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

Corresponding author: Douglas J. Wieczorek, University of Iowa, 3-315 BSB, 51 Newton Rd., Iowa City, IA 52242., wieczorekd{at}mail.medicine.uiowa.edu (E-mail)

Communicating editor: G. R. SMITH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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 cosQ⁺ 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.

BACTERIOPHAGE is a tailed, double-stranded DNA virus that serves as an important model for assembly of large DNA viruses such as herpes and poxviruses. The mature chromosome is a linear chromosome 48.5 kb in length with 12-bp cohesive ends at the 5' ends of the strands (SANGER et al. 1982 ). Upon injection into the cell, the cohesive ends anneal and are ligated, circularizing the chromosome. The site of the annealed cohesive ends is part of cos, the cohesive end site that contains the DNA sites required for DNA packaging. Early during infection, the chromosome is replicated bidirectionally to produce a number of progeny rings. Later, rolling circle replication and recombination give rise to end-to-end multimers of chromosomes called concatemers (FURTH and WICKNER 1983 ). Packaging of chromosomes from concatemeric DNA leads to assembly of infectious virions. DNA is recognized by the interaction of terminase, the DNA packaging enzyme, with cos.

A model for DNA packaging is as follows (FEISS 1986 ; BECKER and MURIALDO 1990 ; CATALANO et al. 1995 ). Terminase, composed of gpNu1 and gpA, the large and small subunits, respectively, binds a randomly chosen cos. Terminase introduces staggered nicks at the cosN subsite of cos, resulting in the nicked complex. Through an ATP hydrolysis-dependent process, terminase separates the strands of DNA at the cos site, forming right and left chromosome ends, and remains bound to the left end forming the stable intermediate, complex I. Complex I binds an empty shell precursor, the prohead, forming the ternary DNA-terminase-prohead assembly known as complex II. ATP-dependent DNA translocation into the prohead follows complex II formation; terminase is part of the translocation complex. When the translocation complex encounters the next cos along the concatemer, terminase again introduces staggered nicks at cosN, a step known as terminal cos-cleavage, followed by ATP-dependent DNA strand separation. Packaging is thus terminated. Terminase undocks from the DNA-filled head but remains bound to the left end of the next chromosome in the concatemer, forming complex I, and sponsoring processive packaging of the next chromosome along the concatemer. An average of two to three chromosomes are packaged processively by terminase (EMMONS 1974 ; FEISS et al. 1985 ). Phage construction is completed with the addition of accessory head and tail proteins to the DNA-filled capsid.

cos is composed of three subsites: cosN, cosB, and cosQ (Fig 1). cosN is the site where terminase's gpA subunit generates the cohesive ends (DAVIDSON and GOLD 1992 ; RUBINCHIK et al. 1994 ; HWANG and FEISS 1996 ). cosB, the terminase binding site, is located to the right of cosN. cosB is required for the initiation of chromosome packaging and contains three sequences, R1, R2, and R3, which are bound by gpNu1 (BEAR et al. 1983 ; SHINDER and GOLD 1988 ). Between R3 and R2 is I1, a binding site for Escherichia coli integration host factor (IHF). It is believed that the introduction of a sharp 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 and FEISS 1993 ; XIN et al. 1993 ).

View larger version (18K): In this window In a new window Download PPT slide   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 rotational 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 ).

cosQ, located to the left of cosN, is necessary for the termination of chromosome packaging. cosQ is a 7-bp segment with the sequence 5' GGGTCCT 3' (WIECZOREK and FEISS 2001 ). cosQ has no role in packaging initiation, but mutations within cosQ cause defective packaging termination (CUE and FEISS 1993 ). CUE and FEISS 1998 showed that cosQ is required for the nicking of the bottom DNA strand at cosNR.

CUE and FEISS 1997 found several types of suppressors of the leaky cosQ1 mutation. The first was a local suppressor, named cosQ2, affecting a base pair within the cosQ site. The second suppressor was an increase in the length of the phage chromosome to near the capacity of the head. Length suppression resulted when plasmid integration into the nonessential b region of the chromosome increased the length of the cosQ1 chromosome to 50–51 kb. CUE and FEISS 1997 proposed that the rate of DNA translocation slows as more DNA is packaged into the prohead. The increase in chromosome length is proposed to slow the rate of translocation so that the translocation complex can recognize the mutant cosQ site, leading to more efficient termination. SMITH et al. 2001 demonstrated that the packaging rate of 29 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 suppressors, which are trans-acting, involves mutations in the B gene, which encodes the portal protein. Again, it was proposed that the portal protein acts either as a sensing mechanism to measure the rate of translocation or to identify the cosQ site. These suppressors may allow the mutant cosQ site to be more efficiently recognized by terminase due to the slowing of the rate of translocation or to be recognized more efficiently by the portal protein itself.

's portal protein, gpB, is a 533-amino-acid protein, of which the N-terminal 21 amino acids are cleaved to produce the mature protein, gpB* (WALKER et al. 1982 ). Twelve subunits of gpB* are assembled into a dodecameric ring with a central channel. The ring forms the unique portal vertex of the prohead that is believed to be the site of DNA entry into the prohead and DNA exit during ejection, as well as the site for tail attachment and shell assembly (TSUI and HENDRIX 1980 ; KOCHAN and MURIALDO 1983 ; KOCHAN et al. 1984 ). The portal serves also as a docking site for the terminase-containing DNA translocation complex. Mutational and suppression analysis has confirmed that terminase's gpA interacts directly with the prohead's portal vertex, an interaction essential for DNA packaging (YEO and FEISS 1995A , YEO and FEISS 1995B ). It remains unclear which proteins of the translocation complex recognize the cosQ site during termination of chromosome packaging. In the work reported here, we identified trans-acting cosQ suppressors in the B gene.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media:
Luria broth (LB), Luria agar (LA), and SOB were prepared as described in SAMBROOK et al. 1989 . Tryptone broth (TB), tryptone agar (TA), and tryptone broth soft agar (TBSA) were prepared as described by ARBER et al. 1983 except that MgSO₄ was added to a final concentration of 10 mM. When required, kanamycin was added to the media at a final concentration of 50 µg/ml, while ampicillin was added at a final concentration of 200 µg/ml for pUC19-based vectors.

Strains:
The standard strain used was -P1:5R cI857 Kn^r nin5; it is designated simply as in the text. This strain carries a 10-kb segment of phage P1 DNA encoding functions for plasmid replication and partitioning (STERNBERG and AUSTIN 1983 ). The -P1:5R cI857 Kn^r nin5 prophage replicates as a single-copy plasmid using the P1 replication machinery. The cI857 mutation renders the repressor heat labile, allowing prophage induction at 42°. The -P1:5R cI857 Kn^r nin5 also carries a 1.3-kb kanamycin-resistance cassette (PAL and CHATTORAJ 1988 ) and has a genome size of 46.2 kb. The -P1 cosQ derivative of -P1:5R cI857 Kn^r nin5 contains a 14-bp deletion of cosQ from 48,470 to 48,483 (CUE and FEISS 1993 ). The standard bacterial hosts used were MF1427, a galK derivative of the E. coli C strain C1a (SIX and KLUG 1973 ), and MF2049, a ⁺ lysogen of MF1427.

General recombinant DNA techniques:
Enzymes for recombinant DNA manipulations were purchased from New England Biolabs (Beverly, MA) and Boehringer Mannheim (Indianapolis) and used according to the manufacturer's recommendations. For cloning segments of DNA, the commercial vector pUC19 was used (YANISCH-PERRON et al. 1985 ). Plasmid DNA, PCR purifications, and DNA fragments purified from agarose gels were prepared with reagents purchased from QIAGEN (Valencia, CA). Preparation of competent cells and transformations were performed as described by HANAHAN 1983 . DNA sequencing reactions were performed using dye terminator cycle sequencing chemistry with AmpliTaq DNA polymerase, FS enzyme (PE Biosystems, Foster City, CA). The reactions were analyzed with an Applied Biosystems model 373 stretch fluorescent automated sequencer at the University of Iowa DNA facility.

Sequence designations:
All references to sequence are based on the numbering convention described by DANIELS et al. 1983 . Numbering of the sequence begins with the first base of the left cohesive end and continues along the top strand in a 5' to 3' direction. The position of each restriction cut site is given as the first nucleotide of the recognition sequence.

Introduction of B suppressor mutations into the genome:
Plasmids bearing DNA fragments containing the B suppressor mutations were introduced, by transformation, into MF1427 lysogenic for Bam1 or Bam7, two prophages containing amber mutations in the B gene. Transformed lysogens were selected by plating at 31° on LA containing kanamycin (selects for prophage) and ampicillin (selects for plasmid). The transformed lysogens were grown overnight in LB plus antibiotics at 31°. Overnight cultures were diluted 1:125 into LB and grown to 3 x 10⁷ cells/ml at 31°. Prophages were induced by incubation at 42° for 20 min, followed by incubation at 37° for 60 min. The lysates were treated with CHCl₃ and titered on MF1427. Plaque-forming recombinants were selected and single-plaque purified. Lysogens of the recombinants were constructed by infecting MF1427 with virions isolated from plaques. PCR amplification followed by restriction enzyme analysis and DNA sequencing were performed to verify the recombinants.

Phage yield determinations:
MF1427 lysogenized with or a derivative were grown overnight with aeration in LB plus kanamycin at 31°. The cultures were diluted 1:125 into LB and grown to 3 x 10⁷ cells/ml at 31°. Portions of each culture were removed at this time, diluted 1:10,000 in 10 mM MgSO₄, and plated on LA plus kanamycin. Plates were incubated overnight at 31° to determine the number of viable lysogens in each culture. Prophages were induced as described above, and lysates were titered on MF1427. Kanamycin resistance (Kn^r) transducing particles were titered by diluting lysates in 10 mM MgSO₄, and diluted lysates were used to infect MF2049 grown in TB plus 0.2% maltose. The infected cells were incubated at 31° for 60 min and then plated on LA containing kanamycin at 31°.

Test for the identification of trans-acting suppressors:
MF1427 was lysogenized with cosQ4 pseudorevertants and transformed with a 3.3-kb cosQ4 cosmid. The cosmid is a pUC19 derivative containing a 514-bp DNA insert from base pair 48,446 to 458, which includes the cosQ4 mutation. The cosmid also contains an ampicillin resistance gene (Ap^r), and the ability to package a concatemer of these cosmids was determined by an in vivo packaging assay to measure the number of Ap^r transducing particles. Lysates of the transformed lysogens were prepared as described above, and the Ap^r transducing particles were titered on LA containing ampicillin at 31°.

E. coli mutD mutagenesis:
MF2449 or MS1414, C1a zae-13:: Tn10 mutD (FOWLER et al. 1974 , constructed by M. Sunshine, Iowa City, Iowa) was lysogenized with non-plaque-forming mutants of cosQ by Kn^r transduction as previously described. Prophages were induced and multiple independent lysates were titered on MF1427 to minimize the number of sibling mutants. Plaque-forming recombinants were selected and single-plaque purified. Lysogens of the recombinants were constructed by infecting MF1427 with virions isolated from plaques. PCR amplification followed by restriction enzyme analysis and DNA sequencing were performed to determine the cosQ sequence.

PCR mutagenesis:
PCR mutagenesis of the B gene using purified ⁺ DNA as a template was performed using the Diversify PCR random mutagenesis kit from CLONTECH Laboratories (Palo Alto, CA). Mutagenesis was optimized at an approximate frequency of 2.3–2.7 mutations per 1000 bp for an average target frequency of 4 mutations per B gene (1598 bp). The frequency and randomness of mutagenesis were confirmed through DNA sequencing of three full-length clones of the mutagenized B gene. Mutagenized clones of the B gene were produced in multiple independent reactions to minimize the number of sibling mutants, and a library of clones was introduced, by transformation, into MF1427 lysogenic for non-plaque-forming mutants of cosQ. Transformed lysogens were selected by plating at 31° on LA containing kanamycin and ampicillin. For each cosQ mutant, 5000–8000 transformed colonies were scraped from the plates and resuspended in LB. The pooled transformed lysogens were grown overnight, and prophages were induced and lysates titered on MF1427 as described previously. Plaque-forming recombinants were selected and single-plaque purified. Lysogens of these recombinants were constructed by infecting MF1427 with virions isolated from the plaques. PCR amplification followed by restriction enzyme analysis and/or DNA sequencing was performed to determine cosQ and B gene sequences.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We recently used saturation mutagenesis to define cosQ as the 7-bp segment from base pairs 48,473 to 48,479 (WIECZOREK and FEISS 2001 ). That work generated a complete collection of the 21 possible single-base pair cosQ mutations. The mutations were crossed into prophages. The phage background used was -P1:5R cI857 Kn^r nin5. -P1:5R cI857 Kn^r nin5 has phage P1's plasmid replication system, so that the prophage is a plasmid; there is also a kanamycin-resistance cassette that permits selection for lysogens. For simplicity, -P1:5R cI857 Kn^r nin5 is called .

The cosQ mutants fall into four groups on the basis of virus yield (WIECZOREK and FEISS 2001 ), as follows (Table 1). requires a minimum yield of 5.5 phage per cell to form a plaque (WIECZOREK and FEISS 2001 ); and cosQ⁺ has a yield of 85 phage per induced lysogen. Six mutations are slight, with virus yields of >10.0. Four mutations are mild, giving yields between 5.5 and 10. Three mutations are moderate, giving yields between 1.0 and 5.5, while eight severe mutants have yields of <1.0. The 11 phages with moderate and severe cosQ mutations are inviable, i.e., unable to form plaques. We screened plaque-forming revertants of these inviable cosQ mutants for cosQ suppressors. Plaque-forming revertants were selected from unmutagenized, mutD-mutagenized, and in vitro-mutagenized stocks of cosQ mutants. Although most revertants were true revertants, pseudorevertants with cis- and trans-acting suppressors were found (Table 2). The cis-acting suppressors will be presented in a separate report.

Spontaneous revertants of cosQ4:
Revertants of a nonmutagenized moderate mutant, cosQ4 (T_48,479A), were screened for trans-acting suppressors as follows. cosQ4 exhibits a reversion frequency of 1 x 10^-6. Lysogens of revertants were transformed with an Ap^r cosmid containing the cosQ4 mutation. The cosmid contains an ampicillin resistance gene (Ap^r), and the ability of cosQ4 to package the cosQ4 cosmid was determined by assaying the titer of Ap^r transducing particles in lysates of the cosQ4 revertants. We reasoned that cosQ4 revertants with trans-acting suppressors would be able to package the mutant cosmid more efficiently than true revertants or phages with cis-acting suppressors.

The cosmid packaging assay was used to screen 143 cosQ4 revertants. Revertants with at least a 2.5-fold increase in the number of Ap^r transducing particles, i.e., cosQ4 cosmid-containing phages, relative to the cosQ4 control, were chosen as possibly carrying trans-acting suppressors. Three of the 26 revertants chosen, rev98, rev22, and rev33, were initially sequenced across cos and found to contain the cosQ4 mutation and no suppressor in cosQ. Because the cosQ4 parent is inviable, we assayed phage titers as kanamycin-transducing phages. The cosQ4 rev98, cosQ4 rev22, and cosQ4 rev33 phages had yields 3- to 8-fold higher than the cosQ4 yield (Table 3).

To map the suppressors, plasmids bearing MluI restriction fragments of cosQ4 rev98, cosQ4 rev22, and cosQ4 rev33 prophage DNAs were prepared. Crosses of the plasmids vs. cosQ4 were used to screen for inserts carrying suppressors. For all three revertants, a suppressor was present in the MluI fragment from base pair 458 to 5548. The rev98 suppressor was further narrowed to the EcoO109I-RsrII fragment from base pair 2815 to 3800. The rev22 and rev33 suppressors were in the RsrII-AvaI fragment from base pair 3800 to 4720. All three suppressors mapped to the B gene. The characteristics of the mutations are described in Fig 2.

View larger version (38K): In this window In a new window Download PPT slide   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).

Two other trans-acting cosQ suppressors in the B gene were described previously. CUE and FEISS 1997 identified Bms6 and Bms8 as suppressors of cosQ1. The Bms8 mutation had previously been identified as a suppressor of a mutation altering the prohead binding domain of the gpA subunit of terminase (YEO and FEISS 1995B ). Given that all five trans-acting cosQ suppressors were in B, we sequenced the B genes of additional revertants of cosQ4. Rev31 was found to contain a single mutation in B, and its ability to suppress cosQ4 was confirmed by marker rescue crosses (data not shown). Rev66 was identical to rev33. In spite of the screen for trans-acting suppressors, 4 revertants were B⁺ and were found to contain the local suppressor T_48,479C, and the 17 others were true revertants. Overall, the in vivo cosmid packaging screen resulted in the identification of four new B gene suppressors of cosQ4 (Fig 2) and a local suppressor.

Spontaneous revertants of other cosQ mutations:
We screened plaque-forming revertants in unmutagenized lysates of phages with eight of the severe cosQ mutations: G_48,473T, G_48,473C, G_48,474A, G_48,474T, G_48,474C, C_48,478A, C_48,478T, and C_48,478G (Table 1). Lysogens of plaque-forming revertants were screened by sequencing cos to identify true revertants and revertants with local, cis-acting suppressors in cosQ. Of 423 revertants of the eight mutants, a total of 15 cis-acting revertants were found, all suppressors of only two cosQ point mutations, cosQ G_48,473T and cosQ G_48,473C (Table 2). No trans-acting revertants were recovered.

E. coli mutD mutagenesis:
Because the high percentage of true revertants hampered the isolation of pseudorevertants, we searched for pseudorevertants in lysates of mutD-mutagenized cosQ mutants with the following moderate and severe mutations: G_48,474C, C_48,478A, C_48,478T, C_48,478G, G_48,473T, G_48,473C, G_48,474A, G_48,474T, G_48,475C (cosQ3), and T_48,479G (cosQ5). On average, the E. coli mutD mutagenesis resulted in 500-fold increase in the titer of plaque-forming revertants when compared to the titer of nonmutagenized control lysates (0–500 pfu/ml). Lysogens of revertants were first screened by using PCR to generate cos-containing segments and then digested with the EcoO109I restriction enzyme. Since cosQ⁺ is identical to the EcoO109I recognition sequence, digestion with EcoO109I indicates reversion to cosQ⁺.

We again found that most cosQ revertants were true revertants. Numerous cis-acting suppressors, within cosQ, were also identified by sequencing cos. One trans-acting suppressor of cosQ5 (T_48,479G) was identified. This results in a change in the same codon as the Brev22 suppressor. These suppressors are located just four amino acids away from Bms6 at codon 450 (Fig 2).

Segment-specific PCR mutagenesis:
When it became clear that most of the spontaneous and mutD-induced revertants were true revertants, we employed PCR mutagenesis to target our mutagenesis to specific genes known to be involved in DNA packaging. An obvious candidate was the B gene, since B was already known to contain a variety of suppressors of multiple cosQ mutations. Mutagenized clones of the B gene were amplified, and a library of 1000 clones was pooled for transformation into a cosQ4 (T_48,479A) lysogen. Plasmid vs. cosQ4 crosses were carried out by prophage induction, and 40 plaque formers were selected for analysis. Crosses with mutagenized B produced an 1000-fold increase in revertants when compared with nonmutagenized B control crosses.

Each of the 40 plaque formers was sequenced across cosQ to confirm the presence of the parental cosQ4 mutation. Then the B gene was sequenced. Seven suppressors were identified; two were identical to Brev22, and one was identical to Brev33. Rev24 contained a new single B mutation, and its ability to suppress cosQ4 was confirmed by marker rescue (data not shown). Two revertants, Rev13 and Rev17, were sequenced and found to be sibs with three B mutations; when separated from each other, the suppressor mutation was found to result in a substitution of lysine for methionine in codon 397. These new cosQ suppressors were located close to the previously identified Brev31. In sum, nine total B suppressors have been identified, including the two described by CUE and FEISS 1997 . Two clusters involving six of these trans-acting cosQ suppressors were identified in B (Fig 2), affecting codons 394–397 and 450–454. The virus yields of all of the suppressors of cosQ mutations in gene B are shown in Table 3.

We also used PCR mutagenesis to target the terminase genes, Nu1 and A. Mutagenized clones of the Nu1 and A genes were amplified, and a pool of 1000 clones was used to transform lysogens of cosQ3 (G_48,475C), cosQ4 (T_48,479A), cosQ5 (T_48,479G), G_48,473T, G_48,473C, G_48,474A, and G_48,474T. Lysogens with plasmids bearing unmutagenized wild-type Nu1 and A were included as controls. There was no difference in the titers of plaque-forming phages in the lysates from cells with mutagenized and nonmutagenized terminase genes, indicating that the mutagenesis had not produced any cosQ suppressors in the terminase genes. To show that the PCR mutagenesis had worked, we crossed the mutagenized and nonmutagenized terminase plasmids with cosN G₂C C₁₁G, a phage carrying mutations known to be suppressed by mutations in A (SIPPY ARENS et al. 1999 ). The mutagenized pool of terminase plasmids gave a 6.5-fold increase in the titer of revertants from the cells with the unmutagenized terminase plasmid pool.

Studies on the allelic specificity of cosQ suppressors in B:
To ask whether the trans-acting cosQ suppressors were allele specific, we first constructed cos⁺ prophages containing the four B gene suppressors of cosQ4, and virus yield assays were performed (Table 4). All of the cosQ⁺ Brev phages were healthy, and each had a yield larger than that of the corresponding cosQ4 Brev phage. The results indicate the suppressors are not allele specific. Previous work also showed the Bms6 and Bms8 mutations did not impair growth of cosQ⁺ phages (CUE and FEISS 1997 ).

View this table: In this window In a new window   Table 4. Relative virus yields of Brev single and double mutants

It has been proposed that the cosQ suppressors in B act by slowing the rate of DNA translocation into the prohead, thereby making recognition of a mutant cosQ more efficient (CUE and FEISS 1997 ). We wished to ask if multiple suppressors would have additive effects on cosQ suppression. To test for additive effects, phages with two cosQ suppressors in the cosQ4 background were constructed. Virus yield studies showed clearly that the suppressors had additive effects (Table 4). We also asked whether multiple suppressors would have effects on the growth of cosQ⁺. Yield studies showed that the multiple suppressors tested had at best minimal effects on cosQ⁺ growth.

To test for allele specificity, we crossed phages with three different lethal cosQ point mutations, cosQ3 (G_48,475C), cosQ4 (T_48,479A), and cosQ5 (T_48,479G), with plasmids carrying each of the nine cosQ suppressors in B and looked for viable recombinants (Table 5). Suppression of cosQ1 (C_48,477T), which produces tiny plaques, by Bms6 and Bms8 was confirmed by measuring the number of large plaque variants produced from the cross. All cosQ suppressors in B were able to suppress multiple cosQ point mutations, demonstrating that each of the suppressors is an allele-nonspecific cosQ suppressor.

View this table: In this window In a new window   Table 5. Suppression of cosQ mutants by suppressors in gene B

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mutagenesis strategy and results:
We employed three strategies in looking for cosQ suppressors. Revertants from unmutagenized and E. coli mutD-mutagenized stocks of cosQ mutants were mostly revertants to cosQ⁺, making it hard to find cosQ suppressors. The excess of true revertants is likely because they have a yield higher than that of pseudorevertants. Segment-specific PCR mutagenesis was used to target gene B; this approach yielded a large majority of trans-acting suppressors in B.

Identification of trans-acting suppressors in gpB:
We obtained 7 new suppressors of cosQ affecting the portal protein, gpB, by isolating revertants of unmutagenized, E. coli mutD-mutagenized, and PCR-mutagenized stocks of cosQ mutants. Previously, 2 suppressors of cosQ1, Bms6 and Bms8, were isolated by CUE and FEISS 1997 . Although the sample size is small, of the 9 trans-acting suppressors, 2 were independently recovered twice. Using the Poisson distribution and the m = 1 and m = 2 terms of 7 and 2, respectively, provides a rough estimate that there is a total of 22 potential B gene suppressors of cosQ (BENZER 1961 ).

The nine cosQ suppressors are not uniformly distributed throughout B; two clusters involving six of the cosQ suppressors are seen (Fig 2). The first lies in the region from amino acid residues 394 to 397. This cluster contains cosQ4 Brev31, cosQ4 Brev24, and cosQ4 Brev17 at codons 394, 395, and 397, respectively. The amino acid sequence immediately following the methionine at codon 397 is the sequence 398-Gly-Arg-Arg-Lys-401. Of particular note are the two arginines followed by a lysine, three highly basic residues. Interestingly, one of the suppressors, Brev17, substitutes an additional basic residue, lysine, for the neutral methionine residue at codon 397. The changes in gpB created by the cosQ suppressor mutations are various. In addition to the charge change of Brev17, M₃₉₇K, others include the polar to nonpolar S₄₅₀F change of Bms6; the nonpolar to polar changes Y₃₉₅H, P₃₃₁S, A₄₅₄T, and A₄₉₁S of Brev24, Bms8, Brev22, and Brev33, respectively; and the nonpolar to nonpolar changes I₈₁V, A₃₉₄V, and A₄₅₄V of Brev98, Brev31, and Brev1, respectively. Clearly, structural information about the locations of the affected residues and the effects of the amino acid changes on the structure are needed to make detailed statements about how the gpB changes suppress cosQ mutations.

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 (VALPUESTA and CARRASCOSA 1994 ). Since portals do appear to have a similar structure, it is likely that they are descendants of a common ancestor, but have diverged sufficiently so that no sequence identity is seen (HENDRIX et al. 1999 ). The molecular structure of the dodecameric 29 portal protein, gp10, has recently been solved by X-ray crystallography (SIMPSON et al. 2000 ). The structure of the 309-residue gp10 protein exhibits three cylindrical regions: the narrow end, which protrudes from the vertex and is encircled by the pentameric pRNA; the central channel; and the wide end, which contacts the inside of the prohead (SIMPSON et al. 2000 ; Fig 3). The inside of the channel has a preponderance of negative charges at its wide end, which are proposed to repel the DNA, permitting its smooth passage during packaging and ejection. Also, the amino terminus of gp10 has been shown to interact with DNA to be packaged (HERRANZ et al. 1986 , HERRANZ et al. 1990 ; DONATE et al. 1992 , DONATE et al. 1993 ; TURNQUIST et al. 1992 ; VALPUESTA et al. 1992 ; ZACHARY and BLACK 1992 ; VALLE et al. 1996 , VALLE et al. 1999 ).

View larger version (61K): In this window In a new window Download PPT slide   Figure 3. General model of the structure of the tailed, dsDNA bacteriophage portal. The model is based on the known structures of the portals of bacteriophages 29 and SPP1 (ORLOVA et al. 1999 ; SIMPSON et al. 2000 ). 29 and SPP1 exhibit similar structures consisting of three domains: (1) a wing domain (WD), or wide end, that contacts the prohead; (2) a stem domain (SD) or central channel that has been suggested to be the docking region for terminase and the packaging complex and the region through which it is presumed the DNA translocates through into the prohead; and (3) the tentacle domain (TD). In SPP1, the TD is thought to be the region of the "headful sensor" that is necessary for packaging termination. In 29, two stretches of residues, disordered in the crystal structure, occur in the DNA channel near the border between the central and wide regions of the connector, similar to the location of the TD of SPP1, and were proposed to be ideal for interacting with the negatively charged DNA. The possibility exists that this is the headful sensor of 29. These stretches of disordered residues are similar to the sequence immediately following the cluster of cosQ suppressors identified in the portal protein of . Thus, this cluster may reside near a comparable headful sensor in necessary for packaging termination. A second cluster was proposed to reside in a region that is near the gauge that measures DNA packaging, resulting in a conformational change of this gauge, or might simply lie in the channel through which the DNA passes, resulting in steric hindrance in the DNA channel of the portal and causing the slowing of the translocating DNA. The direction of DNA entry is given by the arrow.

Studies on the phages SPP1 and P22 have shown that mutations altering the portal proteins affect DNA packaging, as follows. Bacillus subtilis bacteriophage SPP1 uses a headful packaging strategy in which the DNA cleavage that terminates DNA packaging is triggered when the head shell is filled with DNA; the downstream cleavage, which is not sequence specific, generates a terminal redundancy. TAVARES et al. 1992 identified three mutations, called siz mutations, that affect the 503-residue SPP1 portal protein, gp6, and result in a 4–6% decrease in the amount of DNA packaged. Each siz mutation substitutes a basic residue: Two are E K changes (residues 251 and 424) and the other is an N K change (residue 365). Like SPP1, the Salmonella phage P22 packages DNA by the headful mechanism. Mutations in the 725-residue P22 portal protein gene 1 have been described that are opposite in effect to the SPP1 siz mutants, in that 4% excess DNA is packaged (CASJENS et al. 1992 ). The two P22 gene 1 mutations both produce V M changes (residues 64 and 303). In both the SPP1 and P22 cases, the authors argue that headful packaging requires a sensing mechanism that detects when the head is filled. The sensor is proposed to detect some parameter correlated with head filling such as an increase in DNA packing density, a decrease in the rate of DNA translocation, or an increase in the energy required to continue packaging (Fig 3; CASJENS et al. 1992 ; TAVARES et al. 1992 ; ORLOVA et al. 1999 ).

In the 29 portal protein, gp10, two stretches of residues that are disordered in the crystal structure (residues 229–246 and 287–309) occur in the DNA channel near the border between the central and wide regions of the connector. These disordered residues, located in the C terminus of gp10, include the amino acid sequences 233-Glu-Lys-Lys-Glu-Arg-237 and 289-Arg-Arg-Glu-291, which are proposed to interact with the negatively charged sugar-phosphate DNA backbone. These segments with tandem basic residues are similar to residues that follow the cluster of suppressors we identified in the portal protein of , 398-Gly-Arg-Arg-Lys-401. This highly basic stretch acquires a further net positive charge from the Brev17 suppressor change. The gp10 amino terminus also contains a basic segment: 1-Ala-Arg-Lys-Arg-4. This basic sequence is necessary for DNA packaging and is essential for efficient DNA binding (DONATE et al. 1992 ). It is proposed that this region interacts with the negatively charged DNA. It is possible that the region we have identified in the 533-residue portal protein of , 398-Gly-Arg-Arg-Lys-401, performs a similar role (Fig 3). Deletion studies and site-specific mutagenesis of this region would be helpful in determining whether this region is necessary for DNA packaging and reveal any inherent DNA-binding activity for gpB.

As with phages SPP1 and P22, DNA packaging also requires a headful sensor since the efficiency of the terminal cutting depends on the extent of DNA packaging (FEISS and SIEGELE 1979 ). For the siz mutations of SPP1, which cause substitutions of basic residues in gp6, it is tempting to propose that electrostatic interactions slow DNA translocation so that the sensor triggers cleavage when the triggering rate of translocation is reached prematurely. Similarly, the P22 mutations may raise the threshold required to trigger cleavage.

The second cluster of cosQ suppressors lies in gpB residues 450–454 (Fig 2). This cluster contains cosQ1 Bms6 (S₄₅₀F), cosQ4 Brev22 (A₄₅₄T), and cosQ5 Brev1 (A₄₅₄V). 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 ų vs. 73 ų for serine (RICHARDS 1974 ). Threonine (Brev22) and valine (Brev1) have Van der Waals volumes of 93 ų and 105 ų, respectively, vs. 67 ų 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 (Fig 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 (Fig 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, consistent 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 packaged DNA and in the termination of DNA packaging, the steric hindrance model presumes the portal has a passive, more general role in packaging termination.

We looked for additive effects produced by combining suppressors. Of the double mutants constructed in the cosQ⁺ background, none was found to have more than a minor decrease in virus yield. The fact that these suppressors are mild suppressors may make it difficult to observe significant additive effects on phage production in this background. Since the time to package a genome is very fast, (1–2 min; HOHN 1975 ) in comparison with the length of the virus growth cycle (60 min), a suppressor that slows packaging to an extent sufficient for cosQ suppression might not greatly affect virus yield.

In contrast, each of the double mutants constructed in the cosQ4 background resulted in a significant increase in phage yield compared to the effects of the individual cosQ suppressors, signifying the additive effects of the B gene suppressors on phage production in the cosQ4 background. Thus, it appears that Brev98 acts to suppress cosQ4 in a manner independent from suppression by Brev22, Brev33, and Brev31. It is interesting to note that Brev98 is the only suppressor thus far identified that is located in the amino third of the protein. Each of the other suppressors identified has been localized to the carboxy third of the B protein. We propose that cosQ suppressors in gene B enhance recognition of a mutant cosQ by slowing packaging. Since the B suppressor mutations cause such a variety of residue changes, no single explanation for how each works is obvious. The failure of the B suppressors to suppress all cosQ mutations is most likely due to the mild strength of the suppressors and the severity of the individual cosQ mutations. Clearly, structural information about wild-type and mutant gpB proteins is required, along with molecular information on the effects of the suppressors on DNA packaging.

Despite our intense efforts, the trans-acting suppressors we identified were all allele-nonspecific suppressors of cosQ mutations. This inability to detect allele-specific suppressors may be because the component that recognizes cosQ has multiple roles in DNA packaging. Thus, mutations in the domain of this component that suppresses cosQ mutations may disrupt other necessary functions. For instance, it is not surprising that there was a failure to identify suppressors of cosQ mutations in terminase. gpA has numerous roles in DNA packaging, including interacting with gpNu1, nicking at cosN, hydrolyzing ATP (to separate the cohesive ends and presumably for translocating DNA), gpA dimerization, and interaction with the portal. Because of its numerous roles, it is quite possible that if gpA also recognizes cosQ, however unlikely, any mutation in gpA that suppresses a cosQ mutation may have detrimental effects on other gpA functions, rendering the protein nonfunctional.

	ACKNOWLEDGMENTS

We thank our co-workers, Alok Dhar, Carol Duffy, Qi Hang, Jason Luke, Jenny Meyer, and Jean Sippy for advice and interest during the course of this work. The work involving the effects of multiple B suppressors on cosQ⁺ growth constitutes part of the undergraduate honors thesis of Lisa Didion. This work was supported by National Institutes of Health (NIH) research grant GM-51611, NIH genetics research training grant T32GM08629 (D.W.), and NIH Iowa kidney disease, hypertension, and cell biology research training grant DK07690-10 (D.W.).

Manuscript received November 5, 2001; Accepted for publication February 18, 2002.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ARBER, W., L. ENQUIST, B. HOHN, N. E. MURRAY and K. MURRAY, 1983 Experimental methods for use with lambda, pp. 433–466 in Lambda II, edited by R. W. HENDRIX, J. W. ROBERTS, F. W. STAHL and R. A. WEISBERG. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

BEAR, S., D. COURT, and D. I. FRIEDMAN, 1983  An accessory role for integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging. J.Virol. 52:966-972.

BECKER, A. and H. MURIALDO, 1990  Bacteriophage DNA: the beginning of the end.. J. Bacteriol. 172:2819-2824[Free Full Text].

BENZER, S., 1961  On the topography of the genetic fine structure. Proc. Natl. Acad. Sci. USA 47:403-415[Free Full Text].

CASJENS, S., E. WYCKOFF, M. HAYDEN, L. SAMPSON, and K. EPPLER et al., 1992  Bacteriophage P22 portal protein is part of a gauge that regulates packing density of intravirion DNA. J. Mol. Biol. 224:1055-1074[Medline].

CATALANO, C. E., D. CUE, and M. FEISS, 1995  Virus DNA packaging: the strategy used by phage . Mol. Microbiol. 16:1075-1086[Medline].

CUE, D. and M. FEISS, 1993  A site required for the termination of packaging of the phage chromosome. Proc. Natl. Acad. Sci. USA 90:9290-9294[Abstract/Free Full Text].

CUE, D. and M. FEISS, 1997  Genetic evidence that recognition of cosQ, the signal for termination of phage DNA packaging, depends on the extent of head filling. Genetics 147:7-17[Abstract].

CUE, D. and M. FEISS, 1998  Termination of packaging of the bacteriophage chromosome: cosQ is required for nicking the bottom strand of cosN.. J. Mol. Biol. 280:11-29[Medline].

DANIELS, D., J. SCHROEDER, W. SZYBALSKI, F. SANGER, A. COULSEN et al., 1983 Completed annotated lambda sequence, pp. 519–676 in Lambda II, edited by R. W. HENDRIX, J. W. ROBERTS, F. W. STAHL and R. A. WEISBERG. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

DAVIDSON, A. and M. GOLD, 1992  Mutations abolishing the endonuclease activity of bacteriophage terminase lie in two distinct regions of the A gene, one of which may encode a "leucine zipper" DNA binding domain. Virology 189:21-30[Medline].

DONATE, L. E., J. M. VALPUESTA, A. ROCHER, E. MENDEZ, and F. ROJO et al., 1992  Role of the amino-terminal domain of bacteriophage 29 connector in DNA binding and packaging. J. Biol Chem. 267:10919-10924[Abstract/Free Full Text].

DONATE, L. E., J. M. VALPUESTA, C. MIER, F. ROJO, and J. L. CARRASCOSA, 1993  Characterization of an RNA-binding domain in the bacteriophage 29 connector. J. Biol. Chem. 268:20198-20204[Abstract/Free Full Text].

EMMONS, S. W., 1974  Bacteriophage lambda derivatives carrying two copies of the cohesive end site.. J. Mol. Biol. 83:511-525[Medline].

FEISS, M., 1986  Terminase and the recognition, cutting, and packaging of chromosomes. Trends Genet. 2:100-104.

FEISS, M. and D. A. SIEGELE, 1979  Packaging of the bacteriophage chromosome: dependence of cos cleavage on chromosome length. Virology 92:190-200[Medline].

FEISS, M., J. SIPPY, and G. MILLER, 1985  Processive action of terminase during sequential packaging of bacteriophage chromosomes. J. Mol. Biol. 186:759-771[Medline].

FOWLER, R. G., G. E. DEGNEN, and E. C. COX, 1974  Mutational specificity of a conditional Escherichia coli mutator, mutD5.. Mol. Gen. Genet. 133:179-191[Medline].

FURTH, M. E., and S. H. WICKNER, 1983 Lambda DNA replication, pp. 145–173 in Lambda II, edited by R. W. HENDRIX, J. W. ROBERTS, F. W. STAHL and R. A. WEISBERG. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

HANAHAN, D., 1983  Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580[Medline].

HENDRIX, R. W., M. C. M. SMITH, R. N. BURNS, M. E. FORD, and G. F. HATFULL, 1999  Evolutionary relationships among diverse bacteriophages and prophages: all the world is a phage. Proc. Natl. Acad. Sci. USA 96:2192-2197[Abstract/Free Full Text].

HERRANZ, L., M. SALAS, and J. L. CARRASCOSA, 1986  Interaction of the bacteriophage 29 connector protein with the viral DNA. Virology 155:289-292[Medline].

HERRANZ, L., J. BORDAS, E. TOWNS-ANDREWS, E. MENDEZ, and P. USOBIAGA et al., 1990  Conformational changes in bacteriophage 29 connector prevent DNA-binding activity. J. Mol. Biol. 213:263-273[Medline].

HOHN, B., 1975  DNA as a substrate for packaging into bacteriophage lambda in vitro.. J. Mol. Biol. 98:93-106[Medline].

HWANG, Y. and M. FEISS, 1996  Mutations affecting the high affinity ATPase center of gpA, the large subunit of bacteriophage terminase, inactivate the endonuclease activity of terminase. J. Mol. Biol. 261:524-535[Medline].

KOCHAN, J. and H. MURIALDO, 1983  Early intermediates in bacteriophage lambda prohead assembly. Virology 131:100-115[Medline].

KOCHAN, J., J. L. CARRASCOSA, and H. MURIALDO, 1984  Bacteriophage lambda preconnectors: purification and structure. J. Mol. Biol. 174:433-447[Medline].

KOSTURKO, L., E. DAUB, and H. MURIALDO, 1989  The interaction of E. coli integration host factor and lambda cos DNA multiple complex formation and protein-induced bending. Nucleic Acids Res. 17:329-334.

MENDELSON, I., M. GOTTESMAN, and A. B. OPPENHEIM, 1991  HU and integration host factor function as auxiliary proteins in cleavage of phage lambda cohesive ends by terminase. J. Bacteriol. 173:1670-1676[Abstract/Free Full Text].

ORLOVA, E. V., P. DUBE, E. BECKMANN, F. ZEMLIN, and R. LURZ et al., 1999  Structure of the 13-fold symmetric portal protein of bacteriophage SPP1. Nat. Struct. Biol. 6:842-846[Medline].

PAL, S. K. and D. K. CHATTORAJ, 1988  P1 plasmid replication: initiator sequestration is inadequate to explain control by initiator-binding sites. J. Bacteriol. 172:2819-2824.

RICHARDS, F. M., 1974  The interpretation of protein structures: total volume, group volume distributions and packing density. J. Mol. Biol. 82:1-14[Medline].

RUBINCHIK, S., W. PARRIS, and M. GOLD, 1994  The in vitro endonuclease activity of gene product A, the large subunit of the bacteriophage terminase, and its relationship to the endonuclease activity of the holoenzyme. J. Biol. Chem. 269:13575-13585[Abstract/Free Full Text].

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Cloning: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SANGER, F., A. R. COULSON, G. F. HONG, D. F. HILL, and G. B. PETERSEN, 1982  Nucleotide sequence of bacteriophage lambda DNA. J. Mol. Biol. 162:729-773[Medline].

SHINDER, G. and M. GOLD, 1988  The Nu1 subunit of bacteriophage lambda binds to specific sites in cos DNA. J. Virol. 62:387-392[Abstract/Free Full Text].

SIMPSON, A. A., Y. TAO, P. G. LEIMAN, M. O. BADASSO, and Y. HE et al., 2000  Structure of the bacteriophage 29 DNA packaging motor. Nature 408:745-750[Medline].

SIPPY ARENS, J., Q. HANG, Y. HWANG, B. TUMA, and S. MAX et al., 1999  Mutations that extend the specificity of the endonuclease activity of terminase. J. Bacteriol. 181:218-224[Abstract/Free Full Text].

SIX, E. and C. A. C. KLUG, 1973  Bacteriophage P4: a satellite virus depending on a helper such as prophage P2. Virology 51:327-344[Medline].

SMITH, D. E., S. J. TANS, S. B. SMITH, S. GRIMES, and D. L. ANDERSON et al., 2001  The bacteriophage 29 portal motor can package DNA against a large internal force. Nature 413:748-752[Medline].

STERNBERG, N. and S. AUSTIN, 1983  Isolation and characterization of P1 minireplicons, -P1:5R and -P1:5L. J. Bacteriol. 153:800-812[Abstract/Free Full Text].

TAVARES, P., M. A. SANTOS, R. LURZ, G. MORELLI, and H. DE LENCASTRE et al., 1992  Identification of a gene in Bacillus subtilis bacteriophage SPP1 determining the amount of packaged DNA. J. Mol. Biol. 225:81-92[Medline].

TSUI, L. and R. W. HENDRIX, 1980  Head-tail connector of bacteriophage lambda. J. Mol. Biol. 142:419-438[Medline].

TURNQUIST, S., M. SIMON, E. ENGELMAN, and D. ANDERSON, 1992  Supercoiled DNA wraps around the bacteriophage 29 head-tail connector. Proc. Natl. Acad. Sci. USA 89:10479-10483[Abstract/Free Full Text].

VALLE, M., J. M. VALPUESTA, J. L. CARRASCOSA, J. TAMAYO, and R. GARCIA, 1996  The interaction of DNA with bacteriophage phi 29 connector: a study by AFM and TEM. J. Struct. Biol. 116:390-398[Medline].

VALLE, M., L. KREMER, C. MARTINEZ-A, F. RONCAL, and J. M. VALPUESTA et al., 1999  Domain architecture of the bacteriophage 29 connector protein. J. Mol. Biol. 288:899-909[Medline].

VALPUESTA, J. M. and J. L. CARRASCOSA, 1994  Structure of viral connectors and their function in bacteriophage assembly and DNA packaging. Q. Rev. Biophys. 27:107-155[Medline].

VALPUESTA, J. M., M. SERRANO, L. E. DONATE, L. HERRANZ, and J. L. CARRASCOSA, 1992  DNA conformational change induced by the bacteriophage 29 connector. Nucleic Acids Res. 20:5549-5554[Abstract/Free Full Text].

WALKER, J. E., A. D. AUFRETT, A. CARNE, A. GURNETT, and P. HANISCH et al., 1982  Solid-phase sequence analysis of polypeptides eluted from polyacrylamide gels. An aid to interpretation of DNA sequences exemplified by the Escherichia coli unc operon and bacteriophage lambda. Eur. J. Biochem. 123:253-260[Medline].

WIECZOREK, D. J. and M. FEISS, 2001  Defining cosQ, the site required for termination of bacteriophage DNA packaging. Genetics 158:495-506[Abstract/Free Full Text].

XIN, W. and M. FEISS, 1993  Function of IHF in DNA packaging. I. Identification of the strong binding site for integration host factor and the locus for intrinsic bending in cosB. J. Mol. Biol. 230:492-504[Medline].

XIN, W., Z.-H. CAI, and M. FEISS, 1993  Function of IHF in DNA packaging. II. Effects of mutations altering the IHF binding site and the intrinsic bend in cosB on development. J. Mol. Biol. 230:505-515[Medline].

YANISCH-PERRON, C., J. VIEIRA, and J. MESSING, 1985  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119[Medline].

YEO, A. and M. FEISS, 1995a  Mutational analysis of the prohead binding domain of the large subunit of terminase, the bacteriophage DNA packaging enzyme. J. Mol. Biol. 245:126-140[Medline].

YEO, A. and M. FEISS, 1995b  Specific interaction of terminase, the DNA packaging enzyme of bacteriophage , with the portal protein of the prohead. J. Mol. Biol. 245:141-150[Medline].

ZACHARY, A. and L. W. BLACK, 1992  Isolation and characterization of a portal protein-DNA complex from dsDNA bacteriophage. Intervirology 33:6-16[Medline].

This article has been cited by other articles:

				D. J. Wieczorek and M. Feiss Genetics of cosQ, the DNA-Packaging Termination Site of Phage {lambda}: Local Suppressors and Methylation Effects Genetics, September 1, 2003; 165(1): 11 - 21. [Abstract] [Full Text] [PDF]