Bacteriophage Φ29 Early Protein p17

Gene 17 of the Bacillus subtilis phage Φ29 is expressed early after infection, and it has been shown to be required at the very beginning of phage replication under conditions of low but not high multiplicity of infection. It has been proposed that, at the beginning of the infection, protein p17 could be recruiting limiting amounts of initiation factors at the viral origins. Once the infection process is established and the replication proteins reach optimal concentration, protein p17 becomes dispensable. In this paper we focused, on the one hand, on the study of protein p17 dimerization and the role of a putative coiled-coil region. On the other hand, we focused on its interaction with the viral origin-binding protein p6. Based on our results we propose that protein p17 function is to optimize binding of protein p6 at the viral DNA ends, thus favoring the initiation of replication and negatively modulating its own transcription.

Gene 17 of the Bacillus subtilis phage ⌽29 is expressed early after infection, and it has been shown to be required at the very beginning of phage replication under conditions of low but not high multiplicity of infection. It has been proposed that, at the beginning of the infection, protein p17 could be recruiting limiting amounts of initiation factors at the viral origins. Once the infection process is established and the replication proteins reach optimal concentration, protein p17 becomes dispensable. In this paper we focused, on the one hand, on the study of protein p17 dimerization and the role of a putative coiled-coil region. On the other hand, we focused on its interaction with the viral origin-binding protein p6. Based on our results we propose that protein p17 function is to optimize binding of protein p6 at the viral DNA ends, thus favoring the initiation of replication and negatively modulating its own transcription.
Processes like DNA replication, transcription, compaction, or recombination occur in all organisms, and all of them have to be regulated and coordinated in order to obtain biological activity. Factors involved in these regulations are found in both eukaryotes and prokaryotes. In eukaryotes there are proteins like HMG-1 (renamed HMGB1) and HMG-2 that enhance the DNA binding of a variety of proteins (1)(2)(3)(4). In prokaryotes, there are histone-like proteins that can function in global regulation, acting as transcriptional modulators in multiprotein complexes with DNA. For example, H-NS influences transcription of a number of genes involved in diverse biological processes (5)(6)(7)(8)(9). Another histone-like protein, HU, has been shown to stimulate the action of different DNA-binding proteins (10 -12), to repress promoters together with other proteins forming high order multiprotein complex (13), to participate in the initiation of DNA replication assisting other replication proteins (14), or to be involved in post-transcriptional regulation (15). Generally, regulation requires homo-and/or hetero-protein-protein interactions, and different domains involved in these associations have been described: coiled-coiled (16), the RING-B box coiled-coil, renamed recently TRIM (17), EHV1 motif (18), or the Eps15 homology domain (19).
Bacillus subtilis phage ⌽29 has a linear double-stranded DNA with a terminal protein (TP) 1 covalently linked to the 5Ј ends. ⌽29 DNA replication starts by recognition of the origins of replication, i.e. the TP-containing DNA ends, by a TP/DNA polymerase heterodimer (20). Protein p6 is a viral nonspecific DNA-binding protein that is involved in different DNA processes forming a nucleoprotein complex that plays an essential role in the initiation of ⌽29 DNA replication activating the replication origins (21,22). This complex has been shown to cover most of the ⌽29 genome, suggesting that p6 may also have a structural role in organizing the genome (23). Also, it has been demonstrated that protein p6 is involved in regulating the viral transcription; binding of protein p6 to the right genomic end represses transcription from the early C2 promoter (see Fig. 1) (24 -26). In addition, it cooperates with the regulatory viral protein p4 in the switch from early to late transcription (27,28); as a result, the A2b and A2c early promoters are repressed, and the late A3 promoter is activated. Thus, proteins like p4 or p6 help to coordinate and regulate the main viral processes. On the other hand, under conditions of limiting amounts of the viral DNA and replication proteins (DNA polymerase, TP, single-stranded DNA-binding protein, and protein p6), in vitro ⌽29 DNA amplification is stimulated by the presence of the gene 17 product, protein p17 (29). Gene 17, located at the right end of the ⌽29 DNA molecule (30), encodes a 19-kDa protein involved in the in vivo viral DNA replication. In fact, when a B. subtilis non-suppressor strain (su Ϫ ) was infected with the ⌽29 mutant sus17 (112), a reduced phage yield and viral DNA synthesis were observed (30 -32). Moreover, when the infection was in solid medium, ⌽29 mutant sus17 (112) showed a "leaky" phenotype, characterized by the late appearance of very tiny lysis plaques, suggesting that protein p17 can be partially dispensable (33). We have shown that gene 17 is dispensable under conditions of high m.o.i., but it is indispensable at low m.o.i., which are probably the natural conditions for infection (29).
The C2 promoter controls protein p17 transcription and is strongly inhibited by the viral histone-like protein p6, both in vivo and in vitro (24 -26). This fact is probably because of the formation of a p6-DNA nucleoprotein complex at the right end of the viral genome (34). Thus, protein p17 is synthesized very early after infection, and its synthesis declines later on due to the inhibition of the C2 promoter by protein p6. 1 The abbreviations used are: TP, terminal protein; m.o.i., multiplicity of infection; GGH, tripeptide glycine-glycine-histidine; DSS, disuccinimidyl suberate; IPTG, isopropylthio-␤-D-galactoside; wt, wild type; PVDF, polyvinylidene fluoride; OD, oligomerization domain; DBD, DNA binding domain; M w,a , apparent weight average molecular weights.
At late times after ⌽29 infection, the number of copies of protein p6 in B. subtilis cells has been calculated to be enough to cover the entire viral DNA progeny (35). The self-association ability of protein p6 was studied by analytical ultracentrifugation at the in vivo protein p6 concentration. Protein p6 shows a monomer-dimer equilibrium that shifts to higher order association at the highest concentrations. These oligomeric structures have been proposed to act as a scaffold for the DNA into the appropriate configuration (36).
We were interested in analyzing the role of p17 at early infection times, when protein p6 is still limiting in the host cell. In these conditions, protein p17 seems to be required for viral DNA replication (29). To understand further the function of protein p17, we studied its capacity to self-interact and to bind to the viral DNA-binding protein p6 under conditions that can parallel those of the beginning of infection: limiting amount of protein p6 and high amount of protein p17.
In this paper we show that protein p17 self-associates both in vivo and in vitro, independently from other viral or host factors. Protein prediction suggests the existence of a 28-amino acidlong coiled-coil region, possibly involved in in vivo self-association. We also show that protein p17 interacts with the viral protein p6, facilitating the binding of protein p6 to the ⌽29 DNA ends, thus probably favoring the initiation of replication and negatively modulating its own transcription.

EXPERIMENTAL PROCEDURES
Materials-GGH cross-linking agent was purchased from Sigma, and DSS was from Pierce. Pre-stained high molecular weight protein markers were from Invitrogen. IPTG and ampicillin were from Sigma. Restriction enzymes HindIII, EcoRV, and BamHI, T4 ligase, Vent polymerase, and DNase I were from New England Biolabs; oligonucleotides were from Genset SA; dNTPs, [␣-32 P]dATP (3000 Ci/mmol), and [␥-32 P]ATP (3000 Ci/mmol) were from Amersham Biosciences.
In Vivo Protein p17 Cross-linking-B. subtilis 110NA cells were grown at 30°C in LB medium to an A 560 of 0.4 and infected with wt ⌽29 at m.o.i. 5 as described (29). Infected cells were harvested 30 min after infection, washed once with 50 mM Hepes, pH 8.0, and concentrated 20-fold in the permeabilization buffer (50 mM Hepes, pH 8.0, 10 mM EDTA, pH 8.0, 20% sucrose). Cross-linking reaction was carried out in 100 l with DSS at the indicated concentration, incubated for 20 min at room temperature, and quenched with 50 mM Tris-HCl, pH 7.5. After addition of 25 l of SDS-PAGE loading buffer (38), samples were sonicated, and 15 l of each reaction mixture was run onto 12% Tris-glycine SDS-PAGE to be electrophoretically transferred to PVDF membrane (Immobilon-P, Millipore) and probed with polyclonal antibody ␣p17 as described (29).
Construction of Phage cI Repressor Fusions-Plasmid pBF21 containing the cI gene under control of a tandem lacUV5 promoteroperator region (39) was digested with HindIII and EcoRV to remove the cI gene fragment encoding for the oligomerization domain (OD). Plasmid p⌬cI17w and mutants p⌬cIL70A, p⌬cIL70R, and p⌬cIL84P were constructed by PCR from gene 17 cloned in plasmid pET17 (29). A silent mutation in gene 17 was introduced to eliminate an internal HindIII restriction site in a two-step PCR procedure. PCR conditions are as follows: 1 M oligonucleotides, 0.1 g of plasmid pET17 as template, 100 M dNTPs, and 2 units of Vent polymerase on its reaction buffer. Gene 17 was first amplified in two steps as follows: the first step with primer 5ЈG17 (TAGGAaagcttACACATGAATAACTAT) containing a HindIII external restriction site, and 3ЈconsLys (CCTGGCTACAAG-TTTATTGATCTC) having a single nucleotide substitution to eliminate the HindIII restriction site internal to the gene; the second step with primer 5ЈconsLys (GAGATCAATAAACTTGTAGCCAAG) having a single mutation to substitute the HindIII site internal to the gene, and TG17 (GTTGTAACGgatatcTTACTTGTT) to introduce an EcoRV external site. Fragments were purified from agarose gel and used in a second step of PCR with external primers 5ЈG17 and TG17. The PCR product, consisting in gene 17 lacking internal HindIII site, was purified from agarose gel, and after digestion with HindIII and EcoRV was cloned into digested pBF21, to generate plasmid p⌬cI17w, that contains a chimerical gene encoding for the DNA binding domain from the cI repressor cI(DBD) fused with the gene coding for protein p17. Gene 17 point mutants of lysine at positions 70 and 84 were obtained with the same two-step PCR procedure, using the following internal primers: for mutant L70A, primers L70A (GTTGAAGAGGCAGGCAC-ACAA) and L70Ac (TTGTGTGCCTGCCTCTTCAAC) were used; for mutant L70R, primers L70R (GTTGAAGAGCGAGGCACACAA) and L70Rc (TTGTGTGCCTCGCTCTTCAAC); and for mutant L84P, primers L84P (TTAGAAGATCGAGACGGTGAAA) and L84Pc (TTTCACC-GTCTCGATCTTCTAA). Amplified fragments were cloned as described above for gene 17 lacking the internal HindIII restriction site.
Phage Development-Escherichia coli 71-18 lacIq cells were electrotransformed with the plasmids described above and grown at 37°C up to A 620 of 0.6 in LB medium containing ampicillin (100 g/ml). The production of chimeric proteins was induced by addition of 250 l of 100 mM IPTG to 0.4 ml of culture. After induction, cells were infected with 146 hypervirulent phage (40) essentially as described (39). At the indicated times, 0.4-ml aliquots were centrifuged, and the plaque-forming unit/ml was determined in each supernatant using E. coli 71-18 lacIq as recipient strain.
Sedimentation Equilibrium Analysis-The experiments were performed in a Beckman Optima XL-A analytical ultracentrifuge using an An60Ti rotor. Protein p17 was equilibrated in 50 mM Tris-HCl, pH 7.5 (90 l of 70 M), and centrifuged at 15,000 rpm, and absorbance scans at 280 nm were taken at sedimentation equilibrium. The equilibrium temperature was 4°C. High speed sedimentation (42,000 rpm) was conducted afterward for base-line correction.
Whole-cell apparent weight average molecular weights (M w,a ) were determined by fitting a sedimentation equilibrium model for a single sedimenting solute to individual data sets with the program EQASSOC (supplied by Beckman Instruments; see Ref. 41). The partial specific volume of protein p17 was 0.737 ml/g, and the monomer relative molecular mass was taken as 19,173, both of them calculated from the amino acid composition of the protein, deduced from the gene 17 sequence (42). A value of 5,800 M Ϫ1 cm Ϫ1 was used for the extinction coefficient of protein p17 at 280 nm (43).
In Vitro Cross-linking-Purified protein p17 in phosphate buffer, pH 7.0 (15 M final concentration), was incubated in 50 l containing 100 M GGH-Ni(II) as cross-linking agent, at the indicated NaCl concentrations, for 5 min at room temperature. The reaction was quenched with 20 mM Tris-HCl, pH 7.5, and, after addition of SDS-PAGE loading buffer, loaded onto 12% SDS-PAGE gel, and after electrophoresis stained with Coomassie Blue. When cross-linking was performed in the presence of protein p6 (12 M final concentration, equimolar to that of protein p17), the gel was electrophoretically transferred to PVDF membrane (Immobilon-P, Millipore TM ) and probed with polyclonal antibodies as described (29).
DNase I Footprinting-DNase I footprinting was carried out essentially as described (44). Right and left ⌽29 DNA terminal fragments, obtained from clones in pBluescript vectors (obtained from V. Gonzalez-Huici), were end-labeled at the 3Ј end by Klenow filling of BamHI protruding strand. In a final volume of 25 l containing 10 mM MgCl 2 , DNA fragments were added to protein p6 or to protein p6 preincubated with protein p17 at the indicated concentrations and incubated for 5 min at room temperature, before addition of 2 ng of DNase I to initiate the reaction. The digestion was allowed to proceed for 5 min at room temperature and stopped with 20 mM EDTA final concentration. DNA was ethanol-precipitated in the presence of carrier tRNA and run onto 8 M urea 6% PAGE.

RESULTS AND DISCUSSION
Protein p17 Self-association-The viral protein p17 is required in vivo to infect B. subtilis at low m.o.i. and shows a stimulatory effect on in vitro ⌽29 DNA amplification. As a step to understand protein p17 function, we have studied its oligomerization state. Extracts of B. subtilis cells infected with wt ⌽29 were collected and treated with DSS as cross-linking agent and analyzed by Western blotting with ␣ p17 polyclonal antibodies as described under "Experimental Procedures." Fig. 2 shows protein p17 dimers formation at increasing DSS concentration. In addition, higher molecular weight complexes containing protein p17 could be detected in the presence of increasing concentrations of DSS. The high molecular weight material could correspond to protein p17 homocomplexes, although we cannot rule out the formation of heterocomplexes with other proteins.
To check whether protein p17 self-association capacity was influenced by other viral and/or host components, we performed in vitro analytical ultracentrifugation using purified protein p17 (see "Experimental Procedures"). The theoretical value for the wt protein p17 monomer is 19,173, calculated from the amino acid composition of the protein deduced from the gene 17 sequence (42). Purified protein p17, at a concentration of 70 M, was shown to be a dimer in solution with an average molecular weight (M w,a ) of 39,304 Ϯ 400 (Fig. 3). Thus, we can conclude that protein p17 self-associates independently from other components.
The in vivo accumulated protein p17 was calculated to be 15,000 molecules at 30 min post-infection (29), which is almost 45 M intracellular protein p17 concentration taking into account the cell volume of 10 Ϫ15 liters determined (35). Our in vitro results at 70 M protein concentration demonstrate that protein p17 forms dimers, in agreement with the finding of dimers in vivo.
Data obtained by cross-linking with GGH-Ni(II) suggested that oligomerization of protein p17 was favored at high salt concentration. Fig. 4A shows in vitro increasing of p17 dimer as well as oligomer formation from 50 to 200 mM salt concentra- tion. To confirm this effect on protein p17 self-association, we performed sedimentation equilibrium studies at 50 and 200 mM NaCl. The results presented in Fig. 4B show that at 50 mM NaCl the best fit function (line) is for a single solute of a M w,a ϭ 44,400 Ϯ 200 and at 200 mM NaCl for a single solute of a M w,a ϭ 51,100 Ϯ 100. As NaCl favors self-association, we can sug-gest that protein p17 self-association is not dependent on charged residues but possibly on hydrophobic ones.
Residues Involved in Self-association of Protein p17-A secondary structure prediction program to look for coiled-coil domains (COILS) (16) revealed that the region of p17 spanning amino acids Leu 63 to Glu 92 has a high probability of forming a coiled-coil structure (see Fig. 5A). This structure is formed by repetition of seven amino acids, adopting an helical conformation, in which hydrophobic residues are arranged on one face of the helix (positions a and d of the repetition), forming a spine (see Ref. 16 for a review). The hypothetical coiled-coil region of protein p17 is shown in Fig. 5B. These types of structures (see also leucine zipper motifs) (45,46) are involved in proteinprotein interactions, homo-and/or hetero-dimerization, by the facing of hydrophobic spines belonging to two (or more) helices in a parallel or antiparallel orientation.
To test for the involvement of the coiled-coil region in protein p17 self-association, we analyzed its behavior in an in vivo system based on the cI repressor dimerization (39). The cI repressor is a protein that binds to DNA through a DNA binding domain (DBD) at the N terminus and dimerizes through an oligomerization domain (OD) at the C terminus. Dimerization is required for an efficient binding to the operator, so that  FIG. 7. In vitro cross-linking between proteins p17 and p6. Cross-linking reactions between pure proteins p17 and p6 were run on Tris-glycine 15% acrylamide SDS-PAGE, transferred onto membrane, then blotted with ␣p6 and, upon stripping, with ␣p17. The left panel shows the result of ␣p6 labeling, and the right panel shows that of ␣p17. Monomers of protein p17 and p6 are indicated with a bar, and the putative heterodimer and heterotrimer are indicated with an asterisk. E. coli cells carrying the cIwt gene are immune to hypervirulent phage 146 infection, whereas those carrying only the cI(DBD) are sensitive to the infection (see Fig. 6A). Substitution of cI(OD) with another protein provides an assay for its self-association in vivo. By using the plasmid pBF21 (39), expressing the cI repressor under control of the lac promoter, we constructed plasmid p⌬cI17w which encodes for a fusion protein containing the cI(DBD) at the N terminus and protein p17 at the C terminus. E. coli cells bearing plasmid p⌬cI17w were induced by IPTG to express the fusion protein and were assayed for hypervirulent phage 146 development. Cells expressing fusion protein ⌬cI17w were immune to phage infection; this result suggests that protein p17 was able to selfassociate efficiently in this in vivo system (see Fig. 6A). It is interesting to notice that phage development was slower in the case of fusion protein ⌬cI17w than in the case of the control cIwt, suggesting that substitution of the cI(OD) with protein p17 at the C terminus gave rise to the formation of a complex of high efficiency in repression. However, the mechanism by which this occurs is unknown. Expression of the fusion protein ⌬cI17w was confirmed by Western blot analysis using ␣p17 antibody (not shown).
In order to get more information on specific residues involved in the coiled-coil region of protein p17, we constructed point mutants to be tested in the in vivo system described above. Because hydrophobic residues of the helical turn have been shown to be responsible for protein-protein interaction (16), and because hydrophobic forces seem to be involved in p17 self-association (Fig. 4, A and B), we mutated Leu 70 into Ala and Arg and Leu 84 into Pro. Fig. 6B shows the time course development of phage 146 resulting from infection of E. coli cells expressing different mutant fusion proteins versus the fusion protein carrying wt protein p17 (⌬cI17w). Substitution of Leu 70 into Arg and Leu 84 into Pro showed a lytic phenotype. These results indicate that repression has not been carried out, suggesting that mutant protein self-association is affected. On the other hand, change of Leu 70 into Ala showed a phenotype similar to that found in cells carrying ⌬cI17w. Change of Leu 84 into Pro could destroy completely the helical coiled-coil structure and destabilize the protein local structure. The change of Leu 70 into Arg may alter the surrounding charges at the helical spine, making self-interaction more difficult, whereas change of Leu 70 into Ala seems not to have an effect in protein p17 self-association. These results suggest that Leu 70 and Leu 84 could be involved in protein p17 self-association.
Protein p17 Interacts in Vitro with the Viral Protein p6 -To further investigate protein p17 function, we studied its role in the viral infection system. It is known that (i) protein p17 is synthesized early from the C2 promoter; (ii) the viral histonelike protein p6 represses the C2 promoter and activates initiation of ⌽29 DNA replication by forming a nucleoprotein complex; and (iii) protein p17 stimulates in vitro phage DNA replication at low doses of DNA and initiation proteins (DNA polymerase, TP, single-stranded DNA-binding protein, and protein p6). To underlie the mechanisms by which protein p17 stimulates viral DNA replication, and because protein p17 does not bind DNA (not shown), we performed protein-protein in vitro cross-linking experiments using protein p17 and different proteins involved in ⌽29 initiation of replication. Equimolar concentrations of proteins p17 and p6 were cross-linked and analyzed by Western blotting as described under "Experimental Procedures." The same blotting was first analyzed with ␣p6 antibodies and then, after stripping, with ␣p17 antibodies to compare hetero-oligomers bands. Fig. 7 shows that when the two proteins are present, two extra bands are formed (indicated with an asterisk) that can be explained as the result of an interaction between proteins p6 and p17. It should be noticed that under the experimental conditions used, protein p6 migrates with an approximate molecular mass of 13 kDa (the theoretical one is of 11.8 kDa) and protein p17 as 23 kDa. The lower extra band of ϳ36 kDa can be due to the formation of an heterodimer between one molecule of protein p6 and one of protein p17. The upper extra band (ϳ48 kDa) could result from interaction of two molecules of protein p6 and one of protein p17. The presence of the hetero-complexes supports the idea of a direct interaction among proteins p17 and p6.
To investigate further the possible effect of protein p17 on protein p6 function, we performed DNase I footprinting experiments of protein p6 in the absence or in the presence of protein p17 with the left or the right viral DNA ends. As shown in Fig.  8, the presence of protein p6 at the viral DNA ends produces a characteristic pattern of DNase I-hypersensitive bands regularly spaced every 24 bp and protected regions in between. In the presence of protein p17 less amount of protein p6 is needed to obtain the characteristic pattern at both DNA ends. This result suggests that protein p17, through its interaction with protein p6, enhances its binding to the DNA.
Conclusion-In this study we show that protein p17 selfassociates both in vivo and in vitro. This association seems to involve hydrophobic forces and to occur through an ␣-helical coiled-coil sequence located between residues Leu 63 and Glu 92 . In addition, by using an in vivo system we showed that residues Leu 70 and Leu 84 , which are located at the hydrophobic d position of the heptad repeats, are essential for self-association. We have also demonstrated that protein p17 interacts with the viral histone-like protein p6 enhancing its DNA binding ability. Because it has been described that binding of protein p6 to DNA has a dynamic nature (23), we suggest that protein p17 could play an important role at the initial steps of replication (29) in favoring p6 binding to DNA, when its concentration is still limiting. This binding could help to activate viral DNA replication and also to repress the C2 promoter, giving rise to a negative autoregulation of protein p17.