCl2). Pellets were resuspended in 2  SDS-PAGE sample buffer, heated for three minutes to 100  C, and proteins were resolved on SDS-15 % PAGE. Gels were stained with Coomassie blue, dried, and autoradiographed. For preparation of protein extracts of E. coli BL21(DE3)pLysS carrying the desired plasmids, the corresponding strains were grown at 37  C in 5 ml cultures of LB medium supplemented with 100 mg/ml ampicillin and 30 mg/ml chloramphenicol. Protein production was induced by addition of 5 mM IPTG when the cultures reached an optical density equivalent to 108 cfu/ml. Two hours after induction the cells were collected by centrifugation (ten minutes, 4  C, 8000 g) and resuspended in 500 ml of resuspension buffer. After ®ve minutes incubation at room temperature, 1 ml of lysis buffer was added. Samples were kept on ice for ten minutes, then 1 ml of DNAase (Benzonase, Merck) was added to reduce the viscosity of the lysate, and incubation was continued for additional 20 minutes. Cell debris was removed by centrifugation (30 minutes, 4  C, 14,000 g). The supernatant was used either immediately for immunoprecipitation or kept at ÿ20  C. The NaCl concentration in E. coli extracts was reduced to 150 mM by dilution with lysis buffer lacking NaCl. Immunoprecipitation was performed as described for B. subtilis extracts of infected cells with the exception that washing solution II was replaced by washing solution I. Detection of proteins after SDS-PAGE was performed by Western blot analysis using the ECL-Kit (Amersham) according to the protocol of the supplier.

Assembly of Bacteriophage SPP1 Procapsid

Rabbit polyclonal anti-sera (Becker et al., 1997) were used for all immunoprecipitations. Sera were preadsorbed with extracts of B. subtilis and E. coli cells to prevent cross-reaction with bacterial proteins. Extracts were prepared from cell cultures entering stationary phase and incubated with sera as described (Sambrook et al., 1989) except that in case of B. subtilis 1 mg/ml lysozyme was also added to ensure cell lysis. In vitro assembly reaction In vitro assembly assays were performed as described by DroÈge & Tavares (2000).

Acknowledgments This work was partially supported by grant PRAXIS/ PCNA/P/BIO/61/96 to P.T. and grant PB 96-0817 from DGCICYT to J.C.A.

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Edited by J. Karn (Received 13 September 1999; received in revised form 13 December 1999; accepted 13 December 1999)

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