Alterations of the Portal Protein, Gpb, of Bacteriophage Suppress

Alterations of the Portal Protein, gpB, of Bacteriophage ␭ Suppress Mutations in cosQ , the Site Required for Termination of DNA Packaging

Douglas J. Wieczorek,1 Lisa Didion and Michael Feiss Genetics Ph.D. Program and Department of Microbiology, University of Iowa, Iowa City, Iowa 52242 Manuscript received November 5, 2001 Accepted for publication February 18, 2002

ABSTRACT The cosQ site of bacteriophage ␭ is required for DNA packaging termination. Previous studies have shown that cosQ mutations can be suppressed in three ways: by a local suppressor within cosQ, an increase in the length of the ␭ chromosome, and missense mutations affecting the prohead’s portal protein, gpB. In the present work, revertants of a set of lethal cosQ mutants were screened for suppressors. Seven new cosQ suppressors affected gene B, which encodes the portal protein of the prohead. All seven were allele- nonspeciﬁc suppressors of cosQ mutations. Experiments with several phages having two cosQ suppressors showed that the suppression effects were additive. Furthermore, these double suppressors had minimal effects on the growth of cosQϩ phages. These trans-acting suppressors affecting the portal protein are proposed to allow the mutant cosQ site to be more efﬁciently recognized, due to the slowing of the rate of translocation.

ACTERIOPHAGE ␭ is a tailed, double-stranded head, forming the ternary DNA-terminase-prohead as- B DNA virus that serves as an important model for sembly known as complex II. ATP-dependent DNA assembly of large DNA viruses such as herpes and poxvi- translocation into the prohead follows complex II for- ruses. The mature ␭ chromosome is a linear chromo- mation; terminase is part of the translocation complex. some 48.5 kb in length with 12-bp cohesive ends at When the translocation complex encounters the next the 5Ј ends of the strands (Sanger et al. 1982). Upon cos along the concatemer, terminase again introduces injection into the cell, the cohesive ends anneal and staggered nicks at cosN, a step known as terminal cos- are ligated, circularizing the chromosome. The site of cleavage, followed by ATP-dependent DNA strand sepa- the annealed cohesive ends is part of cos, the cohesive ration. Packaging is thus terminated. Terminase un- end site that contains the DNA sites required for ␭ DNA docks from the DNA-ﬁlled head but remains bound to packaging. Early during infection, the ␭ chromosome the left end of the next chromosome in the concatemer, is replicated bidirectionally to produce a number of forming complex I, and sponsoring processive packag- progeny rings. Later, rolling circle replication and re- ing of the next chromosome along the concatemer. combination give rise to end-to-end multimers of ␭ chro- An average of two to three chromosomes are packaged mosomes called concatemers (Furth and Wickner processively by terminase (Emmons 1974; Feiss et al. 1983). Packaging of ␭ chromosomes from concatemeric 1985). Phage construction is completed with the addi- DNA leads to assembly of infectious virions. ␭ DNA is tion of accessory head and tail proteins to the DNA- recognized by the interaction of terminase, the DNA ﬁlled capsid. packaging enzyme, with cos. cos is composed of three subsites: cosN, cosB, and cosQ A model for ␭ DNA packaging is as follows (Feiss (Figure 1). cosN is the site where terminase’s gpA sub- 1986; Becker and Murialdo 1990; Catalano et al. unit generates the cohesive ends (Davidson and Gold 1995). Terminase, composed of gpNu1 and gpA, the 1992; Rubinchik et al. 1994; Hwang and Feiss 1996). large and small subunits, respectively, binds a randomly cosB, the terminase binding site, is located to the right chosen cos. Terminase introduces staggered nicks at the of cosN. cosB is required for the initiation of chromosome cosN subsite of cos, resulting in the nicked complex. packaging and contains three sequences, R1, R2, and Through an ATP hydrolysis-dependent process, termi- R3, which are bound by gpNu1 (Bear et al. 1983; nase separates the strands of DNA at the cos site, forming Shinder and Gold 1988). Between R3 and R2 is I1, a right and left chromosome ends, and remains bound binding site for Escherichia coli integration host factor to the left end forming the stable intermediate, complex (IHF). It is believed that the introduction of a sharp I. Complex I binds an empty shell precursor, the pro- bend at I1 by IHF facilitates cooperative interactions between terminase subunits bound to the R boxes of cosB (Kosturko et al. 1989; Mendelson et al. 1991; Xin 1Corresponding author: Department of Microbiology, University of Iowa, 3-315 BSB, 51 Newton Rd., Iowa City, IA 52242. and Feiss 1993; Xin et al. 1993). E-mail: [email protected] cosQ, located to the left of cosN, is necessary for the

Genetics 161: 21–31 (May 2002) 22 D. J. Wieczorek, L. Didion and M. Feiss

Figure 1.—Structure of cos and the terminase genes. The Nu1 and A genes encode the small (gpNu1) and large (gpA) subunits of terminase, W is an accessory protein involved in head- tail joining, B encodes the portal protein, and C is a capsid component involved in the production of the connector. cos is a tripartite structure consisting of cosQ, required for packaging termination, cosN, the nicking site, and cosB, required for packaging initiation. The three R sequences of cosB are gpNu1 binding sites. I1 is a binding site for E. coli integration host factor (IHF). The sequences of cosQ and cosN are shown at the bottom, denoted by boxes. cosN exhibits partial twofold rota- tional symmetry with the center of symmetry indicated by the dot. Numbering of the ␭ sequence and the positions of terminase nicking at cosN, N1, and N2 are also shown (reviewed in Catalano et al. 1995). termination of chromosome packaging. cosQ is a 7-bp or to be recognized more efficiently by the portal pro- segment with the sequence 5Ј GGGTCCT 3Ј (Wiecz- tein itself. orek and Feiss 2001). cosQ has no role in packaging ␭’s portal protein, gpB, is a 533-amino-acid protein, initiation, but mutations within cosQ cause defective of which the N-terminal 21 amino acids are cleaved to packaging termination (Cue and Feiss 1993). Cue and produce the mature protein, gpB* (Walker et al. 1982). Feiss (1998) showed that cosQ is required for the nicking Twelve subunits of gpB* are assembled into a dodecam- of the bottom DNA strand at cosNR. eric ring with a central channel. The ring forms the Cue and Feiss (1997) found several types of suppres- unique portal vertex of the prohead that is believed to sors of the leaky cosQ1 mutation. The first was a local be the site of DNA entry into the prohead and DNA suppressor, named cosQ2, affecting a base pair within exit during ejection, as well as the site for tail attachment the cosQ site. The second suppressor was an increase in and shell assembly (Tsui and Hendrix 1980; Kochan the length of the phage chromosome to near the capac- and Murialdo 1983; Kochan et al. 1984). The portal ity of the head. Length suppression resulted when plas- serves also as a docking site for the terminase-containing mid integration into the nonessential b region of the ␭ DNA translocation complex. Mutational and suppres- chromosome increased the length of the ␭ cosQ1 chro- sion analysis has confirmed that terminase’s gpA inter- mosome to 50–51 kb. Cue and Feiss (1997) proposed acts directly with the prohead’s portal vertex, an interac- that the rate of DNA translocation slows as more DNA tion essential for DNA packaging (Yeo and Feiss is packaged into the prohead. The increase in chromo- 1995a,b). It remains unclear which proteins of the trans- some length is proposed to slow the rate of translocation location complex recognize the cosQ site during termi- so that the translocation complex can recognize the nation of chromosome packaging. In the work reported mutant cosQ site, leading to more efficient termination. here, we identified trans-acting cosQ suppressors in the Smith et al. (2001) demonstrated that the packaging B gene. rate of φ29 decreases as the prohead is filled due to an internal force within the prohead that builds owing to the confinement of the DNA. The third class of suppres- MATERIALS AND METHODS sors, which are trans-acting, involves mutations in the B Media: Luria broth (LB), Luria agar (LA), and SOB were gene, which encodes the portal protein. Again, it was prepared as described in Sambrook et al. (1989). Tryptone proposed that the portal protein acts either as a sensing broth (TB), tryptone agar (TA), and tryptone broth soft agar mechanism to measure the rate of translocation or to (TBSA) were prepared as described by Arber et al. (1983) except that MgSO4 was added to a final concentration of 10 identify the cosQ site. These suppressors may allow the mm. When required, kanamycin was added to the media at a mutant cosQ site to be more efficiently recognized by final concentration of 50 ␮g/ml, while ampicillin was added terminase due to the slowing of the rate of translocation at a final concentration of 200 ␮g/ml for pUC19-based vectors. Trans-Acting cosQ Suppressors 23

Strains: The standard ␭ strain used was ␭-P1:5R cI857 Knr were incubated at 31Њ for 60 min and then plated on LA nin5; it is designated simply as ␭ in the text. This strain carries containing kanamycin at 31Њ. a 10-kb segment of phage P1 DNA encoding functions for Test for the identification of trans-acting suppressors: plasmid replication and partitioning (Sternberg and Austin MF1427 was lysogenized with ␭ cosQ4 pseudorevertants and 1983). The ␭-P1:5R cI857 Knr nin5 prophage replicates as a transformed with a 3.3-kb ␭ cosQ4 cosmid. The cosmid is a single-copy plasmid using the P1 replication machinery. The pUC19 derivative containing a 514-bp ␭ DNA insert from base cI857 mutation renders the repressor heat labile, allowing pair 48,446 to 458, which includes the cosQ4 mutation. The prophage induction at 42Њ. The ␭-P1:5R cI857 Knr nin5 also cosmid also contains an ampicillin resistance gene (Apr), and carries a 1.3-kb kanamycin-resistance cassette (Pal and Chat- the ability to package a concatemer of these cosmids was kb. The ␭-P1 determined by an in vivo packaging assay to measure the 46.2ف toraj 1988) and has a genome size of ⌬cosQ derivative of ␭-P1:5R cI857 Knr nin5 contains a 14-bp number of Apr transducing particles. Lysates of the trans- deletion of cosQ from 48,470 to 48,483 (Cue and Feiss 1993). formed lysogens were prepared as described above, and the The standard bacterial hosts used were MF1427, a galK deriva- Apr transducing particles were titered on LA containing ampi- tive of the E. coli C strain C1a (Six and Klug 1973), and cillin at 31Њ. MF2049, a ␭ϩ lysogen of MF1427. E. coli mutD mutagenesis: MF2449 or MS1414, C1a zae-13:: General recombinant DNA techniques: Enzymes for re- Tn10 mutD (Fowler et al. 1974, constructed by M. Sunshine, combinant DNA manipulations were purchased from New Iowa City, Iowa) was lysogenized with non-plaque-forming mu- England Biolabs (Beverly, MA) and Boehringer Mannheim tants of ␭ cosQ by Knr transduction as previously described. (Indianapolis) and used according to the manufacturer’s rec- Prophages were induced and multiple independent lysates ommendations. For cloning segments of ␭ DNA, the commer- were titered on MF1427 to minimize the number of sibling cial vector pUC19 was used (Yanisch-Perron et al. 1985). mutants. Plaque-forming recombinants were selected and sin- Plasmid DNA, PCR purifications, and DNA fragments purified gle-plaque purified. Lysogens of the recombinants were con- from agarose gels were prepared with reagents purchased structed by infecting MF1427 with virions isolated from from QIAGEN (Valencia, CA). Preparation of competent cells plaques. PCR amplification followed by restriction enzyme and transformations were performed as described by Hana- analysis and DNA sequencing were performed to determine han (1983). DNA sequencing reactions were performed using the cosQ sequence. dye terminator cycle sequencing chemistry with AmpliTaq PCR mutagenesis: PCR mutagenesis of the B gene using DNA polymerase, FS enzyme (PE Biosystems, Foster City, CA). purified ␭ϩ DNA as a template was performed using the Diver- The reactions were analyzed with an Applied Biosystems model sify PCR random mutagenesis kit from CLONTECH Labora- 373 stretch fluorescent automated sequencer at the University tories (Palo Alto, CA). Mutagenesis was optimized at an ap- of Iowa DNA facility. proximate frequency of 2.3–2.7 mutations per 1000 bp for an Sequence designations: All references to ␭ sequence are average target frequency of 4 mutations per B gene (1598 bp). based on the numbering convention described by Daniels et The frequency and randomness of mutagenesis were con- al. (1983). Numbering of the ␭ sequence begins with the first firmed through DNA sequencing of three full-length clones base of the left cohesive end and continues along the top of the mutagenized B gene. Mutagenized clones of the B gene strand in a 5Ј to 3Ј direction. The position of each restriction were produced in multiple independent reactions to minimize cut site is given as the first nucleotide of the recognition the number of sibling mutants, and a library of clones was sequence. introduced, by transformation, into MF1427 lysogenic for non- Introduction of B suppressor mutations into the ␭ genome: plaque-forming mutants of ␭ cosQ. Transformed lysogens were Plasmids bearing ␭ DNA fragments containing the B suppres- selected by plating at 31Њ on LA containing kanamycin and transformed 8000–5000ف ,sor mutations were introduced, by transformation, into MF1427 ampicillin. For each ␭ cosQ mutant lysogenic for ␭ Bam1 or ␭ Bam7, two ␭ prophages containing colonies were scraped from the plates and resuspended in amber mutations in the B gene. Transformed lysogens were LB. The pooled transformed lysogens were grown overnight, selected by plating at 31Њ on LA containing kanamycin (selects and prophages were induced and lysates titered on MF1427 for prophage) and ampicillin (selects for plasmid). The trans- as described previously. Plaque-forming recombinants were formed lysogens were grown overnight in LB plus antibiotics selected and single-plaque purified. Lysogens of these recom- at 31Њ. Overnight cultures were diluted 1:125 into LB and binants were constructed by infecting MF1427 with virions -ϫ 107 cells/ml at 31Њ. Prophages were induced isolated from the plaques. PCR amplification followed by re 3ف grown to by incubation at 42Њ for 20 min, followed by incubation at 37Њ striction enzyme analysis and/or DNA sequencing was per- for 60 min. The lysates were treated with CHCl3 and titered formed to determine cosQ and B gene sequences. on MF1427. Plaque-forming recombinants were selected and single-plaque purified. Lysogens of the recombinants were constructed by infecting MF1427 with virions isolated from RESULTS plaques. PCR amplification followed by restriction enzyme analysis and DNA sequencing were performed to verify the We recently used saturation mutagenesis to define recombinants. cosQ as the 7-bp segment from ␭ base pairs 48,473 to ␭ Phage yield determinations: MF1427 lysogenized with or 48,479 (Wieczorek and Feiss 2001). That work gener- a derivative were grown overnight with aeration in LB plus ated a complete collection of the 21 possible single-base kanamycin at 31Њ. The cultures were diluted 1:125 into LB ϫ 107 cells/ml at 31Њ. Portions of each pair cosQ mutations. The mutations were crossed into 3ف and grown to culture were removed at this time, diluted 1:10,000 in 10 mm ␭ prophages. The ␭ phage background used was ␭-P1:5R r r MgSO4, and plated on LA plus kanamycin. Plates were incu- cI857 Kn nin5. ␭-P1:5R cI857 Kn nin5 has phage P1’s bated overnight at 31Њ to determine the number of viable plasmid replication system, so that the prophage is a lysogens in each culture. Prophages were induced as described plasmid; there is also a kanamycin-resistance cassette above, and lysates were titered on MF1427. Kanamycin resis- ␭ tance (Knr) transducing particles were titered by diluting ly- that permits selection for lysogens. For simplicity, -P1:5R r ␭ sates in 10 mm MgSO4, and diluted lysates were used to infect cI857 Kn nin5 is called . MF2049 grown in TB plus 0.2% maltose. The infected cells The cosQ mutants fall into four groups on the basis 24 D. J. Wieczorek, L. Didion and M. Feiss

TABLE 1 Groupings of cosQ mutations from base pair 48,473 to 48,479 based on virus yields

Virus yielda,b Mutant phenotype Mutationsc Ͻ 1.00 (8) Severe G48,473T, G48,473C, G48,474 A, G48,474T, G48,474C, C48,478 A, C48,478T, C48,478G 1.00–5.49 (3) Moderate G48,475C(cosQ3), T48,479 A(cosQ4), T48,479G(cosQ5) 5.50–10.0 (4) Mild G48,473 A(cosQ2), T48,476 A, T48,476C, C48,477G Ͼ 10.0 (6) Slight G48,475 A, G48,475T, T48,476G, C48,477 A, C48,477T(cosQ1), T48,479C Summarized from Wieczorek and Feiss (2001). a The number of ␭ cosQ mutants in the respective groups is shown in parentheses. b Plaque formation on MF1427 requires a minimum virus yield of 5.50 (Wieczorek and Feiss 2001). c All mutations at positions 48,468–48,472 and 48,480–48,483 resulted in virus yields Ͼ60.

of virus yield (Wieczorek and Feiss 2001), as follows had yields 3- to 8-fold higher than the ␭ cosQ4 yield .(phage (Table 3 5.5ف Table 1). ␭ requires a minimum yield of) per cell to form a plaque (Wieczorek and Feiss 2001); To map the suppressors, plasmids bearing MluI re- phage per induced striction fragments of ␭ cosQ4 rev98, ␭ cosQ4 rev22, and 85ف and ␭ cosQϩ has a yield of lysogen. Six mutations are slight, with virus yields of ␭ cosQ4 rev33 prophage DNAs were prepared. Crosses Ͼ10.0. Four mutations are mild, giving yields between of the plasmids vs. ␭ cosQ4 were used to screen for 5.5 and 10. Three mutations are moderate, giving yields inserts carrying suppressors. For all three revertants, a between 1.0 and 5.5, while eight severe mutants have suppressor was present in the MluI fragment from ␭ yields of Ͻ1.0. The 11 phages with moderate and severe base pair 458 to 5548. The rev98 suppressor was further cosQ mutations are inviable, i.e., unable to form plaques. narrowed to the EcoO109I-RsrII fragment from base pair We screened plaque-forming revertants of these inviable 2815 to 3800. The rev22 and rev33 suppressors were in cosQ mutants for cosQ suppressors. Plaque-forming re- the RsrII-AvaI fragment from base pair 3800 to 4720. All vertants were selected from unmutagenized, mutD-muta- three suppressors mapped to the ␭ B gene. The charac- genized, and in vitro-mutagenized stocks of ␭ cosQ mu- teristics of the mutations are described in Figure 2. tants. Although most revertants were true revertants, Two other trans-acting cosQ suppressors in the B gene pseudorevertants with cis- and trans-acting suppressors were described previously. Cue and Feiss (1997) identi- were found (Table 2). The cis-acting suppressors will be fied Bms6 and Bms8 as suppressors of cosQ1. The Bms8 presented in a separate report. mutation had previously been identified as a suppressor Spontaneous revertants of ␭ cosQ4: Revertants of a of a mutation altering the prohead binding domain of ␭ nonmutagenized moderate mutant, cosQ4 (T48,479 A), the gpA subunit of terminase (Yeo and Feiss 1995b). were screened for trans-acting suppressors as follows. Given that all five trans-acting cosQ suppressors were in ϫ 10Ϫ6. B, we sequenced the B genes of additional revertants of 1ف ␭ cosQ4 exhibits a reversion frequency of Lysogens of revertants were transformed with an Apr ␭ cosQ4. Rev31 was found to contain a single mutation cosmid containing the cosQ4 mutation. The cosmid con- in B, and its ability to suppress cosQ4 was confirmed tains an ampicillin resistance gene (Apr), and the ability by marker rescue crosses (data not shown). Rev66 was of ␭ cosQ4 to package the cosQ4 cosmid was determined identical to rev33. In spite of the screen for trans-acting by assaying the titer of Apr transducing particles in ly- suppressors, 4 revertants were Bϩ and were found to ␭ ␭ sates of the cosQ4 revertants. We reasoned that cosQ4 contain the local suppressor T48,479C, and the 17 others revertants with trans-acting suppressors would be able were true revertants. Overall, the in vivo cosmid packag- to package the mutant cosmid more efficiently than ing screen resulted in the identification of four new B true revertants or phages with cis-acting suppressors. gene suppressors of cosQ4 (Figure 2) and a local sup- The cosmid packaging assay was used to screen 143 pressor. ␭ cosQ4 revertants. Revertants with at least a 2.5-fold Spontaneous revertants of other cosQ mutations: We increase in the number of Apr transducing particles, i.e., screened plaque-forming revertants in unmutagenized cosQ4 cosmid-containing phages, relative to the ␭ cosQ4 lysates of phages with eight of the severe cosQ mutations:

control, were chosen as possibly carrying trans-acting G48,473T, G48,473C, G48,474 A, G48,474T, G48,474C, C48,478 A, C48,478T,

suppressors. Three of the 26 revertants chosen, rev98, and C48,478G (Table 1). Lysogens of plaque-forming re- rev22, and rev33, were initially sequenced across cos and vertants were screened by sequencing cos to identify true found to contain the cosQ4 mutation and no suppressor revertants and revertants with local, cis-acting suppres- in cosQ. Because the ␭ cosQ4 parent is inviable, we assayed sors in cosQ. Of 423 revertants of the eight mutants, a phage titers as kanamycin-transducing phages. The ␭ total of 15 cis-acting revertants were found, all suppres- ␭ ␭ cosQ4 rev98, cosQ4 rev22, and cosQ4 rev33 phages sors of only two cosQ point mutations, cosQ G48,473T and Trans-Acting cosQ Suppressors 25

TABLE 2 Analysis of the cosQ mutagenesis strategy

Revertants with suppressors Strategy and Revertants True cosQ mutation analyzed revertantsa trans-acting cis-acting Spontaneous reversion b G48,473T978413 c G48,473C17152 G48,474 A 118 118 G48,474T3232 G48,474C1313 C48,478 A6565 C48,478T4545 C48,478G3636 d e f g T48,479 A(cosQ4) 143 (26) 17 5 4 E. coli mutD mutagenesis h G48,473T47416 i G48,473C472819 G48,474 A4747 G48,474T4747 G48,474C4747 j G48,475C(cosQ3)381 37 C48,478 A4646 C48,478T4646 C48,478G4646 k T48,479G(cosQ5)46442 PCR mutagenesis l m T48,479 A(cosQ4)4017 1 a ␭ cosQϩ revertants. b Twelve insertions of A, one insertion of T, between base pairs 48,475 and 48,476. c Two insertions of A between base pairs 48,475 and 48,476. d Revertants identiﬁed by in vivo cosmid packaging assay. e Twenty-six of 143 pseudorevertants selected by minimum of 2.5-fold increase in the production of Apr transducing particles. f Four unique suppressors: Brev98, Brev22, Brev33, and Brev31. g All four contained the revertant T48,479C. h One cosQ1, four insertions of T between base pairs 48,475 and 48,476. i Four cosQ1, one cosQ1cosQ2 double mutant, 14 insertions of T between base pairs 48,475 and 48,476. j All contained the mutation T48,479C. k One unique suppressor, Brev1. l Two unique suppressors, Brev17 and Brev24. m Length suppression by 3.0-kb partial duplication of the head genes.

cosQ G48,473C (Table 2). No trans-acting revertants were identical to the EcoO109I recognition sequence, diges- recovered. tion with EcoO109I indicates reversion to cosQϩ. E. coli mutD mutagenesis: Because the high percent- We again found that most cosQ revertants were true age of true revertants hampered the isolation of pseudorevertants. Numerous cis-acting suppressors, within revertants, we searched for pseudorevertants in lysates cosQ, were also identiﬁed by sequencing cos. One trans- of mutD-mutagenized cosQ mutants with the following acting suppressor of cosQ5 (T48,479G) was identiﬁed. This moderate and severe mutations: G48,474C, C48,478 A, C48,478T, results in a change in the same codon as the Brev22

C48,478G, G48,473T, G48,473C, G48,474 A, G48,474T, G48,475C(cosQ3), suppressor. These suppressors are located just four and T48,479G(cosQ5). On average, the E. coli mutD muta- amino acids away from Bms6 at codon 450 (Figure 2). fold increase in the titer of Segment-specific PCR mutagenesis: When it became-500ف genesis resulted in plaque-forming revertants when compared to the titer clear that most of the spontaneous and mutD-induced -pfu/ml). revertants were true revertants, we employed PCR muta 500–0ف) of nonmutagenized control lysates Lysogens of revertants were first screened by using PCR genesis to target our mutagenesis to specific genes to generate cos-containing segments and then digested known to be involved in DNA packaging. An obvious with the EcoO109I restriction enzyme. Since cosQϩ is candidate was the B gene, since B was already known 26 D. J. Wieczorek, L. Didion and M. Feiss ␭ ␭ TABLE 3 G48,474 A, and G48,474T. Lysogens with plasmids bearing Relative virus yields of cosQ suppressors involving the B gene unmutagenized wild-type Nu1 and A were included as controls. There was no difference in the titers of plaque- Burst Knr transductants/ forming phages in the lysates from cells with mutagen- Prophage sizea induced lysogena,b ized and nonmutagenized terminase genes, indicating that the mutagenesis had not produced any cosQ sup- ␭ wild type 1.00 1.00 ␭ cosQ1 0.07 0.13 pressors in the terminase genes. To show that the PCR ␭ cosQ4 Ͻ10Ϫ7 0.06 mutagenesis had worked, we crossed the mutagenized ␭ cosQ5 Ͻ10Ϫ7 0.02 and nonmutagenized terminase plasmids with ␭ cosN ␭ c,d cosQ1 Bms6 0.21 ND G2CC11G, a phage carrying mutations known to be sup- ␭ cosQ1 Bms8 c,d 0.12 ND pressed by mutations in A (Sippy Arens et al. 1999). ␭ d cosQ4 Brev22 0.36 0.47 The mutagenized pool of terminase plasmids gave a ␭ d fold increase in the titer of revertants from the-6.5ف cosQ4 Brev31 0.23 0.23 ␭ cosQ4 Brev33 d 0.20 0.20 ␭ cosQ4 Brev98 d 0.14 0.21 cells with the unmutagenized terminase plasmid pool. ␭ cosQ4 Brev17 d 0.28 ND Studies on the allelic specificity of cosQ suppressors ␭ cosQ4 Brev24 d 0.22 ND in B: To ask whether the trans-acting cosQ suppressors ␭ cosQ5 Brev1d 0.23 ND were allele specific, we first constructed ␭ cosϩ prophages containing the four B gene suppressors of cosQ4, and a Relative to the ␭ wild-type burst size of 86.9 in plaque- r virus yield assays were performed (Table 4). All of the forming units assay and 92.7 in Kn transduction assay. ϩ b ND, not determined. cosQ Brev phages were healthy, and each had a yield c Described in Cue and Feiss (1997). larger than that of the corresponding cosQ4 Brev phage. d Mutations are described in Figure 2. The results indicate the suppressors are not allele specific. Previous work also showed the Bms6 and Bms8 mutations did not impair growth of cosQϩ phages (Cue to contain a variety of suppressors of multiple cosQ muta- and Feiss 1997). tions. Mutagenized clones of the B gene were amplified, It has been proposed that the cosQ suppressors in B ف and a library of 1000 clones was pooled for transforma- act by slowing the rate of DNA translocation into the ␭ ␭ tion into a cosQ4 (T48,479A) lysogen. Plasmid vs. cosQ4 prohead, thereby making recognition of a mutant cosQ crosses were carried out by prophage induction, and 40 more efficient (Cue and Feiss 1997). We wished to ask plaque formers were selected for analysis. Crosses with if multiple suppressors would have additive effects on ف mutagenized B produced an 1000-fold increase in re- cosQ suppression. To test for additive effects, phages vertants when compared with nonmutagenized B con- with two cosQ suppressors in the ␭ cosQ4 background trol crosses. were constructed. Virus yield studies showed clearly that Each of the 40 plaque formers was sequenced across the suppressors had additive effects (Table 4). We also cosQ to confirm the presence of the parental cosQ4 muta- asked whether multiple suppressors would have effects tion. Then the B gene was sequenced. Seven suppressors on the growth of ␭ cosQϩ. Yield studies showed that the were identified; two were identical to Brev22, and one multiple suppressors tested had at best minimal effects was identical to Brev33. Rev24 contained a new single on ␭ cosQϩ growth. B mutation, and its ability to suppress cosQ4 was con- To test for allele specificity, we crossed phages with

ﬁrmed by marker rescue (data not shown). Two re- three different lethal cosQ point mutations, cosQ3 (G48,475C), vertants, Rev13 and Rev17, were sequenced and found cosQ4 (T48,479A), and cosQ5 (T48,479G), with plasmids car- to be sibs with three B mutations; when separated from rying each of the nine cosQ suppressors in B and looked each other, the suppressor mutation was found to result for viable recombinants (Table 5). Suppression of cosQ1

in a substitution of lysine for methionine in codon 397. (C48,477T), which produces tiny plaques, by Bms6 and These new cosQ suppressors were located close to the Bms8 was confirmed by measuring the number of large previously identified Brev31. In sum, nine total B sup- plaque variants produced from the cross. All cosQ suppressors have been identified, including the two de- pressors in B were able to suppress multiple cosQ point scribed by Cue and Feiss (1997). Two clusters involving mutations, demonstrating that each of the suppressors six of these trans-acting cosQ suppressors were identified is an allele-nonspecific cosQ suppressor. in B (Figure 2), affecting codons 394–397 and 450–454. The virus yields of all of the suppressors of cosQ muta- DISCUSSION tions in gene B are shown in Table 3. We also used PCR mutagenesis to target the terminase Mutagenesis strategy and results: We employed three genes, Nu1 and A. Mutagenized clones of the Nu1 and strategies in looking for cosQ suppressors. Revertants clones from unmutagenized and E. coli mutD-mutagenized 1000ف A genes were amplified, and a pool of ␭ ␭ ␭ ϩ was used to transform lysogens of cosQ3 (G48,475C), stocks of cosQ mutants were mostly revertants to cosQ , ␭ ␭ ␭ cosQ4 (T48,479A), cosQ5 (T48,479G), G48,473T, G48,473C, making it hard to find cosQ suppressors. The excess of Trans-Acting cosQ Suppressors 27

Figure 2.—Locations of suppressors of cosQ mutations affecting the portal protein, gpB. (a) Amino acid sequence of gpB. The 533-amino-acid sequence of gpB is shown. The locations of the nine identified suppressors of cosQ mutations are labeled and depicted in boldface type. (b) Suppressors of cosQ. “Rev” mutations were isolated as suppressors of the indicated cosQ mutation. Isolated suppressors of cosQ1 (C48,477T), Bms6 and Bms8; isolated suppressors of cosQ4 (T48,479A), Brev98, Brev31, Brev24, Brev22, and Brev33; the isolated suppressor of cosQ5 (T48,479G), Brev1. Base pair locations of mutations are given. Note that two unique suppressors isolated, cosQ4 Brev22 and cosQ5 Brev1, affect the same amino acid residue 454, but different base pair positions (base pairs 4195 and 4196). true revertants is likely because they have a yield higher contains cosQ4 Brev31, cosQ4 Brev24, and cosQ4 Brev17 than that of pseudorevertants. Segment-specific PCR at codons 394, 395, and 397, respectively. The amino mutagenesis was used to target gene B; this approach acid sequence immediately following the methionine at yielded a large majority of trans-acting suppressors in B. codon 397 is the sequence 398-Gly-Arg-Arg-Lys-401. Of Identification of trans-acting suppressors in gpB: We particular note are the two arginines followed by a lysine, obtained 7 new suppressors of cosQ affecting the ␭ portal three highly basic residues. Interestingly, one of the protein, gpB, by isolating revertants of unmutagenized, suppressors, Brev17, substitutes an additional basic resi- E. coli mutD-mutagenized, and PCR-mutagenized stocks due, lysine, for the neutral methionine residue at codon of ␭ cosQ mutants. Previously, 2 suppressors of cosQ1, 397. The changes in gpB created by the cosQ suppressor Bms6 and Bms8, were isolated by Cue and Feiss (1997). mutations are various. In addition to the charge change

Although the sample size is small, of the 9 trans-acting of Brev17,M397K, others include the polar to nonpolar suppressors, 2 were independently recovered twice. Us- S450F change of Bms6; the nonpolar to polar changes ϭ ϭ ing the Poisson distribution and the m 1 and m 2Y395H, P331S, A454T, and A491SofBrev24, Bms8, Brev22, terms of 7 and 2, respectively, provides a rough estimate and Brev33, respectively; and the nonpolar to nonpolar that there is a total of 22 potential B gene suppressors changes I81V, A394V, and A454VofBrev98, Brev31, and of cosQ (Benzer 1961). Brev1, respectively. Clearly, structural information about The nine cosQ suppressors are not uniformly distrib- the locations of the affected residues and the effects of uted throughout B; two clusters involving six of the cosQ the amino acid changes on the structure are needed to suppressors are seen (Figure 2). The ﬁrst lies in the make detailed statements about how the gpB changes region from amino acid residues 394 to 397. This cluster suppress cosQ mutations. 28 D. J. Wieczorek, L. Didion and M. Feiss

TABLE 4 TABLE 5 Relative virus yields of ␭ Brev single and double mutants Suppression of ␭ cosQ mutants by suppressors in gene B

␭ cosQ4 ␭ cosϩ Suppressor ␭ cosQ1 a ␭ cosQ3 ␭ cosQ4 ␭ cosQ5 Prophage backgrounda backgrounda Bms6 ϩ ϩϩϪ ␭ wild type NAb 1.00 Bms8 ϩ ϪϩϪ ␭ cosQ4 0.04 NAb Brev98 ND ϩϩϪ ␭ Brev22 0.39 0.98 Brev22 ND ϩϩϩ ␭ Brev33 0.15 0.88 Brev33 ND ϩϩϪ ␭ Brev31 0.14 0.68 Brev31 ND Ϫϩϩ ␭ Brev98 0.11 0.85 Brev17 ND ϩϩϩ ␭ Brev98rev22 0.49 0.93 Brev24 ND ϩϩϪ ␭ Brev98rev33 0.49 0.65 Brev1 ND ϩϩϩ ␭ Brev98rev31 0.53 0.63 ϩ denotes suppression as indicated by plaque formation a Relative to ␭ wild-type transducing titer. for viable recombinants of cosQ3, cosQ4, and cosQ5 or the b NA, not applicable. production of large plaque variants of cosQ1; Ϫ denotes no suppression. a ND, not determined. Portals have been purified from a variety of bacteriophages, and although little sequence identity is seen, each is a gear-shaped multimer with a central channel Mutations in the 725-residue P22 portal protein gene 1 (Valpuesta and Carrascosa 1994). Since portals do have been described that are opposite in effect to the ف appear to have a similar structure, it is likely that they are SPP1 siz mutants, in that 4% excess DNA is packaged descendants of a common ancestor, but have diverged (Casjens et al. 1992). The two P22 gene 1 mutations → sufficiently so that no sequence identity is seen (Hen- both produce V M changes (residues 64 and 303). drix et al. 1999). The molecular structure of the dode- In both the SPP1 and P22 cases, the authors argue that cameric φ29 portal protein, gp10, has recently been headful packaging requires a sensing mechanism that solved by X-ray crystallography (Simpson et al. 2000). detects when the head is filled. The sensor is proposed The structure of the 309-residue gp10 protein exhibits to detect some parameter correlated with head filling three cylindrical regions: the narrow end, which pro- such as an increase in DNA packing density, a decrease trudes from the vertex and is encircled by the pentam- in the rate of DNA translocation, or an increase in the eric pRNA; the central channel; and the wide end, which energy required to continue packaging (Figure 3; Cas- contacts the inside of the prohead (Simpson et al. 2000; jens et al. 1992; Tavares et al. 1992; Orlova et al. 1999). φ Figure 3). The inside of the channel has a preponder- In the 29 portal protein, gp10, two stretches of resi- ance of negative charges at its wide end, which are dues that are disordered in the crystal structure (resi- proposed to repel the DNA, permitting its smooth pas- dues 229–246 and 287–309) occur in the DNA channel sage during packaging and ejection. Also, the amino near the border between the central and wide regions terminus of gp10 has been shown to interact with DNA of the connector. These disordered residues, located in to be packaged (Herranz et al. 1986, 1990; Donate et the C terminus of gp10, include the amino acid se- al. 1992, 1993; Turnquist et al. 1992; Valpuesta et quences 233-Glu-Lys-Lys-Glu-Arg-237 and 289-Arg-Arg- al. 1992; Zachary and Black 1992; Valle et al. 1996, Glu-291, which are proposed to interact with the nega- 1999). tively charged sugar-phosphate DNA backbone. These Studies on the phages SPP1 and P22 have shown that segments with tandem basic residues are similar to resi- mutations altering the portal proteins affect DNA pack- dues that follow the cluster of suppressors we identified aging, as follows. Bacillus subtilis bacteriophage SPP1 in the portal protein of ␭, 398-Gly-Arg-Arg-Lys-401. This uses a headful packaging strategy in which the DNA highly basic stretch acquires a further net positive cleavage that terminates DNA packaging is triggered charge from the Brev17 suppressor change. The gp10 when the head shell is filled with DNA; the downstream amino terminus also contains a basic segment: 1-Ala- cleavage, which is not sequence specific, generates a Arg-Lys-Arg-4. This basic sequence is necessary for DNA terminal redundancy. Tavares et al. (1992) identified packaging and is essential for efficient DNA binding three mutations, called siz mutations, that affect the 503- (Donate et al. 1992). It is proposed that this region residue SPP1 portal protein, gp6, and result in a 4–6% interacts with the negatively charged DNA. It is possible decrease in the amount of DNA packaged. Each siz that the region we have identified in the 533-residue mutation substitutes a basic residue: Two are E → K portal protein of ␭, 398-Gly-Arg-Arg-Lys-401, performs a changes (residues 251 and 424) and the other is an similar role (Figure 3). Deletion studies and site-specific N → K change (residue 365). Like SPP1, the Salmonella mutagenesis of this region would be helpful in de- phage P22 packages DNA by the headful mechanism. termining whether this region is necessary for DNA Trans-Acting cosQ Suppressors 29 packaging and reveal any inherent DNA-binding activity slow DNA translocation so that the sensor triggers cleav- for gpB. age when the triggering rate of translocation is reached As with phages SPP1 and P22, ␭ DNA packaging also prematurely. Similarly, the P22 mutations may raise the requires a headful sensor since the efficiency of the threshold required to trigger cleavage. terminal cutting depends on the extent of DNA packag- The second cluster of cosQ suppressors lies in gpB ing (Feiss and Siegele 1979). For the siz mutations of residues 450–454 (Figure 2). This cluster contains cosQ1 SPP1, which cause substitutions of basic residues in gp6, Bms6 (S450F), cosQ4 Brev22 (A454T), and cosQ5 Brev1 (A454V). it is tempting to propose that electrostatic interactions Although the results of these changes do not affect the overall net charge of the gpB protein, each of the mutations in this second cluster results in the substitution of an amino acid residue that is bulkier than the residue normally found at that position. For Bms6, the phenylalanine has a Van der Waals volume of 135 A˚ 3 vs. 73 A˚ 3 for serine (Richards 1974). Threonine (Brev22) and valine (Brev1) have Van der Waals volumes of 93 A˚ 3 and 105 A˚ 3, respectively, vs. 67 A˚ 3 for the wild- type alanine. This second cluster may reside in a region that is in or near the gauge that measures DNA packaging, resulting in a conformational change of this gauge (Figure 3). On the other hand, the cluster might simply lie in the channel through which the DNA passes, resulting in steric hindrance in the DNA channel of the connector and causing the slowing of the translocating DNA (Figure 3). Suppression by these mutations may occur as a result of this slowed translocation, allowing for more efficient recognition of the mutant cosQ site and leading to more efficient termination of DNA packaging. The third suppressor of the first cluster, Brev31, also results in a substitution of valine for alanine, consis- tent with the steric hindrance hypothesis for the first cluster as well. Whereas the sensor model presumes that the portal protein plays an active role in gauging the Figure 3.—General model of the structure of the tailed, packaged DNA and in the termination of DNA packag- dsDNA bacteriophage portal. The model is based on the ing, the steric hindrance model presumes the portal has known structures of the portals of bacteriophages φ29 and SPP1 (Orlova et al. 1999; Simpson et al. 2000). φ29 and SPP1 a passive, more general role in packaging termination. exhibit similar structures consisting of three domains: (1) a We looked for additive effects produced by combin- wing domain (WD), or wide end, that contacts the prohead; ing suppressors. Of the double mutants constructed in (2) a stem domain (SD) or central channel that has been the ␭ cosQϩ background, none was found to have more suggested to be the docking region for terminase and the than a minor decrease in virus yield. The fact that these packaging complex and the region through which it is pre- sumed the DNA translocates through into the prohead; and suppressors are mild suppressors may make it difficult (3) the tentacle domain (TD). In SPP1, the TD is thought to to observe significant additive effects on phage produc- be the region of the “headful sensor” that is necessary for tion in this background. Since the time to package a ␭ packaging termination. In φ29, two stretches of residues, disor- genome is very fast, (1–2 min; Hohn 1975) in compari- ,(min 60ف) dered in the crystal structure, occur in the DNA channel son with the length of the virus growth cycle near the border between the central and wide regions of the connector, similar to the location of the TD of SPP1, and a suppressor that slows packaging to an extent sufficient were proposed to be ideal for interacting with the negatively for cosQ suppression might not greatly affect virus yield. charged DNA. The possibility exists that this is the headful In contrast, each of the double mutants constructed sensor of φ29. These stretches of disordered residues are simi- in the ␭ cosQ4 background resulted in a significant in- lar to the sequence immediately following the cluster of cosQ crease in phage yield compared to the effects of the suppressors identified in the portal protein of ␭. Thus, this cluster may reside near a comparable headful sensor in ␭ individual cosQ suppressors, signifying the additive ef- necessary for packaging termination. A second cluster was fects of the B gene suppressors on phage production proposed to reside in a region that is near the gauge that in the ␭ cosQ4 background. Thus, it appears that Brev98 measures DNA packaging, resulting in a conformational acts to suppress cosQ4 in a manner independent from change of this gauge, or might simply lie in the channel suppression by Brev22, Brev33, and Brev31. It is interest- through which the DNA passes, resulting in steric hindrance in the DNA channel of the portal and causing the slowing of ing to note that Brev98 is the only suppressor thus far the translocating DNA. The direction of DNA entry is given identified that is located in the amino third of the pro- by the arrow. tein. Each of the other suppressors identified has been 30 D. J. Wieczorek, L. Didion and M. Feiss localized to the carboxy third of the B protein. We packaging of the phage ␭ chromosome. Proc. Natl. Acad. Sci. USA 90: 9290–9294. propose that cosQ suppressors in gene B enhance recog- Cue, D., and M. Feiss, 1997 Genetic evidence that recognition of nition of a mutant cosQ by slowing packaging. Since the cosQ, the signal for termination of phage ␭ DNA packaging, de- ␭ B suppressor mutations cause such a variety of residue pends on the extent of head filling. Genetics 147: 7–17. Cue, D., and M. Feiss, 1998 Termination of packaging of the bacte- changes, no single explanation for how each works is riophage ␭ chromosome: cosQ is required for nicking the bottom obvious. The failure of the B suppressors to suppress strand of cosN. J. Mol. Biol. 280: 11–29. all cosQ mutations is most likely due to the mild strength Daniels, D., J. Schroeder,W.Szybalski,F.Sanger,A.Coulsen et al., 1983 Completed annotated lambda sequence, pp. 519–676 of the suppressors and the severity of the individual cosQ in Lambda II, edited by R. W. Hendrix,J.W.Roberts,F.W. mutations. Clearly, structural information about wild- Stahl and R. A. Weisberg. Cold Spring Harbor Laboratory Press, type and mutant gpB proteins is required, along with Cold Spring Harbor, NY. Davidson, A., and M. Gold, 1992 Mutations abolishing the endonu- molecular information on the effects of the suppressors clease activity of bacteriophage ␭ terminase lie in two distinct on DNA packaging. regions of the A gene, one of which may encode a “leucine Despite our intense efforts, the trans-acting suppres- zipper” DNA binding domain. Virology 189: 21–30. Donate, L. E., J. M. Valpuesta,A.Rocher,E.Mendez,F.Rojo et sors we identified were all allele-nonspecific suppressors al., 1992 Role of the amino-terminal domain of bacteriophage of cosQ mutations. This inability to detect allele-specific φ29 connector in DNA binding and packaging. J. Biol Chem. suppressors may be because the component that recog- 267: 10919–10924. Donate, L. E., J. M. Valpuesta,C.Mier,F.Rojo and J. L. Carras- nizes cosQ has multiple roles in DNA packaging. Thus, cosa, 1993 Characterization of an RNA-binding domain in the mutations in the domain of this component that sup- bacteriophage φ29 connector. J. Biol. Chem. 268: 20198–20204. presses cosQ mutations may disrupt other necessary func- Emmons, S. W., 1974 Bacteriophage lambda derivatives carrying two copies of the cohesive end site. J. Mol. Biol. 83: 511–525. tions. For instance, it is not surprising that there was Feiss, M., 1986 Terminase and the recognition, cutting, and packag- a failure to identify suppressors of cosQ mutations in ing of ␭ chromosomes. Trends Genet. 2: 100–104. terminase. gpA has numerous roles in DNA packaging, Feiss, M., and D. A. Siegele, 1979 Packaging of the bacteriophage ␭ chromosome: dependence of cos cleavage on chromosome including interacting with gpNu1, nicking at cosN, hy- length. Virology 92: 190–200. drolyzing ATP (to separate the cohesive ends and pre- Feiss, M., J. Sippy and G. Miller, 1985 Processive action of termi- sumably for translocating DNA), gpA dimerization, and nase during sequential packaging of bacteriophage ␭ chromosomes. J. Mol. Biol. 186: 759–771. interaction with the portal. Because of its numerous Fowler, R. G., G. E. Degnen and E. C. Cox, 1974 Mutational speci- roles, it is quite possible that if gpA also recognizes cosQ, ficity of a conditional Escherichia coli mutator, mutD5. Mol. Gen. however unlikely, any mutation in gpA that suppresses Genet. 133: 179–191. Furth, M. E., and S. H. Wickner, 1983 Lambda DNA replication, a cosQ mutation may have detrimental effects on other pp. 145–173 in Lambda II, edited by R. W. Hendrix,J.W.Rob- gpA functions, rendering the protein nonfunctional. erts,F.W.Stahl and R. A. Weisberg. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. We thank our co-workers, Alok Dhar, Carol Duffy, Qi Hang, Jason Hanahan, D., 1983 Studies on transformation of Escherichia coli with Luke, Jenny Meyer, and Jean Sippy for advice and interest during the plasmids. J. Mol. Biol. 166: 557–580. course of this work. The work involving the effects of multiple B Hendrix, R. W., M. C. M. Smith,R.N.Burns,M.E.Ford and suppressors on ␭ cosQϩ growth constitutes part of the undergraduate G. F. Hatfull, 1999 Evolutionary relationships among diverse honors thesis of Lisa Didion. This work was supported by National bacteriophages and prophages: all the world is a phage. Proc. Institutes of Health (NIH) research grant GM-51611, NIH genetics Natl. Acad. Sci. USA 96: 2192–2197. Herranz, L., M. Salas and J. L. Carrascosa, 1986 Interaction of research training grant T32GM08629 (D.W.), and NIH Iowa kidney the bacteriophage φ29 connector protein with the viral DNA. disease, hypertension, and cell biology research training grant Virology 155: 289–292. DK07690-10 (D.W.). Herranz, L., J. Bordas,E.Towns-Andrews,E.Mendez,P.Usobiaga et al., 1990 Conformational changes in bacteriophage φ29 connector prevent DNA-binding activity. J. Mol. Biol. 213: 263–273. Hohn, B., 1975 DNA as a substrate for packaging into bacteriophage LITERATURE CITED lambda in vitro. J. Mol. Biol. 98: 93–106. Hwang, Y., and M. Feiss, 1996 Mutations affecting the high affinity Arber,W.,L.Enquist,B.Hohn,N.E.Murray and K. Murray, ATPase center of gpA, the large subunit of bacteriophage ␭ termi- 1983 Experimental methods for use with lambda, pp. 433–466 nase, inactivate the endonuclease activity of terminase. J. Mol. in Lambda II, edited by R. W. Hendrix,J.W.Roberts,F.W. Biol. 261: 524–535. Stahl and R. A. Weisberg. Cold Spring Harbor Laboratory Press, Kochan, J., and H. Murialdo, 1983 Early intermediates in bacterio- Cold Spring Harbor, NY. phage lambda prohead assembly. Virology 131: 100–115. Bear, S., D. Court and D. I. Friedman, 1983 An accessory role Kochan, J., J. L. Carrascosa and H. Murialdo, 1984 Bacterio- for integration host factor: characterization of a lambda mutant phage lambda preconnectors: purification and structure. J. Mol. dependent upon integration host factor for DNA packaging. Biol. 174: 433–447. J.Virol. 52: 966–972. Kosturko, L., E. Daub and H. Murialdo, 1989 The interaction of Becker, A., and H. Murialdo, 1990 Bacteriophage ␭ DNA: the E. coli integration host factor and lambda cos DNA multiple com- beginning of the end. J. Bacteriol. 172: 2819–2824. plex formation and protein-induced bending. Nucleic Acids Res. Benzer, S., 1961 On the topography of the genetic fine structure. 17: 329–334. Proc. Natl. Acad. Sci. USA 47: 403–415. Mendelson, I., M. Gottesman and A. B. Oppenheim, 1991 HU and Casjens, S., E. Wyckoff,M.Hayden,L.Sampson,K.Eppler et al., integration host factor function as auxiliary proteins in cleavage 1992 Bacteriophage P22 portal protein is part of a gauge that of phage lambda cohesive ends by terminase. J. Bacteriol. 173: regulates packing density of intravirion DNA. J. Mol. Biol. 224: 1670–1676. 1055–1074. Orlova, E. V., P. Dube,E.Beckmann,F.Zemlin,R.Lurz et al., 1999 Catalano,C.E.,D.Cue and M. Feiss, 1995 Virus DNA packaging: Structure of the 13-fold symmetric portal protein of bacterio- the strategy used by phage ␭. Mol. Microbiol. 16: 1075–1086. phage SPP1. Nat. Struct. Biol. 6: 842–846. Cue, D., and M. Feiss, 1993 A site required for the termination of Pal, S. K., and D. K. Chattoraj, 1988 P1 plasmid replication: initia- Trans-Acting cosQ Suppressors 31

tor sequestration is inadequate to explain control by initiator- Valle, M., J. M. Valpuesta,J.L.Carrascosa,J.Tamayo and R. binding sites. J. Bacteriol. 172: 2819–2824. Garcia, 1996 The interaction of DNA with bacteriophage phi Richards, F. M., 1974 The interpretation of protein structures: total 29 connector: a study by AFM and TEM. J. Struct. Biol. 116: volume, group volume distributions and packing density. J. Mol. 390–398. Biol. 82: 1–14. Valle, M., L. Kremer,C.Martinez-A., F. Roncal,J.M.Valpuesta φ Rubinchik, S., W. Parris and M. Gold, 1994 The in vitro endonucle- et al., 1999 Domain architecture of the bacteriophage 29 con- ase activity of gene product A, the large subunit of the bacterio- nector protein. J. Mol. Biol. 288: 899–909. phage ␭ terminase, and its relationship to the endonuclease activ- Valpuesta, J. M., and J. L. Carrascosa, 1994 Structure of viral ity of the holoenzyme. J. Biol. Chem. 269: 13575–13585. connectors and their function in bacteriophage assembly and Sambrook, J., E. F. Fritsch and T. Maniatis, 1989 Molecular Clon- DNA packaging. Q. Rev. Biophys. 27: 107–155. ing: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory Valpuesta,J.M.,M.Serrano,L.E.Donate,L.Herranz and J. L. Carrascosa, 1992 DNA conformational change induced by the Press, Cold Spring Harbor, NY. bacteriophage φ29 connector. Nucleic Acids Res. 20: 5549–5554. Sanger, F., A. R. Coulson,G.F.Hong,D.F.Hill and G. B. Petersen, Walker, J. E., A. D. Aufrett,A.Carne,A.Gurnett,P.Hanisch 1982 Nucleotide sequence of bacteriophage lambda DNA. J. et al., 1982 Solid-phase sequence analysis of polypeptides eluted Mol. Biol. 162: 729–773. from polyacrylamide gels. An aid to interpretation of DNA se- Shinder, G., and M. Gold, 1988 The Nu1 subunit of bacteriophage quences exemplified by the Escherichia coli unc operon and bacte- lambda binds to specific sites in cos DNA. J. Virol. 62: 387–392. riophage lambda. Eur. J. Biochem. 123: 253–260. Simpson,A.A.,Y.Tao,P.G.Leiman,M.O.Badasso,Y.He et al., Wieczorek, D. J., and M. Feiss, 2001 Defining cosQ, the site required 2000 Structure of the bacteriophage φ29 DNA packaging motor. for termination of bacteriophage ␭ DNA packaging. Genetics Nature 408: 745–750. 158: 495–506. Sippy Arens, J., Q. Hang,Y.Hwang,B.Tuma,S.Max et al., 1999 Xin, W., and M. Feiss, 1993 Function of IHF in ␭ DNA packaging. Mutations that extend the specificity of the endonuclease activity I. Identification of the strong binding site for integration host of ␭ terminase. J. Bacteriol. 181: 218–224. factor and the locus for intrinsic bending in cosB. J. Mol. Biol. Six, E., and C. A. C. Klug, 1973 Bacteriophage P4: a satellite virus 230: 492–504. depending on a helper such as prophage P2. Virology 51: 327– Xin, W., Z.-H. Cai and M. Feiss, 1993 Function of IHF in ␭ DNA 344. packaging. II. Effects of mutations altering the IHF binding site Smith, D. E., S. J. Tans,S.B.Smith,S.Grimes,D.L.Anderson et and the intrinsic bend in cosB on ␭ development. J. Mol. Biol. al., 2001 The bacteriophage φ29 portal motor can package DNA 230: 505–515. against a large internal force. Nature 413: 748–752. Yanisch-Perron, C., J. Vieira and J. Messing, 1985 Improved M13 Sternberg, N., and S. Austin, 1983 Isolation and characterization phage cloning vectors and host strains: nucleotide sequences of of P1 minireplicons, ␭-P1:5R and ␭-P1:5L. J. Bacteriol. 153: 800– the M13mp18 and pUC19 vectors. Gene 33: 103–119. 812. Yeo, A., and M. Feiss, 1995a Mutational analysis of the prohead Tavares, P., M. A. Santos,R.Lurz,G.Morelli,H.De Lencastre binding domain of the large subunit of terminase, the bacteriophage ␭ DNA packaging enzyme. J. Mol. Biol. 245: 126–140. et al., 1992 Identification of a gene in Bacillus subtilis bacterio- Yeo, A., and M. Feiss, 1995b Specific interaction of terminase, the phage SPP1 determining the amount of packaged DNA. J. Mol. DNA packaging enzyme of bacteriophage ␭, with the portal pro- Biol. 225: 81–92. tein of the prohead. J. Mol. Biol. 245: 141–150. Tsui, L., and R. W. Hendrix, 1980 Head-tail connector of bacterio- Zachary, A., and L. W. Black, 1992 Isolation and characterization phage lambda. J. Mol. Biol. 142: 419–438. of a portal protein-DNA complex from dsDNA bacteriophage. Turnquist, S., M. Simon,E.Engelman and D. Anderson, 1992 Intervirology 33: 6–16. Supercoiled DNA wraps around the bacteriophage φ29 head-tail connector. Proc. Natl. Acad. Sci. USA 89: 10479–10483. Communicating editor: G. R. Smith