DNA Packaging Specificity of Bacteriophage N15 with an Excursion into the Genetics of a Cohesive End Mismatch

Michael Feiss; Jea Young Min; Sawsan Sultana; Priyal Patel; Jean Sippy

doi:10.1371/journal.pone.0141934

Abstract

During DNA replication by the λ-like bacteriophages, immature concatemeric DNA is produced by rolling circle replication. The concatemers are processed into mature chromosomes with cohesive ends, and packaged into prohead shells, during virion assembly. Cohesive ends are generated by the viral enzyme terminase, which introduces staggered nicks at cos, an approx. 200 bp-long sequence containing subsites cosQ, cosN and cosB. Interactions of cos subsites of immature concatemeric DNA with terminase orchestrate DNA processing and packaging. To initiate DNA packaging, terminase interacts with cosB and nicks cosN. The cohesive ends of N15 DNA differ from those of λ at 2/12 positions. Genetic experiments show that phages with chromosomes containing mismatched cohesive ends are functional. In at least some infections, the cohesive end mismatch persists through cyclization and replication, so that progeny phages of both allelic types are produced in the infected cell. N15 possesses an asymmetric packaging specificity: N15 DNA is not packaged by phages λ or 21, but surprisingly, N15-specific terminase packages λ DNA. Implications for genetic interactions among λ-like bacteriophages are discussed.

Citation: Feiss M, Young Min J, Sultana S, Patel P, Sippy J (2015) DNA Packaging Specificity of Bacteriophage N15 with an Excursion into the Genetics of a Cohesive End Mismatch. PLoS ONE 10(12): e0141934. https://doi.org/10.1371/journal.pone.0141934

Editor: Mark J. van Raaij, Centro Nacional de Biotecnologia - CSIC / CIF Q2818002D, SPAIN

Received: August 5, 2015; Accepted: October 14, 2015; Published: December 3, 2015

Copyright: © 2015 Feiss et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This project was supported by funding from National Science Foundation awards MCB-0717620 and MCB-1158495, the National Institutes of Health, United States awards 5R01GM088186 and 5R01GM051611 (www.nih.gov/), and the University of Iowa Department of Microbiology.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Large DNA viruses, such as tailed bacteriophages and herpes viruses, use an ATP-powered motor to translocate viral DNA into the preformed empty shell, called the prohead or procapsid [1–3]. Recent structural and bioinformatic studies demonstrate that the DNA packaging machinery of these viruses is descended from that of an ancient common ancestor [4]. For example, the prohead shell is an icosahedral lattice principally constructed of many copies of the major capsid protein whose fold is conserved [5]. Similarly, one of the prohead’s 5-fold vertexes, the unique portal vertex, contains the radially disposed dodecameric portal protein. The portal protein contains a channel through which DNA enters and exits the shell interior [6–9]. Terminase, also conserved in the herpes viruses and tailed bacteriophages, is usually a hetero-oligomer of small (TerS) and large (TerL) subunits [2,10–12]. TerS carries out viral DNA recognition. TerL is a motor protein whose N-terminal ATPase domain powers translocation of the DNA into the prohead. TerL also contains the C-terminal endonuclease domain that cuts concatemeric DNA into unit-length virion chromosomes.

TerS molecules contain three domains, as follows. An α-helical central domain oligomerizes TerS into cylindrical, 8- to 12-mer oligomers, depending on the virus. At the C-terminus is a β-barrel structure. The C-terminus contains a functional TerL binding domain, as shown for λ and 21 [13,14], P22 [15,16], and T4 [17,18]. In the case of P22’s TerL, short α-helical extensions at the extreme C-terminus may tether TerS to TerL [19]. At the TerS N-terminus is a DNA binding motif. In the cases of λ [20], Sf6 [21], T4 [18,22], and SF6 [23,24], the DNA binding motif is likely a classic helix-turn-helix. For SF6 TerS, the DNA binding domains are tethered to the central cylinder by linkers hence are highly mobile. It is proposed in a number of these cases that the DNA is wrapped around the TerS oligomer into a nucleosome-like structure by interactions with multiple TerS DNA binding domains [15,17,25,26]. Phage λ TerS is unusual in that in addition to oligomerizing, the DNA binding domain forms a tight dimer [20], as discussed further below.

Among the tailed dsDNA bacteriophages, pac phages use a headful mechanism to package virion DNA from concatemers, i.e., immature end-to-end multimers of virus DNA produced by rolling circle replication. An initial cut is made near a pac site, the TerS recognition site, and packaging is terminated by a non-specific cut when the prohead is full. The first termination cut is also the start of packaging of the next chromosome along the concatemer, as packaging is processive. The resulting viral chromosomes have a terminal redundancy, and individual chromosomes are circular permutations of the unique viral sequence. In contrast, virion DNAs of cos phages have a unique DNA sequence with short complementary cohesive ends. cos phages produce virion DNAs through TerL’s introduction of precisely staggered nicks in concatemeric DNA. The interaction of TerS with pac or cos recognition sites varies from phage to phage. pac phage P22’s TerS recognition site is a simple, 22 bp, asymmetric sequence located in the TerS gene [27]. In contrast, for pac phage SPP1, there are multiple TerS binding sites flanking the site where the initial cut is made [25]. For T4, the headful mechanism is used, but initiation is complex and not well understood [28].

For cos phage λ and its close relatives, virion chromosomes have cohesive ends, i.e., complementary, 5’-ended, 12 base-long, single-strand extensions that anneal, circularizing the DNA, upon entry into a host cell. The DNA segment containing the DNA packaging signals and the annealed cohesive ends is called cos. At late times during the lytic cycle, recombination and rolling circle replication produce concatemeric DNA [29]. During packaging, concatemeric DNA is recognized and processed by terminase into monomeric virion chromosomes. TerS^λ and TerL^λ are gene products of the Nu1 and A genes, respectively. TerS^λ’s winged helix-turn-helix (wHTH) domain, which specifically binds cos, forms a tight dimer [20]. The simplest form of λ terminase is the protomer, a TerS₂:TerL₁ heterotrimer. Protomers further assemble into [TerS₂:TerL₁]₄ tetramers [30,31]. TerS^λ recognizes λDNA by specific binding to cosB, a cos subsite adjacent to cosN, the nicking site at which TerL^λ introduces staggered nicks to generate the cohesive ends of mature virion chromosomes. Assembly of TerS^λ on cosB^λ positions TerL^λ’s endonuclease to introduce the staggered nicks at cosN [32–34]. When cosB is deleted or re-positioned, cosN nicking is inefficient and inaccurate, indicating that anchoring by TerS is critical for nick introduction by TerL [33–36].

Packaging is initiated when terminase binds and nicks a cos on a concatemer. Following cosN nicking and cohesive end separation, terminase forms a tight complex, Complex I, on the cosB-containing chromosomal end [37,38]. Complex I then docks on the portal protein, gpB, of the prohead [39,40]. Following docking of Complex I on the portal, TerL^λ’s ATPase is activated and ATP hydrolysis-powered translocation of the DNA into the shell ensues [41,42]. Terminase remains docked on the portal during translocation. When the next cos along the concatemer is encountered, terminase (1) nicks cosN, (2) dissociates from the portal, and (3) remains bound to the cosB-containing end of the next chromosome along the concatemer [43]. By remaining bound to the next chromosome along the concatemer as a Complex I, terminase processively packages downstream chromosomes. A third sub-site, cosQ, is essential for recognition and nicking of the downstream cos during termination of packaging [44,45]. In sum, cosN and cosB are required to initiate λDNA packaging, cosQ and cosN are required for termination, and all three subsites are required for processivity [46].

The 120 bp-long cosB^λ is complex, containing three TerS binding sites, R3, R2, and R1 (Fig 1) [38]. Between R3 and R2 is a binding site [47], I1, for IHF, the E. coli site-specific DNA bending protein [48–51]. IHF bends DNA into an approx. 180° hairpin [52]. At cos, the IHF-induced bend at I1 positions R3 and R2 such that the wHTH motifs of dimeric TerS can be docked into the major grooves [20]. Complex I likely includes this nucleoprotein structure [53].

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Fig 1. Comparison of the coses and terminase small subunit genes of λ, 21 and N15.

A. Complex cos structure of phages λ, 21, Monarch. R3, R2 and R1 sequences are TerS binding sites, and I1 is an IHF binding site. B. Simple cos of N15 and relatives [58]. rR2 is a proposed accessory TerS binding site in the cos of N15 and relatives.

https://doi.org/10.1371/journal.pone.0141934.g001

In this paper, we refer to the λ-like phages as those with λ-like cohesive ends, i.e., 12 base-long, 5’ extensions with significant identity to λ's cohesive ends. This group includes λ, 434, ϕ80, 21, N15, Monarch, and gifsy-1 [54]. In the cosNs of this group, bp differences in the right half of the cohesive end sequences, i.e., bp 7–12, are common. For example N15’s cohesive ends differ from λ’s at bp 9 and 12, and gifsy-1’s differ at positions 8, 9, and 11. In contrast, the 7 bp cosQ site is highly conserved. The cosBs of the λ-like phages differ both in structural complexity and in packaging specificity, as follows.

With respect to cosB structure, DNA sequence analysis suggest that many λ-like phages have a complex cosB similar to cosB^λ, with three R sequences with R2 oriented opposite to R3 and R1, and with a likely IHF binding site between R3 and R2. Recently, N15 was shown to have a different, simpler cosB consisting of a single critical TerS binding site located approximately at the position of the R3 in complex cosBs. A second, inverted sequence that closely matches R3^N15, called rR2, is located within the TerS gene, about 200 bp distal to R3^N15, and though not critical for DNA packaging, is proposed to play a non-essential, accessory role in DNA packaging. The R1 sequence of cosB^λ plays such a role [55]. Examination of prophages in Genebank identified an additional five prophages that have the R3 and rR2 sequences found in N15’s cosB [56]. In sum, two strikingly different cosB structures, complex and simple, are found in the λ-like phages.

In addition to structural differences, packaging specificity differences are found in the λ-like phages, as exemplified by the well-characterized specificities of λ and 21. λ and 21 have identical cosQ and cosN sequences, yet each package self-DNA >10³-fold more efficiently than that of the other phage. Examination of the recognition helix and cosB R site sequences shows significant differences that account for the specificity difference (Fig 2) [20,57]. Genetic analysis identified recognition helix:R site contacts that differ between λ and 21, plus clashes involved in discrimination by TerS^λ and TerS²¹ against the heterologous DNA [56]. Looking at TerS^N15 and R3^N15 suggests that N15, and its relatives with simple cosBs might have a novel packaging specificity distinct from the λ and 21 specificities (Fig 2)[58]. Interestingly, a second group of N15-like prophages, exemplified by Monarch, have complex, tripartite cosBs. TerS^Monarch and TerS^N15 have identical recognition α-helixes and strong R site identity, suggesting that both subgroups share the same packaging specificity. Here we present a study of N15’s packaging specificity. Because of the strategy used, a preliminary analysis of the genetics of phage chromosomes with mismatched cohesive ends, λ versus N15, was carried out.

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Fig 2. Alignments of the cosB and TerS DNA packaging recognition elements of λ-like bacteriophages.

A. Alignment of R3 sequences of 21, Monarch, N15, and λ. Numbering is rightwards from the first base of the left cohesive end of each phage. Putative R3 sequences are highlighted in grey. The boundaries and locations of the R3 elements are only approximate and are mostly based on sequence conservation among the R3, R2 and R1 elements. Residues 56 and 59 in R3^λ and R3²¹ are important for recognition by TerS and are highlighted in green [56]. The CG→TA change at R3^λ bp 58 (highlighted in green) renders λ IHF-dependent for plaque formation [49,70][62]. B. Alignments of the left ends of TerS subunits. Proposed recognition helixes are highlighted in grey and residues 20 and 24 of TerS²¹ and TerS^λ, known to be involved in packaging recognition and discrimination, are highlighted in green.

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Results

Genetics of a cohesive end mismatch: Phages with mismatched cohesive ends are viable; the mismatches can persist to post-DNA replication

Strategy for studying DNA packaging specificity.

To ask about the effects of cosN and cosB differences on DNA packaging, we used a helper phage/passive prophage approach (Fig 3). In these experiments, the DNA packaging substrate, a passive prophage, is provided to the DNA packaging machinery of a lytically growing “helper” phage, as follows. Two prophages integrated in tandem at attB contain between them a chromosome that mimics a chromosome in a concatemer. Terminase from a helper phage can initiate packaging at the cos of the upstream prophage, translocate DNA and terminate packaging at the cos of the downstream prophage. In our experiments, the helper phage was a heat-inducible (cI857) derivative of a λ strain that forms a plasmid prophage. Helpers with λ, 21 or N15 DNA packaging specificity were used. The 21-and N15-specific helpers were viable hybrid derivatives of λ in which a left chromosome end, the DNA segment including cosB^λ and much of the TerS^λ-encoding Nu1 gene, was replaced with the functionally analogous segment from phages 21 or N15, respectively. The 21- and N15-specific helpers are called λ21^hy51 [59] and λN15^hy4[58], respectively. These helper phages are viable hybrid phages in which the left λ DNA end is replaced with the corresponding left end of the 21 and N15 chromosome, respectively. The left end segments, each about 600 bp in length, include 21’s or N15’s cosB and the 5’ end of the TerS-encoding gene which contains the helix-turn-helix DNA binding specificity motif. The corresponding chimeric TerS proteins are called TerS^hy51 and TerS^hy4, respectively. When a helper prophage is heat-induced to carry out lytic growth, the tandem prophages remain repressed because the prophages have thermo-stable imm²¹and imm⁴³⁴ immunity repressors that differ in specificity from the imm^λ helper. The maximum number of packageable passive prophages equals the number of bacterial chromosomes in the cell, so efficient packaging of a prophage gives a yield of 1–4 virions/infected cell; the helper yield was also low, at ~10–20/induced lysogen.

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Fig 3. Helper packaging strategy.

The large and small rectangles represent the envelope and chromosome of an E. coli cell with three prophages. Prophages1 and 2 are inserted in tandem in the bacterial chromosome, and the circle represents a third, heat-inducible plasmid prophage, the helper prophage. Induction of the helper prophage to lytic growth provides packaging components for both helper DNA and prophage DNA, provided the helper’s terminase can initiate packaging at the cos of upstream prophage 1 and terminate packaging at the cos of downstream prophage 2. The DNA packaged from the tandem prophages is represented by the horizontal arrow. The tandem prophages are not heat-inducible and remain repressed during the experiment.

https://doi.org/10.1371/journal.pone.0141934.g003

Genetics of mismatched cohesive ends.

We did preliminary helper packaging experiments asking if the helper packaging approach was appropriate for N15 specificity studies. First, using in vivo phage crossing techniques, we made λ att⁺ N15^hy4, phage ϕ1182 (Table 1), for dilysogen construction. ϕ1182 was used to lysogenize MF532, which contains a partial λ imm⁴³⁴ ind^-prophage which includes the prophage segment from attL through cos^λ. MF532 (ϕ1182) contains the passive prophage structure: imm²¹ cosQ^N15N^N15B^N15 → imm⁴³⁴ ind^- cosQ^λN^λB^λ, where the arrow indicates the direction of DNA packaging. Packaging of the passive prophage by the λ N15^hy4 helper was expected to package a passive chromosome with mismatched cohesive ends. That is, initiation at the cos^N15 of the upstream prophage and termination at the cos^λ of the downstream prophage generates a helper-packaged chromosome with an N15 left cohesive end (designated as ceL^N15) and a λ right cohesive end (ceR^λ; Fig 4). It was uncertain whether phages carrying the mismatched passive chromosome would be viable. Would the chromosome with a 2-bp cohesive end mismatch be able to circularize? Would the λ N15^hy4 terminase, after initiating packaging at the cos^N15 of the upstream prophage, be able to terminate packaging at the cos^λ of the downstream prophage? Although it was unknown whether N15-specific terminase would terminate packaging at cos^λ, previous work showed that λ and 21 terminases could terminate packaging at either cos^λ or cos²¹. In fact, previous work indicated that termination appears to not depend on the presence of cosB [46]. Another termination issue was whether the helper’s N15-specific terminase would show preference for the downstream cosN sequence. Previous work demonstrated that TerL^λ efficiently nicks a cosN with bp changes in the right half of the cohesive end sequence [60], indicating cosN specificity was also not a likely issue.

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Fig 4. Annealed cohesive ends of phage chromosomes with cohesive end mismatches.

A. The mismatch of a phage DNA with a ceL^N15/ceR^λ mismatch. B. The mismatch of a phage DNA with a ceL^λ/ceR^N15 mismatch. Mismatched bp are underlined and in bold. Highly conserved bp flanking the nick sites are highlighted in grey.

https://doi.org/10.1371/journal.pone.0141934.g004

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Table 1. Bacteria and Phages.

https://doi.org/10.1371/journal.pone.0141934.t001

Packaging of MF532 (ϕ1182)’s passive prophage by the λN15^hy4 helper produced a modest yield (0.1 phages/induced cell) bearing passive prophages with mismatched cohesive ends. This yield is about 10% of the yield one might expect. The low yield of this ceL^N15 → ceR^λ chromosome might be due to inefficiency at any of the stages of DNA packaging, i.e., initiation, termination, or reduced viability of the virions carrying chromosomes with mismatched cohesive ends, or the infirm recA^- uvrB^- host cells. Plaque-formation by ceL^N15/ceR^λ mismatch phages indicates that the cohesive end mismatch is not lethal, i.e., the mismatch does not block cyclization of the injected DNA. Finding that phages with mismatched cohesive ends are viable indicated that none of the confounding possibilities prevented packaging of mismatched chromosomes into virions that were competent to carry out successful infections. Below we report experiments directly testing both cosN specificity and termination efficiency of λ- and N15-specific terminases at heterologous coses.

The cyclized DNA of the mismatch phages from MF532 (ϕ1182) contains a cosN with mismatches at positions 9 and 12 (Fig 4A). The mismatched bp might or might not be subject to repair prior to DNA replication. If the mismatches are repaired, then all the progeny of an infection by a mismatch phage will either be cosN^λ or cosN^N15, depending on the direction of the repair process. If the cosN mismatch is not repaired, then a mixed burst of both cosN^λ and cosN^N15 progeny phages are expected from an infected cell. To ask if the mismatched bp were or were not repaired, four well-isolated plaques produced by mismatch phages were suspended in buffer, replated, and individual plaques were subjected to DNA sequence analysis to determine the cohesive end sequence. We found that in the progeny of two of the four infections by mismatch phages, both cosN^λ and cosN^N15 phages were present (Table 2). We conclude that in at least some infections, the mismatch is not repaired and DNA replication produces a progeny phage burst in which some are cosN^λand some are cosN^N15. In practical terms, the MF532 (ϕ1182) helper packaging produced phages that were imm⁴³⁴ ind^- cosQ^λ cosB^N15 and either cosN^λ (ϕ1197) or cosN^N15 (ϕ1199).

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Table 2. Both cohesive end alleles are found in the progeny of phages with mismatched cohesive ends.

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A second dilysogen, MF713 (ϕ1199), was constructed. MF711 contains a partial λ b511 prophage containing the prophage segment extending rightward from cos^λ. MF713 (ϕ1199) has the prophage structure: cosQ^λN^λB^λ →cosQ^λN ^N15B ^N15. Packaging MF713 (ϕ1199)’s passive prophage by the λ helper is expected to produce phages bearing chromosomes with the reciprocal mismatch, i.e., a ceL^λ/ceR^N15 mismatch (Fig 4B). Using the λ-specific helper, the yield was 0.67 phages bearing passive prophage chromosomes/induced cell, indicating that the reciprocal cohesive end mismatch is also not lethal. To ask about persistence of the mismatch, the progeny of five of these ceL^λ/ceR^N15 mismatch phages were examined. Four isolates produced only progeny with cosN^λ, and a fifth isolate produced a burst containing both cosN^λand cosN^N15 phages (Table 2). As was the case for ceL^N15/ceR^λ mismatch phages, some ceL^λ/ceR^N15 mismatch phages give rise to both cosN^λ phages and cosN^N15 phages. In practical terms, the MF713 (ϕ1199) helper packaging experiment produced a λ imm434 ind^- b511 phage pair with cosQ^λ cosB^λ and with either cosN^λ (ϕ1203) or cosN^N15(ϕ1201) that were useful for dilysogen constructions for experiments presented below.

Although the data are limited, there is a preponderance of cosN^λ progeny; the reason for this asymmetry is unknown. In both orientations, both cohesive end alleles are found in progeny phages, indicating that at least in some infections, mismatches are not resolved when the cohesive ends anneal and are ligated. Rather the mismatch persists until the mismatch is resolved by DNA replication. In sum, the cohesive end mismatch does not block cyclization, and in at least some cases, the mismatch persists until after the mismatch-containing parental chromosome has replicated. The limited data also are consistent with the scenario that in some infections by a mismatch chromosome, repair may resolve the mismatch prior to replication.

Quantitating effects of a cohesive end mismatch on infectious phage production.

To ask about the quantitative effects of a cohesive end mismatch on infectious phage production, MF4942, with the prophage structure imm²¹ cosQ^λN^λB^λ→ λ imm⁴³⁴ cosQ^λ N^N15 B, and MF4943, with the prophage structure λ imm²¹ cosQ^λN^λB^λ→ λ imm⁴³⁴ cosQ^λN^λB^λ, were constructed and used in helper packaging experiments (see S1 Table for details). With the λ-specific helper, packaging the ceL^N15/ceR^λ mismatched chromosome, the yield was about 25% that of a chromosome with matched ceL^λ/ceR^λcohesive ends (Table 3, compare lines 1 and 3), indicating that the mismatch reduced the level of viable virion production by four-fold. Remarkably, the λ-N15^hy4 helper phage was able to package λ DNA rather efficiently, at about 50% the efficiency of the λ-specific helper (Table 3, compare lines 1 and 2 and lines 3 and 4). As with the λ-specific helper, the yield of viable ceL^N15/ceR^λ mismatched chromosome-carrying phages was about 25% that of phages with matched cohesive ends (Table 3, compare lines 2 and 4).

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Table 3. Effect of a cohesive end mismatch on viable phages production.

https://doi.org/10.1371/journal.pone.0141934.t003

To summarize, the λ/N15 cohesive end mismatch reduced the recovery of viable packaged chromosomes roughly fourfold, and the N15-specific helper packaged λ-specific DNA at about 50% efficiency.

N15’s Packaging Specificity

cosN specificity.

One explanation for the reduction in yield for mismatched phages is that packaging is inefficient due to reduced efficiency of cos cleavage. For example, since both the λ and N15^hy4 terminases have TerL^λ, both terminases might cleave cosN^λ more efficiently than cosN^N15. Alternatively, the TerS^N15hy4:cosB^λ interaction might affect TerL^λ positioning and the efficiency of cosN cleavage. In either case, λ terminase might act more efficiently in phages with cosB^λ than N15^hy4 terminase. To investigate this point, the yields of phage pairs expressing λ or N15^hy4 terminase, with cosB^λ and either cosN^λ or cosN^N15, were determined. The yields were found not to depend on cosN: for both λ and λ N15^hy4 phages, the yield did not significantly differ between cosN^λ and cosN^N15 (Table 4). These results do not support the idea that λ- and N15-specific terminases are cosN-specific. An alternative explanation for the yield reduction caused by a cohesive end mismatch is that DNA packaging is fully efficient, but the infectivity of the virions with mismatched cohesive ends is reduced. The reduction in yield might be due to less efficient cyclization or a lethal event caused by attempted mismatch repair. Finding a lack of cosN specificity is not surprising, as follows. First, the λ and N15 TerLs are quite similar, with 64.9% identity and 78.5% similarity. Second, the base pair differences in the cohesive end sequences are located in the right half of the cohesive end sequence, where sequence changes do not have measurable effects on cos cleavage [60].

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Table 4. The terminases of λ and λ N15^hy4 do not show cosN specificity.

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Termination specificity.

Previous work showed that λ- and 21-specific terminases efficiently terminate DNA packaging at the downstream cos regardless of cosB specificity [61]. Later work showed that cosQ, cosN and I2, the latter a segment between cosN and cosB, were required for efficient packaging termination [46]. To ask if λ and N15^hy4 terminases were also cosB-non-specific for packaging termination, dilysogens were constructed in which the initial cos was cosB^N15 and the downstream cos was cosB^N15 (MF4944) or cosB^λ (MF4945). In a second pair of dilysogens, the initial cos was cosB^λ, and the downstream cos was either cosB^λ (MF4946) or cosB^N15 (MF4947). These dilysogens were then further lysogenized with the λ- and N15-specific helpers and used in helper packaging experiments. Neither helper displayed any cosB specificity for termination at the downstream cos (Table 5). These results extend the earlier observations that packaging termination is not dependent on cosB specificity [46]. These results are not unexpected given the strong sequence conservation for cosQ, cosN and I2 in λ, 21 and N15.

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Table 5. Packaging termination does not depend on cosB specificity^{^a}.

https://doi.org/10.1371/journal.pone.0141934.t005

Packaging specificities of λ, 21 and N15.

The packaging specificities of λ and 21 are due to the TerS-cosB interaction at the start of DNA packaging. To study N15’s packaging initiation specificity, a set of three dilysogens was constructed in which the passive prophage’s upstream initiation cos was cosQ^λN^λB^λ (MF4948), cosQ^λN^λB²¹ (MF4949), or cosQ^λN^λB^N15 (MF4950). In each strain the downstream termination cos was Q^λN^λB^λ. These dilysogens were then lysogenized with the λ-, 21-, or N15-specific helper phages. As in previous helper packaging experiments, the λ- and 21-specific terminases failed to package each other’s chromosomes (Table 6). Further, neither the λ-specific or 21-specific helpers packaged the N15-specific chromosome, suggesting that N15 has a unique packaging specificity. The inability of λ and 21 to package N15-specific chromosomes is not unexpected, given the apparent simplicity of cos^N15 compared to the elaborate structures of cos^λand cos²¹. The possibility remained that N15 and λ might have the same packaging specificity, but that λ terminase was unable to utilize cosB^N15 because of the lack of I1, R2 and R1. In previous work, it was shown that several Nu1 missense mutations affecting TerS^λ (gpNu1) suppressed a variety of cosB^λ defects. The Nu1ms1 mutation which causes the TerS change L40F, for example, suppresses a number of cosB mutations, including the Δ[R2,1] mutation. The Δ[R2,1] deletion takes out cosB^λ bp 92–164, a segment containing R1, R2 and part of I1 [62]. Because Nu1ms1 terminase sponsors packaging of DNA containing only R3 of cosB^λ, we wondered if Nu1ms1 terminase would sponsor packaging of N15-specific DNA. We used the N15-specific, λ-specific, and Nu1ms1 helper phages to ask if Nu1ms1 terminase could sponsor packaging of N15-specific chromosomes in MF4950. While the N15-specific helper gave a yield of 3.5 prophages/induced lysogen, the prophage yields were 2.9 x 10^-4 and 1.35 x 10^-4 for the λ wild type and λ Nu1ms1 phages, respectively. As observed above with other dilysogens, the N15-specific helper packaged λ-specific chromosomes with reasonable efficiency, i.e., about 50% the efficiency of packaging self-specific N15 chromosomes. In sum, N15 has an asymmetrically unique packaging specificity, with N15 DNA’s specificity being distinct from those of λ and 21, but surprisingly, N15-specific terminase is able to package λ DNA reasonably efficiently.

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Table 6. Packaging specificities of phages λ, 21 and N15^{^a}.

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Discussion

The goal of this work was to use helper packaging experiments to study N15’s packaging specificity, using λ N15^hy4 [58]. For helper packaging experiments, the λ-versus-N15 cohesive end difference was a complication. That is, the 12 base-long cohesive ends of λ and N15 differ at bp 9 and 12, and initial helper packaging experiments generated chromosomes with a λ/N15 cohesive end mismatch. We found that chromosomes with mismatched cohesive ends are able to cyclize, and the mismatch persists past replication of the chromosome, in at least some infections. The cohesive end mismatch is correlated with a roughly fourfold reduction in viable phage yield. Burst size studies suggest that TerL^λ acts with equal efficiency at cosN^λand cosN^N15, and that cos^N15 is efficiently utilized for packaging termination. The preliminary helper packaging experiments produced progeny phages, enabling helper packaging experiments on N15’s packaging specificity which did not generate mismatched chromosomes. N15’s DNA packaging specificity was found to be distinct from those of λ and 21. That is, neither λ nor 21 packages N15 chromosomes and N15-specific terminase does not sponsor 21 DNA packaging. Surprisingly, N15-specific terminase packages λ DNA at about 50% efficiency.

Functional asymmetry of the cohesive ends of λ-like phages

cosN^λ, while showing significant two-fold rotational symmetry, is remarkably asymmetric functionally, as follows. An early study of cosN mutations found that many mutations affecting bp 7–12 in the right half of cosN had minimal phenotypic effects, whereas mutations affecting symmetric bp in the left half of cosN have severe phenotypic effects [60]. For example, the G2C mutation reduced virus yield about 10-fold, but the symmetrical C₁₁G change had no phenotypic effect. Subsequent biochemical cos cleavage experiments confirmed that cosN is functionally asymmetric, and further showed that the asymmetry reflected the proximity of cosN’s right half to cosB [34]. Studies on the importance of the λ cosN-to-cosB spacing also indicate that the TerS-R3 interaction precisely positions TerL with respect to the cosN nicking sites for proper nicking by the TerL endonuclease [33,36]. These results suggest that anchoring of TerL^λ to cosB makes bottom strand nicking relatively insensitive to base pair changes. The functional asymmetry of cosN correlates well with natural cosN sequence variation, as follows. The cohesive end sequence of ϕD326, determined long ago [63], differs from cosN^λ at positions 9 and 12, as does N15. Similarly, Salmonella phage Gifsy-1 differs from λ at cosN bps 8, 9, and 11. In contrast, the two base pairs flanking the nick positions, 5’-CG-3’ and 5’-CG-3’, are highly conserved, indicating that there is selection against changes of these bps. Similarly, natural variation in cosQ, the packaging termination signal, is not apparent. In sum, the results indicate that sequence maintenance for cosQ and the left half of the cohesive end sequence is under selection but maintenance of bp positions 7–12 is not.

Genetic consequences of the λ versus N15 cohesive end mismatch

Our experiments show phages carrying λ chromosomes with mismatched cohesive ends, at bp positions 9 and 12, are able to circularize and replicate. For such phages, there is a modest, four-fold decrease in yield. The reduced yield is not due to effects of cosN bp changes on terminase efficiency, as both λ and N15^hy4 terminases sponsor robust virus yields with either cosN^λ or cosN^N15. We speculate that the reduced yield of viable phages is due to either inefficient cyclization or lethal attempts at mismatch repair. One candidate for repair of the mismatch is the 5’-to-3’ exonuclease of DNA polymerase I, which could carry out nick translation through the mismatched bp. In at least some of the successful infections by phages with mismatched cohesive ends, the mismatch persists through replication of the parental chromosome, since some singly infected cells yield both cosN^λ and cosN^N15 progeny phages (Table 2). These results are surprising, since bp position 12 is adjacent to the bottom strand nick which presumably must be ligated prior to replication fork passage. The λ and N15^hy4 terminases, with TerS^λ and chimeric TerS^N15hy4 subunits, respectively, do not show cosN specificity. For both, the large subunit is TerL^λ which must be appropriately positioned to nick cosN. Given the functional asymmetry of cosN^λ, efficient cleavage of both cosN^λ and cosN^N15 is as expected.

Packaging specificity in the N15-like phages

N15 is an unusual λ-like phage in having a simple cosB containing a single critical TerS binding site, R3^N15. R3^N15 extends from N15 bp 48–59, and is positioned similarly to R3^λ. In cosB^N15, there is a proposed additional remote site, “rR2”, in opposite orientation to the critical R3^N15 sequence and located in the 1 gene at bp 249–260 [58]. The rR2 sequence strongly matches that of R3^N15 and is found in all N15-like prophages with the simple cosB [58]. The rR2 sequence is not critical for N15 growth. Though not essential for plaque formation, rR2 is speculated to contribute to viral fitness for N15 and its close relatives [58]. Another group of prophages, typified by Monarch, has a strong match to R3^N15 and TerS^N15’s recognition helix, suggesting a shared packaging specificity (Fig 2). The Monarch group, however, does not have the simple cosB^N15. Rather cosB^Monarch is analogous to cosB^λ, with three R sequences and an IHF binding site between R3 and R2, and the rR2 sequence is not present.

Comparing the R3 sequences of 21, N15, Monarch, and λ leads to some conclusions about TerS-R3 interactions (Fig 2). The strong identity of R3^N15 and R3^Monarch is apparent, as is the lack of similarity of R3^N15 and R3^Monarch to R3^λ and R3²¹. In R3^λ and R3²¹, bp 56 and bp 59 are critical for TerS recognition [56]. R3^λ bp 58 is also important for TerS^λ recognition [55]. R3^N15 was identified by the packaging effects of two 6 bp-long block mutations, m1 and m2. In m1 and m2, bp 48–53 and 54–59, respectively, were scrambled, as follows. In m1, all six bp were changed–GAGGTT→TGTAGG, and in m2, four were changed-GTTGTT→TGTTGT [58]. The cosB^N15m1 and m2 mutations reduced λ N15^hy4’s yield by about 7-fold and 100-fold, respectively, indicating bp 54–59 are most important for recognition. That bp 54 to 59 include bp important for TerS interactions agrees with the locations of cosB^λ and cosB²¹ bp 56 and 59, which are important bp for recognition by TerS [56].

Why can N15-specific terminase package λ DNA, but not 21 DNA? Looking at the R3 alignment gives hints, but first it is important to consider the validity of the alignment in Fig 2A. In the alignment, each sequence is equidistant from bp 1, the first base of the left cohesive end. It is possible to shift the N15 and Monarch sequences to increase the sequence identity to R3^λ and R3²¹. However in λ, the cosN-to-R3 distance is critical, because shifting the distance by inserting or deleting more than one or two bp lowers virus yield and results in improper nick introduction in cosN [33,36]. These results indicate that when N15-specific terminase sponsors packaging of λ DNA, TerS^N15hy4 anchors TerL^λ in a manner that closely matches TerS^λ anchoring of TerL^λ. In the cosB alignment, R3^N15 and R3^Monarch have a match to the R3^λ TA bp 56, an important TerS^λcontact, R3^λ bp 56 also plays a role in λ/21 phage specificity, as follows. Genetic analysis indicates that Glu24 of the TerS^λ recognition helix clashes with GC bp 56 of R3²¹, accounting in part for the inability of λ terminase to recognize 21-specific DNA (Fig 2B). The genetics also indicated that Glu24 of TerS^λ makes a favorable contact with TA bp 56 of R3^λ. We speculate that, in the case of N15’s recognition helix, perhaps the Val22 residue, at the position analogous the TerS^λ’s Glu24, makes a favorable contact with the bp 56 TA bp of cosB^λ, but is unable to make a favorable contact with the GC bp 56 of R3²¹. It is also not clear whether the inability of λ terminase to package N15 DNA is due to a defect in cosN cutting, or in a post-cleavage defect such as formation of complex I. More research is required to understand these relationships.

cosB architecture and function

The likely shared packaging specificity of phages N15 and Monarch, and the finding that N15 packages λ DNA raise further interesting questions about cos structure and terminase recognition. The interaction of TerS^λ with cosB^λ includes a proposed IHF-assisted DNA hairpin that juxtaposes the major grooves of R3 and R2 for docking by dimeric gpNu1 [20]. The strong similarity of cosB^λ and cosB^Monarch suggests a hairpin-containing nucleoprotein structure also forms at cosB^Monarch. It is interesting that the TerS^N15 DNA binding domain forms a tight dimer [58], even though cosB^N15 is much simpler than cosB^Monarch. Does TerS^N15 form a similar hairpin structure at cosB^N15, even though there is neither an IHF site nor an R2-equivalent? Elsewhere we propose that cosB^N15 is derived from cosB^Monarch by unequal crossing over between R3 and R1. If so, the TerS^N15 would be derived from TerS^Monarch, and might retain the ability to interact with cosB^Monarch in a manner similar to TerS^λ docking at cosB^λ. A related issue is packaging of λ DNA by N15-specific terminase. Does TerS^N15hy4 utilize all three R sequences of cosB^λ when packaging λ DNA? These interesting questions await further studies.

What evolutionary steps account for the packaging systems of the λ-like phages? It seems clear that λ and 21 are descended from a common ancestor phage, as the basic cos structure is preserved. We earlier presented one possible path, in an early ancestor virus where a single R sequence and a terminase with low sequence specificity acquired greater packaging specificity by acquisition of three R sequences [64]. A scenario for N15 is that N15 has undergone a simplification process, by which a Monarch-like ancestor phage with three R sequences has lost two, and the IHF site, by unequal crossing over between R3 and R1. One would think that such an event would result in a major fitness reduction. It is possible however that the simplification and subsequent refinement, such as the acquisition of rR2, might take place in the prophage state, where fitness for lytic growth is moot [58,65].

Genetic consequences of the asymmetric, shared packaging specificities of λ and N15

The chromosomes of many bacteriophages, including the λ-like phages, are mosaics of DNA segments, indicating that extensive horizontal exchange is part of a phage’s evolutionary history [66]. Enteric bacteria frequently harbor λ-like prophages, so that an infecting λ-like phage has a strong likelihood of replicating in the presence of another λ-like phage chromosome. Of course several significant consequences may result from such an interaction. If a prophage shares the packaging specificity of the infecting phage that enters the lytic cycle, then the packaging machinery of the infecting phage will initiate packaging at the prophage cos. For the λ-like prophages, cos is located roughly in the center of the prophage, so that the packaging initiation encapsidates a DNA segment that will include adjacent bacterial DNA, stalling the motor when the head shell is full, which in turn produces a tailless, non-infectious particle. Production of such inactive phage structures reduces the production efficiency of viable progeny phages. One might think therefore, that it would be advantageous for λ-like phages to have diverse packaging specificities. So far, only modest diversity is observed, though not many examples have been studied. Among characterized λ-like phages with related cos structures, the three packaging specificities of λ, 21 and N15 have been documented. Other natural isolates, such as phages 434 and ϕ80, share λ‘s packaging specificity. N15 is an interesting case, as N15’s packaging machinery retains the ability to package λ DNA, even though N15 has a significantly divergent DNA recognition system.

A second interaction between λ-like phages is recombination. If a packaging event initiated at a prophage cos, is accompanied by a downstream crossover between prophage and lytic phage DNAs, the recombinant is a novel progeny phage that contributes to genetic diversity. Some such genetic exchanges generate recombinants that are more robust in a changing environment. In the case of a cohesive end mismatch, such as in the present λ versus N15 situation, recombination may produce less fit progeny phages. Because the cohesive end mismatch does not persist, the reduced viability of a recombinant phage is transient.

DNA recognition by TerS

TerS molecules interact with their recognition sites through their N-terminal DNA binding domains, and with TerL through their C-terminal domains. These two specificity domains are separated by the long α-helical core domain. In principal, phage diversity might be generated by swapping of the TerS DNA recognition domain for that of any other DNA binding protein with an asymmetric recognition site. In the present case, we note that both the cos and TerS of N15 are recognizably evolutionarily related to those of phage λ [58]. Phage 21’s packaging specificity determinants also share a common ancestor with λ [56]. N15 and 21 were chosen for study as interesting variants of the λ paradigm, and as such these phages do not shed light on extreme possibilities for specificity swapping. We note that in a more systematic study in P22-like phages, considerable horizontal transfer of TerS gene segments has occurred, not only between highly diverged members of the P22 family, but also has occurred between P22-like phages and phages outside the P22-like group [16]. Acquisition of a TerS segment from a non-phage source such as a transcriptional regulatory system has not been observed. Surely further sequence acquisition and study will add to what we know about this issue.

Materials and Methods

Media

Luria broth (LB), LB agar, tryptone broth (TB), tryptone broth agar (TA), and tryptone broth soft agar (TBSA) were prepared as described [67], except TB, TA, and TBSA were supplemented with 0.01 M MgSO₄. For phage infections, TB was supplemented with 0.2% maltose. When required, ampicillin, chloramphenicol, and kanamycin were added at 100 μg/mL, 10 μg/ml, and 50 μg/mL, respectively.

Bacteria, phages and plasmids

These are listed in Table 1. A list of dilysogens constructed for this work is in S1 Table.

Microbiological methods

Phage crosses and infections were done using standard protocols [67].

Helper phages

The helper phages used were in the thermo-inducible λ-P1:5R cI857 Kn^R nin5 genetic background. In λ-P1:5R cI857 Kn^R nin5, the DNA segment containing the λ site-specific recombination system is replaced by the phage P1 plasmid replication and partitioning segment, so the helper phage forms a plasmid prophage [68]. The Kn^R marker in λ-P1:5R cI857 Kn^R nin5 is a substitution for λ DNA between the two λ SalI sites [69]. The SalI sites are located in (1) the bet gene and (2) distal to gam, so that the helper phages are red^-gam^-. When growing in a recA^- host, red^-gam^-phages are unable to generate concatemers through rolling circle replication or recombination [29]. Consequently, the helper phage yield is low, about 10–20 phages/cell in the experiments reported here. For simplicity, λ-P1:5R cI857 Kn^R nin5 will simply be designated λ; this phage was used as the helper with λ packaging specificity. λ 21^hy51 [59] and λ N15^hy4 [58] were the 21- and N15-specific helpers, respectively.

Dilysogens

Dilysogens were constructed with two recA^- strains with deleted prophages, MF713 and MF532 (Table 2). The λ b511 prophage of MF713 is deleted for a DNA segment extending from the bacterial gal operon thru the prophage early genes, including cI, while the segment containing cos, the head and tail genes, the b511 marker and attΔ●B are present. The genetic structure of MF713 is: where λ genes are underlined, slashes indicate the long DNA stretch of the λ head and tail genes, dashed lines indicate E. coli. Lysogenization of MF713 by λ imm⁴³⁴ ind^-cos_i generates the following genetic structure: where cos_i indicates that the downstream cos, at which termination occurs, may have λ, 21 or N15 sequences. The passive packageable prophage is double-underlined. Care was taken to exclude isolates with two copies of the added prophage. For MF713 (λ imm⁴³⁴ cos_i), we confirmed that the chromosomes produced by helper packaging of the passive prophage carried the b511 marker. Generally, 10 isolates were tested by a PCR assay or a test for a b511 phage’s inability to lysogenize.

The λ imm⁴³⁴ prophage of MF532 is deleted rightwards from Nu1 through the head and tail genes, attR, and bacterial DNA including bio, uvrB and chlA. The genetic structure of MF532 is:

Lysogenization of MF532 by λ imm²¹ cos_i gives the genetic structure: where the passive packageable prophage is double-underlined. The cos at which packaging is initiated, cos_i, was either cos^λ, cos²¹ or cos^N15. The cos²¹ is from 21^hy33, which also has gene 1 and a chimeric 2/A gene. The N15^hy4 provided cos^N15. Care was taken to ensure that dilysogen candidates with more than two prophages were eliminated.

For MF532 dilyogens, the upstream prophage carried the immunity of phage 21 (imm²¹) and cos_i. The downstream prophage of MF532 carries imm⁴³⁴ and cos^λ. Helper packaging of the passive prophage of an MF532 dilysogen generates virions carrying chromosomes that are imm⁴³⁴. The immunity of 10 isolates was tested to confirm this expectation.

MF611 (Tables 3 and 6).

To generate this dilysogen set, the recA1 E. coli strain MF611 was sequentially lysogenized by the two phages under study. Then helper phages were added. The genetic structure of dilysogens was determined by examining the genetic content of helper-packaged passive prophages. In all cases care was taken to show that isolates with more than two prophages were excluded from experiments.

MF3510 (Table 5).

MF3510 is a galK^-derivative of MF611. Phages used carried gal or cat markers, and examination of packaged passive prophages was used to eliminate isolates with more than two prophages. In most cases, for all three dilysogen sets, multiple independent dilysogen isolates were used in experiments.

Helper phages.

The helper phage genetic background was λ-P1:5R Kn^R cI857 nin5, and this phage was the λ- specific helper phage. The 21-specific helper phage was a recombinant containing the left chromosome end 21^hy51, which includes cosB²¹ and a chimeric small subunit gene in which the 5’-half was from the 21 1 gene and the 3’ half was from Nu1. The N15-specific helper, λ-P1 N15^hy4, is described in Results. For simplicity, these three helper phages will simply be designated as the λ-, 21-, and N15-specific helpers, resp.

Helper packaging protocol.

Strains were grown overnight in LB+kanamycin, in standing culture, at 31C, and 0.2 ml was used to inoculate 5 ml of LB. Cultures were shaken at 31C for 2 hrs. An aliquot was removed and plated to determine the cell titer. Helper phages were induced by shifting to 42C for 15 min, and the cultures were shaken at 37C for an additional 70 min. Lysis was completed by addition of CHCl₃, debris removed by centrifugation, and the lysates titered at 37C. Helper phages form clear plaques and phages bearing passive prophages form turbid plaques. For lysates with low levels of passive prophage-containing virions, dilutions were plated on C600(λ). If the progeny of phages with passive prophages were to be subjected to further analysis, the indicator strain was MF1427, an E. coli C strain. The level of defective λ-like prophage material in E. coli C is very low, and no cosN^λ is present, so that concern about recombination with defective prophage DNA was eliminated.

Supporting Information

S1 Table. Dilysogens used in Helper Packaging Experiments.

https://doi.org/10.1371/journal.pone.0141934.s001

(DOCX)

Acknowledgments

MF and JS dedicate this work to the late Erich W. Six, for decades our lively colleague and friend. Erich always liked a good phage yarn. This project was supported by funding from National Science Foundation awards MCB- 0717620 and MCB-1158495, National Institutes of Health, United States awards 5R01GM088186 and 5R01GM051611 (www.nih.gov/), and the University of Iowa Department of Microbiology.

Author Contributions

Conceived and designed the experiments: MF. Performed the experiments: MF JYM SS PP JS. Analyzed the data: MF. Contributed reagents/materials/analysis tools: MF. Wrote the paper: MF JS.

References

1. Catalano CE, editor (2005) Viral Genome Packaging Machines: Genetics, Structure and Mechanism. Georgetown TX: Landes Bioscience.
2. Rao VB, Feiss M (2008) The bacteriophage DNA packaging motor. Annual Review of Genetics: Annual Reviews Palo Alto CA. pp. 647.
3. Newcomb WW, Homa FL, Thomsen DR, Trus BL, Cheng N, Steven A, et al. (1999) Assembly of the herpes simplex virus procapsid from purified components and identification of small complexes containing the major capsid and scaffolding proteins. J Virol 73: 4239. pmid:10196320
- View Article
- PubMed/NCBI
- Google Scholar
4. Rixon FJ, Schmid MF (2014) Structural similarities in DNA packaging and delivery apparatuses in Herpesvirus and dsDNA bacteriophages. Curr Opin Virol 5: 105–110. pmid:24747680
- View Article
- PubMed/NCBI
- Google Scholar
5. Baker ML, Jiang W, Rixon FJ, Chiu W (2005) Common ancestry of herpesviruses and tailed DNA bacteriophages. J Virol 79: 14967. pmid:16282496
- View Article
- PubMed/NCBI
- Google Scholar
6. Lebedev AA, Krause MH, Isidro AL, Vagin AA, Orlova EV, Turner J, et al. (2007) Structural framework for DNA translocation via the viral portal protein. The EMBO journal 26: 1984. pmid:17363899
- View Article
- PubMed/NCBI
- Google Scholar
7. Simpson A, Tao Y, Leiman P, Badasso M, He Y, Jardine PJ, et al. (2000) Structure of the bacteriophage phi29 DNA packaging motor. Nature 408: 745. pmid:11130079
- View Article
- PubMed/NCBI
- Google Scholar
8. Agirrezabala X, Martin-Benito J, Valle M, Gonzalez JM, Valencia A, Valpuesta JM, et al. (2005) Structure of the connector of bacteriophage T7 at 8A resolution: structural homologies of a basic component of a DNA translocating machinery. Journal of Molecular Biology 347: 895. pmid:15784250
- View Article
- PubMed/NCBI
- Google Scholar
9. Trus BL, Cheng N, Newcomb WW, Homa FL, Brown JC, Steven AC (2004) Structure and polymorphism of the UL6 portal protein of herpes simplex virus type 1. J Virol 78: 12668. pmid:15507654
- View Article
- PubMed/NCBI
- Google Scholar
10. Casjens SR, Gilcrease EB, Winn-Stapley DA, Schicklmaier P, Schmieger H, Pedulla ML, et al. (2005) The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy. J Bacteriol 187: 1091. pmid:15659686
- View Article
- PubMed/NCBI
- Google Scholar
11. Przech AJ, Yu D, Weller SK (2003) Point mutations in exon I of the herpes simplex virus putative terminase subunit, UL15, indicate that the most conserved residues are essential for cleavage and packaging. J Virol 77: 9613. pmid:12915573
- View Article
- PubMed/NCBI
- Google Scholar
12. Casjens SR (2011) The DNA-packaging nanomotor of tailed bacteriophages. Nature reviewsMicrobiology 9: 647.
- View Article
- Google Scholar
13. Frackman S, Siegele DA, Feiss M (1984) A functional domain of bacteriophage lambda terminase for prohead binding. Journal of Molecular Biology 180: 283. pmid:6096564
- View Article
- PubMed/NCBI
- Google Scholar
14. Wu WF, Christiansen S, Feiss M (1988) Domains for protein-protein interactions at the N and C termini of the large subunit of bacteriophage lambda terminase. Genetics 119: 477. pmid:2969839
- View Article
- PubMed/NCBI
- Google Scholar
15. Nemecek D, Gilcrease EB, Kang S, E PP Jr, Casjens S, Thomas GJ Jr (2007) Subunit conformations and assembly states of a DNA-translocating motor: the terminase of bacteriophage P22. J Mol Biol 374: 817. pmid:17945256
- View Article
- PubMed/NCBI
- Google Scholar
16. Leavitt JC, Gilcrease EB, Wilson K, Casjens SR (2013) Function and horizontal transfer of the small terminase subunit of the tailed bacteriophage Sf6 DNA packaging nanomotor. Virology 440: 117–133. pmid:23562538
- View Article
- PubMed/NCBI
- Google Scholar
17. Sun S, Gao S, Kondabagil K, Xiang Y, Rossmann MG, Rao VB (2012) Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages. Proc Natl Acad Sci U S A 109: 817–822. pmid:22207623
- View Article
- PubMed/NCBI
- Google Scholar
18. Gao S, Rao VB (2011) Specificity of interactions among the DNA-packaging machine components of T4-related bacteriophages. The Journal of biological chemistry 286: 3944. pmid:21127059
- View Article
- PubMed/NCBI
- Google Scholar
19. Roy A, Bhardwaj A, Datta P, Lander GC, Cingolani G (2012) Small terminase couples viral DNA binding to genome-packaging ATPase activity. Structure 20: 1403–1413. pmid:22771211
- View Article
- PubMed/NCBI
- Google Scholar
20. de Beer T, Fang J, Ortega M, Yang Q, Maes L, Duffy C, et al. (2002) Insights into specific DNA recognition during the assembly of a viral genome packaging machine. Mol Cell 9: 981–991. pmid:12049735
- View Article
- PubMed/NCBI
- Google Scholar
21. Zhao H, Kamau YN, Christensen TE, Tang L (2012) Structural and functional studies of the phage Sf6 terminase small subunit reveal a DNA-spooling device facilitated by structural plasticity. J Mol Biol 423: 413–426. pmid:22858866
- View Article
- PubMed/NCBI
- Google Scholar
22. Al-Zahrani AS, Kondabagil K, Gao S, Kelly N, Ghosh-Kumar M, Rao VB (2009) The small terminase, gp16, of bacteriophage T4 is a regulator of the DNA packaging motor. The Journal of biological chemistry 284: 24490. pmid:19561086
- View Article
- PubMed/NCBI
- Google Scholar
23. Benini S, Chechik M, Ortiz Lombardia M, Polier S, Leech A, Shevtsov MB, et al. (2013) The 1.58 A resolution structure of the DNA-binding domain of bacteriophage SF6 small terminase provides new hints on DNA binding. Acta Crystallogr Sect F Struct Biol Cryst Commun 69: 376–381. pmid:23545641
- View Article
- PubMed/NCBI
- Google Scholar
24. Buttner CR, Chechik M, Ortiz-Lombardia M, Smits C, Ebong IO, Chechik V, et al. (2012) Structural basis for DNA recognition and loading into a viral packaging motor. Proc Natl Acad Sci U S A 109: 811–816. pmid:22207627
- View Article
- PubMed/NCBI
- Google Scholar
25. Chai S, Lurz R, Alonso JC (1995) The small subunit of the terminase enzyme of Bacillus subtilis bacteriophage SPP1 forms a specialized nucleoprotein complex with the packaging initiation region. Journal of Molecular Biology 252: 386. pmid:7563059
- View Article
- PubMed/NCBI
- Google Scholar
26. Oliveira L, Tavares P, Alonso JC (2013) Headful DNA packaging: bacteriophage SPP1 as a model system. Virus Res 173: 247–259. pmid:23419885
- View Article
- PubMed/NCBI
- Google Scholar
27. Wu H, Sampson L, Parr R, Casjens S (2002) The DNA site utilized by bacteriophage P22 for initiation of DNA packaging. Molecular microbiology 45: 1631. pmid:12354230
- View Article
- PubMed/NCBI
- Google Scholar
28. Black LW (2015) Old, new, and widely true: The bacteriophage T4 DNA packaging mechanism. Virology.
- View Article
- Google Scholar
29. Furth ME, Wickner SH (1983) Lambda DNA replication. In: Hendrix RW, Roberts JW, Stahl FW, Weisberg RA, editors. Lambda II. Cold Spring Harbor NY: Cold Spring Harbor Press. pp. 145.
30. Maluf N, Gaussier H, Bogner E, Feiss M, Catalano C (2006) Assembly of bacteriophage lambda terminase into a viral DNA maturation and packaging machine. Biochemistry 45: 15259. pmid:17176048
- View Article
- PubMed/NCBI
- Google Scholar
31. Maluf NK, Feiss M (2006) Virus DNA translocation: progress towards a first ascent of mount pretty difficult. Mol Microbiol 61: 1. pmid:16824089
- View Article
- PubMed/NCBI
- Google Scholar
32. Higgins RR, Lucko HJ, Becker A (1988) Mechanism of cos DNA cleavage by bacteriophage lambda terminase: multiple roles of ATP. Cell 54: 765. pmid:2970303
- View Article
- PubMed/NCBI
- Google Scholar
33. Higgins RR, Becker A (1994) The lambda terminase enzyme measures the point of its endonucleolytic attack 47 +/- 2 bp away from its site of specific DNA binding, the R site. Embo J 13: 6162. pmid:7813453
- View Article
- PubMed/NCBI
- Google Scholar
34. Hang JQ, Catalano CE, Feiss M (2001) The functional asymmetry of cosN, the nicking site for bacteriophage lambda DNA packaging, is dependent on the terminase binding site, cosB. Biochemistry 40: 13370. pmid:11683647
- View Article
- PubMed/NCBI
- Google Scholar
35. Higgins RR, Becker A (1994) Chromosome end formation in phage lambda, catalyzed by terminase, is controlled by two DNA elements of cos, cosN and R3, and by ATP. Embo J 13: 6152. pmid:7813452
- View Article
- PubMed/NCBI
- Google Scholar
36. Miller G, Feiss M (1988) The bacteriophage lambda cohesive end site: isolation of spacing/substitution mutations that result in dependence on Escherichia coli integration host factor. Molecular & General Genetics 212: 157.
- View Article
- Google Scholar
37. Becker A, Murialdo H, Gold M (1977) Early events in the in vitro packaging of bacteriophage DNA. Virology 78: 291. pmid:860405
- View Article
- PubMed/NCBI
- Google Scholar
38. Catalano CE, Cue D, Feiss M (1995) Virus DNA packaging: the strategy used by phage lambda. Molecular Microbiology 16: 1075. pmid:8577244
- View Article
- PubMed/NCBI
- Google Scholar
39. Dokland T, Murialdo H (1993) Structural transitions during maturation of bacteriophage lambda capsids. Journal of Molecular Biology 233: 682. pmid:8411174
- View Article
- PubMed/NCBI
- Google Scholar
40. Lander GC, Evilevitch A, Jeembaeva M, Potter CS, Carragher B, Johnson JE (2008) Bacteriophage lambda stabilization by auxiliary protein gpD: timing, location, and mechanism of attachment determined by cryo-EM. Structure (London, England: 1993) 16: 1399.
- View Article
- Google Scholar
41. Yang Q, Catalano CE (2003) Biochemical characterization of bacteriophage lambda genome packaging in vitro. Virology 305: 276. pmid:12573573
- View Article
- PubMed/NCBI
- Google Scholar
42. Dhar A, Feiss M (2005) Bacteriophage lambda terminase: alterations of the high-affinity ATPase affect viral DNA packaging. J Mol Biol 347: 71. pmid:15733918
- View Article
- PubMed/NCBI
- Google Scholar
43. Feiss M, Sippy J, Miller G (1985) Processive action of terminase during sequential packaging of bacteriophage lambda chromosomes. Journal of Molecular Biology 186: 759. pmid:3005594
- View Article
- PubMed/NCBI
- Google Scholar
44. Cue D, Feiss M (1993) A site required for termination of packaging of the phage lambda chromosome. Proc Natl Acad Sci U S A 90: 9290–9294. pmid:8415694
- View Article
- PubMed/NCBI
- Google Scholar
45. Wieczorek DJ, Feiss M (2001) Defining cosQ, the site required for termination of bacteriophage lambda DNA packaging. Genetics 158: 495. pmid:11404316
- View Article
- PubMed/NCBI
- Google Scholar
46. Cue D, Feiss M (2001) Bacteriophage lambda DNA packaging: DNA site requirements for termination and processivity. J Mol Biol 311: 233–240. pmid:11478856
- View Article
- PubMed/NCBI
- Google Scholar
47. Cue D, Feiss M (1993) The role of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, in the nicking reaction. J Mol Biol 234: 594–609. pmid:8254662
- View Article
- PubMed/NCBI
- Google Scholar
48. Kosturko L, Daub E, Murialdo H (1989) The interaction of E. coli integration host factor and lambda cos DNA multicomplex formation and protein-induced bending. Nucleic Acids Res 17: 329.
- View Article
- Google Scholar
49. Bear S, Court D, Friedman D (1984) An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging. J Virol 52: 966. pmid:6238175
- View Article
- PubMed/NCBI
- Google Scholar
50. Xin W, Feiss M (1993) Function of IHF in lambda DNA packaging. I. Identification of the strong binding site for integration host factor and the locus for intrinsic bending in cosB. Journal of Molecular Biology 230: 492. pmid:8385227
- View Article
- PubMed/NCBI
- Google Scholar
51. Ortega ME, Catalano CE (2006) Bacteriophage lambda gpNu1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA: implications for the assembly of a genome-packaging motor. Biochemistry 45: 5180. pmid:16618107
- View Article
- PubMed/NCBI
- Google Scholar
52. Rice PA, Yang S, Mizuuchi K, Nash HA (1996) Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn. Cell 87: 1295. pmid:8980235
- View Article
- PubMed/NCBI
- Google Scholar
53. Yang Q, Hanagan A, Catalano CE (1997) Assembly of a nucleoprotein complex required for DNA packaging by bacteriophage lambda. Biochemistry 36: 2744. pmid:9062101
- View Article
- PubMed/NCBI
- Google Scholar
54. Casjens SR (2008) Diversity among the tailed-bacteriophages that infect the Enterobacteriaceae. Research in microbiology 159: 340. pmid:18550341
- View Article
- PubMed/NCBI
- Google Scholar
55. Cue D, Feiss M (1992) Genetic analysis of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda. J Mol Biol 228: 58–71. pmid:1447794
- View Article
- PubMed/NCBI
- Google Scholar
56. Sippy J, Patel P, Vahanian N, Sippy R, Feiss M (2015) Genetics of critical contacts and clashes in the DNA packaging specificities of bacteriophages lambda and 21. Virology 476: 115–123. pmid:25543962
- View Article
- PubMed/NCBI
- Google Scholar
57. Becker A, Murialdo H (1990) Bacteriophage lambda DNA: the beginning of the end. Journal of Bacteriology 172: 2819. pmid:2140565
- View Article
- PubMed/NCBI
- Google Scholar
58. Feiss M, Geyer H, Klingberg F, Moreno N, Forystek A, Maluf NK, et al. (2015) Novel DNA packaging recognition in the unusual bacteriophage N15. Virology 482: 260–268. pmid:25956737
- View Article
- PubMed/NCBI
- Google Scholar
59. Frackman S, Siegele DA, Feiss M (1985) The terminase of bacteriophage lambda. Functional domains for cosB binding and multimer assembly. J Mol Biol 183: 225. pmid:2989542
- View Article
- PubMed/NCBI
- Google Scholar
60. Xu SY, Feiss M (1991) Structure of the bacteriophage lambda cohesive end site. Genetic analysis of the site (cosN) at which nicks are introduced by terminase. Journal of Molecular Biology 220: 281. pmid:1830343
- View Article
- PubMed/NCBI
- Google Scholar
61. Feiss M, Fisher R, Siegele DA, Widner W (1981) Bacteriophage lambda and 21 packaging specificities. In: DuBow MS, editor. Bacteriophage Assembly. New York: Alan R. Liss Inc. pp. 213.
62. Cue D, Feiss M (1992) Genetic analysis of mutations affecting terminase, the bacteriophage lambda DNA packaging enzyme, that suppress mutations in cosB, the terminase binding site. J Mol Biol 228: 72–87. pmid:1447796
- View Article
- PubMed/NCBI
- Google Scholar
63. Murray K, Murray NE, Bertani G (1975) Base changes in the recognition site for ter functions in lambdoid phage DNA. Nature 254: 262. pmid:1113894
- View Article
- PubMed/NCBI
- Google Scholar
64. Feiss M, Reynolds E, Schrock M, Sippy J (2010) DNA packaging by lambda-like bacteriophages: mutations broadening the packaging specificity of terminase, the lambda-packaging enzyme. Genetics 184: 43. pmid:19841094
- View Article
- PubMed/NCBI
- Google Scholar
65. Little JW (2010) Evolution of complex gene regulatory circuits by addition of refinements. Curr Biol 20: R724–734. pmid:20833317
- View Article
- PubMed/NCBI
- Google Scholar
66. Hendrix RW, Lawrence JG, Hatfull GF, Casjens S (2000) The origins and ongoing evolution of viruses (Review). Trends Microbiol 8: 504. pmid:11121760
- View Article
- PubMed/NCBI
- Google Scholar
67. Arber W, Enquist L, Hohn B, Murray NE, Murray K (1983) Experimental methods for use with lambda. In: Hendrix RW, Roberts JW, Stahl FW, Weisberg RA, editors. Lambda II. Cold Spring Harbor: Cold Spring Harbor Press. pp. 433–466.
68. Sternberg N, Austin S (1983) Isolation and characterization of P1 minireplicons. J Bacteriol 153: 800. pmid:6296053
- View Article
- PubMed/NCBI
- Google Scholar
69. Pal SK, Chattoraj DK (1988) P1 plasmid replication: Initiator sequestration is inadequate to explain control by initiator-binding sites. J Bacteriol 172: 2819.
- View Article
- Google Scholar
70. Granston AE, Alessi DM, Eades LJ, Friedman DI (1988) A point mutation in the Nul gene of bacteriophage lambda facilitates phage growth in Escherichia coli with himA and gyrB mutations. Mol Gen Genet 212: 149–156. pmid:2836702
- View Article
- PubMed/NCBI
- Google Scholar
71. Feiss M, Siegele DA (1979) Packaging of the bacteriophage lambda chromosome: dependence of cos cleavage on chromosome length. Virology 92: 190. pmid:419690
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Catalano CE, editor (2005) Viral Genome Packaging Machines: Genetics, Structure and Mechanism. Georgetown TX: Landes Bioscience.

[ref2] 2. Rao VB, Feiss M (2008) The bacteriophage DNA packaging motor. Annual Review of Genetics: Annual Reviews Palo Alto CA. pp. 647.

[ref3] 3. Newcomb WW, Homa FL, Thomsen DR, Trus BL, Cheng N, Steven A, et al. (1999) Assembly of the herpes simplex virus procapsid from purified components and identification of small complexes containing the major capsid and scaffolding proteins. J Virol 73: 4239. pmid:10196320
View Article
PubMed/NCBI
Google Scholar

[4] View Article

[5] PubMed/NCBI

[6] Google Scholar

[ref4] 4. Rixon FJ, Schmid MF (2014) Structural similarities in DNA packaging and delivery apparatuses in Herpesvirus and dsDNA bacteriophages. Curr Opin Virol 5: 105–110. pmid:24747680
View Article
PubMed/NCBI
Google Scholar

[8] View Article

[9] PubMed/NCBI

[10] Google Scholar

[ref5] 5. Baker ML, Jiang W, Rixon FJ, Chiu W (2005) Common ancestry of herpesviruses and tailed DNA bacteriophages. J Virol 79: 14967. pmid:16282496
View Article
PubMed/NCBI
Google Scholar

[12] View Article

[13] PubMed/NCBI

[14] Google Scholar

[ref6] 6. Lebedev AA, Krause MH, Isidro AL, Vagin AA, Orlova EV, Turner J, et al. (2007) Structural framework for DNA translocation via the viral portal protein. The EMBO journal 26: 1984. pmid:17363899
View Article
PubMed/NCBI
Google Scholar

[16] View Article

[17] PubMed/NCBI

[18] Google Scholar

[ref7] 7. Simpson A, Tao Y, Leiman P, Badasso M, He Y, Jardine PJ, et al. (2000) Structure of the bacteriophage phi29 DNA packaging motor. Nature 408: 745. pmid:11130079
View Article
PubMed/NCBI
Google Scholar

[20] View Article

[21] PubMed/NCBI

[22] Google Scholar

[ref8] 8. Agirrezabala X, Martin-Benito J, Valle M, Gonzalez JM, Valencia A, Valpuesta JM, et al. (2005) Structure of the connector of bacteriophage T7 at 8A resolution: structural homologies of a basic component of a DNA translocating machinery. Journal of Molecular Biology 347: 895. pmid:15784250
View Article
PubMed/NCBI
Google Scholar

[24] View Article

[25] PubMed/NCBI

[26] Google Scholar

[ref9] 9. Trus BL, Cheng N, Newcomb WW, Homa FL, Brown JC, Steven AC (2004) Structure and polymorphism of the UL6 portal protein of herpes simplex virus type 1. J Virol 78: 12668. pmid:15507654
View Article
PubMed/NCBI
Google Scholar

[28] View Article

[29] PubMed/NCBI

[30] Google Scholar

[ref10] 10. Casjens SR, Gilcrease EB, Winn-Stapley DA, Schicklmaier P, Schmieger H, Pedulla ML, et al. (2005) The generalized transducing Salmonella bacteriophage ES18: complete genome sequence and DNA packaging strategy. J Bacteriol 187: 1091. pmid:15659686
View Article
PubMed/NCBI
Google Scholar

[32] View Article

[33] PubMed/NCBI

[34] Google Scholar

[ref11] 11. Przech AJ, Yu D, Weller SK (2003) Point mutations in exon I of the herpes simplex virus putative terminase subunit, UL15, indicate that the most conserved residues are essential for cleavage and packaging. J Virol 77: 9613. pmid:12915573
View Article
PubMed/NCBI
Google Scholar

[36] View Article

[37] PubMed/NCBI

[38] Google Scholar

[ref12] 12. Casjens SR (2011) The DNA-packaging nanomotor of tailed bacteriophages. Nature reviewsMicrobiology 9: 647.
View Article
Google Scholar

[40] View Article

[41] Google Scholar

[ref13] 13. Frackman S, Siegele DA, Feiss M (1984) A functional domain of bacteriophage lambda terminase for prohead binding. Journal of Molecular Biology 180: 283. pmid:6096564
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref14] 14. Wu WF, Christiansen S, Feiss M (1988) Domains for protein-protein interactions at the N and C termini of the large subunit of bacteriophage lambda terminase. Genetics 119: 477. pmid:2969839
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref15] 15. Nemecek D, Gilcrease EB, Kang S, E PP Jr, Casjens S, Thomas GJ Jr (2007) Subunit conformations and assembly states of a DNA-translocating motor: the terminase of bacteriophage P22. J Mol Biol 374: 817. pmid:17945256
View Article
PubMed/NCBI
Google Scholar

[51] View Article

[52] PubMed/NCBI

[53] Google Scholar

[ref16] 16. Leavitt JC, Gilcrease EB, Wilson K, Casjens SR (2013) Function and horizontal transfer of the small terminase subunit of the tailed bacteriophage Sf6 DNA packaging nanomotor. Virology 440: 117–133. pmid:23562538
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref17] 17. Sun S, Gao S, Kondabagil K, Xiang Y, Rossmann MG, Rao VB (2012) Structure and function of the small terminase component of the DNA packaging machine in T4-like bacteriophages. Proc Natl Acad Sci U S A 109: 817–822. pmid:22207623
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref18] 18. Gao S, Rao VB (2011) Specificity of interactions among the DNA-packaging machine components of T4-related bacteriophages. The Journal of biological chemistry 286: 3944. pmid:21127059
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref19] 19. Roy A, Bhardwaj A, Datta P, Lander GC, Cingolani G (2012) Small terminase couples viral DNA binding to genome-packaging ATPase activity. Structure 20: 1403–1413. pmid:22771211
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref20] 20. de Beer T, Fang J, Ortega M, Yang Q, Maes L, Duffy C, et al. (2002) Insights into specific DNA recognition during the assembly of a viral genome packaging machine. Mol Cell 9: 981–991. pmid:12049735
View Article
PubMed/NCBI
Google Scholar

[71] View Article

[72] PubMed/NCBI

[73] Google Scholar

[ref21] 21. Zhao H, Kamau YN, Christensen TE, Tang L (2012) Structural and functional studies of the phage Sf6 terminase small subunit reveal a DNA-spooling device facilitated by structural plasticity. J Mol Biol 423: 413–426. pmid:22858866
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref22] 22. Al-Zahrani AS, Kondabagil K, Gao S, Kelly N, Ghosh-Kumar M, Rao VB (2009) The small terminase, gp16, of bacteriophage T4 is a regulator of the DNA packaging motor. The Journal of biological chemistry 284: 24490. pmid:19561086
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref23] 23. Benini S, Chechik M, Ortiz Lombardia M, Polier S, Leech A, Shevtsov MB, et al. (2013) The 1.58 A resolution structure of the DNA-binding domain of bacteriophage SF6 small terminase provides new hints on DNA binding. Acta Crystallogr Sect F Struct Biol Cryst Commun 69: 376–381. pmid:23545641
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref24] 24. Buttner CR, Chechik M, Ortiz-Lombardia M, Smits C, Ebong IO, Chechik V, et al. (2012) Structural basis for DNA recognition and loading into a viral packaging motor. Proc Natl Acad Sci U S A 109: 811–816. pmid:22207627
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref25] 25. Chai S, Lurz R, Alonso JC (1995) The small subunit of the terminase enzyme of Bacillus subtilis bacteriophage SPP1 forms a specialized nucleoprotein complex with the packaging initiation region. Journal of Molecular Biology 252: 386. pmid:7563059
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref26] 26. Oliveira L, Tavares P, Alonso JC (2013) Headful DNA packaging: bacteriophage SPP1 as a model system. Virus Res 173: 247–259. pmid:23419885
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref27] 27. Wu H, Sampson L, Parr R, Casjens S (2002) The DNA site utilized by bacteriophage P22 for initiation of DNA packaging. Molecular microbiology 45: 1631. pmid:12354230
View Article
PubMed/NCBI
Google Scholar

[99] View Article

[100] PubMed/NCBI

[101] Google Scholar

[ref28] 28. Black LW (2015) Old, new, and widely true: The bacteriophage T4 DNA packaging mechanism. Virology.
View Article
Google Scholar

[103] View Article

[104] Google Scholar

[ref29] 29. Furth ME, Wickner SH (1983) Lambda DNA replication. In: Hendrix RW, Roberts JW, Stahl FW, Weisberg RA, editors. Lambda II. Cold Spring Harbor NY: Cold Spring Harbor Press. pp. 145.

[ref30] 30. Maluf N, Gaussier H, Bogner E, Feiss M, Catalano C (2006) Assembly of bacteriophage lambda terminase into a viral DNA maturation and packaging machine. Biochemistry 45: 15259. pmid:17176048
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref31] 31. Maluf NK, Feiss M (2006) Virus DNA translocation: progress towards a first ascent of mount pretty difficult. Mol Microbiol 61: 1. pmid:16824089
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref32] 32. Higgins RR, Lucko HJ, Becker A (1988) Mechanism of cos DNA cleavage by bacteriophage lambda terminase: multiple roles of ATP. Cell 54: 765. pmid:2970303
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref33] 33. Higgins RR, Becker A (1994) The lambda terminase enzyme measures the point of its endonucleolytic attack 47 +/- 2 bp away from its site of specific DNA binding, the R site. Embo J 13: 6162. pmid:7813453
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref34] 34. Hang JQ, Catalano CE, Feiss M (2001) The functional asymmetry of cosN, the nicking site for bacteriophage lambda DNA packaging, is dependent on the terminase binding site, cosB. Biochemistry 40: 13370. pmid:11683647
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref35] 35. Higgins RR, Becker A (1994) Chromosome end formation in phage lambda, catalyzed by terminase, is controlled by two DNA elements of cos, cosN and R3, and by ATP. Embo J 13: 6152. pmid:7813452
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref36] 36. Miller G, Feiss M (1988) The bacteriophage lambda cohesive end site: isolation of spacing/substitution mutations that result in dependence on Escherichia coli integration host factor. Molecular & General Genetics 212: 157.
View Article
Google Scholar

[131] View Article

[132] Google Scholar

[ref37] 37. Becker A, Murialdo H, Gold M (1977) Early events in the in vitro packaging of bacteriophage DNA. Virology 78: 291. pmid:860405
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref38] 38. Catalano CE, Cue D, Feiss M (1995) Virus DNA packaging: the strategy used by phage lambda. Molecular Microbiology 16: 1075. pmid:8577244
View Article
PubMed/NCBI
Google Scholar

[138] View Article

[139] PubMed/NCBI

[140] Google Scholar

[ref39] 39. Dokland T, Murialdo H (1993) Structural transitions during maturation of bacteriophage lambda capsids. Journal of Molecular Biology 233: 682. pmid:8411174
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref40] 40. Lander GC, Evilevitch A, Jeembaeva M, Potter CS, Carragher B, Johnson JE (2008) Bacteriophage lambda stabilization by auxiliary protein gpD: timing, location, and mechanism of attachment determined by cryo-EM. Structure (London, England: 1993) 16: 1399.
View Article
Google Scholar

[146] View Article

[147] Google Scholar

[ref41] 41. Yang Q, Catalano CE (2003) Biochemical characterization of bacteriophage lambda genome packaging in vitro. Virology 305: 276. pmid:12573573
View Article
PubMed/NCBI
Google Scholar

[149] View Article

[150] PubMed/NCBI

[151] Google Scholar

[ref42] 42. Dhar A, Feiss M (2005) Bacteriophage lambda terminase: alterations of the high-affinity ATPase affect viral DNA packaging. J Mol Biol 347: 71. pmid:15733918
View Article
PubMed/NCBI
Google Scholar

[153] View Article

[154] PubMed/NCBI

[155] Google Scholar

[ref43] 43. Feiss M, Sippy J, Miller G (1985) Processive action of terminase during sequential packaging of bacteriophage lambda chromosomes. Journal of Molecular Biology 186: 759. pmid:3005594
View Article
PubMed/NCBI
Google Scholar

[157] View Article

[158] PubMed/NCBI

[159] Google Scholar

[ref44] 44. Cue D, Feiss M (1993) A site required for termination of packaging of the phage lambda chromosome. Proc Natl Acad Sci U S A 90: 9290–9294. pmid:8415694
View Article
PubMed/NCBI
Google Scholar

[161] View Article

[162] PubMed/NCBI

[163] Google Scholar

[ref45] 45. Wieczorek DJ, Feiss M (2001) Defining cosQ, the site required for termination of bacteriophage lambda DNA packaging. Genetics 158: 495. pmid:11404316
View Article
PubMed/NCBI
Google Scholar

[165] View Article

[166] PubMed/NCBI

[167] Google Scholar

[ref46] 46. Cue D, Feiss M (2001) Bacteriophage lambda DNA packaging: DNA site requirements for termination and processivity. J Mol Biol 311: 233–240. pmid:11478856
View Article
PubMed/NCBI
Google Scholar

[169] View Article

[170] PubMed/NCBI

[171] Google Scholar

[ref47] 47. Cue D, Feiss M (1993) The role of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, in the nicking reaction. J Mol Biol 234: 594–609. pmid:8254662
View Article
PubMed/NCBI
Google Scholar

[173] View Article

[174] PubMed/NCBI

[175] Google Scholar

[ref48] 48. Kosturko L, Daub E, Murialdo H (1989) The interaction of E. coli integration host factor and lambda cos DNA multicomplex formation and protein-induced bending. Nucleic Acids Res 17: 329.
View Article
Google Scholar

[177] View Article

[178] Google Scholar

[ref49] 49. Bear S, Court D, Friedman D (1984) An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging. J Virol 52: 966. pmid:6238175
View Article
PubMed/NCBI
Google Scholar

[180] View Article

[181] PubMed/NCBI

[182] Google Scholar

[ref50] 50. Xin W, Feiss M (1993) Function of IHF in lambda DNA packaging. I. Identification of the strong binding site for integration host factor and the locus for intrinsic bending in cosB. Journal of Molecular Biology 230: 492. pmid:8385227
View Article
PubMed/NCBI
Google Scholar

[184] View Article

[185] PubMed/NCBI

[186] Google Scholar

[ref51] 51. Ortega ME, Catalano CE (2006) Bacteriophage lambda gpNu1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA: implications for the assembly of a genome-packaging motor. Biochemistry 45: 5180. pmid:16618107
View Article
PubMed/NCBI
Google Scholar

[188] View Article

[189] PubMed/NCBI

[190] Google Scholar

[ref52] 52. Rice PA, Yang S, Mizuuchi K, Nash HA (1996) Crystal structure of an IHF-DNA complex: a protein-induced DNA U-turn. Cell 87: 1295. pmid:8980235
View Article
PubMed/NCBI
Google Scholar

[192] View Article

[193] PubMed/NCBI

[194] Google Scholar

[ref53] 53. Yang Q, Hanagan A, Catalano CE (1997) Assembly of a nucleoprotein complex required for DNA packaging by bacteriophage lambda. Biochemistry 36: 2744. pmid:9062101
View Article
PubMed/NCBI
Google Scholar

[196] View Article

[197] PubMed/NCBI

[198] Google Scholar

[ref54] 54. Casjens SR (2008) Diversity among the tailed-bacteriophages that infect the Enterobacteriaceae. Research in microbiology 159: 340. pmid:18550341
View Article
PubMed/NCBI
Google Scholar

[200] View Article

[201] PubMed/NCBI

[202] Google Scholar

[ref55] 55. Cue D, Feiss M (1992) Genetic analysis of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda. J Mol Biol 228: 58–71. pmid:1447794
View Article
PubMed/NCBI
Google Scholar

[204] View Article

[205] PubMed/NCBI

[206] Google Scholar

[ref56] 56. Sippy J, Patel P, Vahanian N, Sippy R, Feiss M (2015) Genetics of critical contacts and clashes in the DNA packaging specificities of bacteriophages lambda and 21. Virology 476: 115–123. pmid:25543962
View Article
PubMed/NCBI
Google Scholar

[208] View Article

[209] PubMed/NCBI

[210] Google Scholar

[ref57] 57. Becker A, Murialdo H (1990) Bacteriophage lambda DNA: the beginning of the end. Journal of Bacteriology 172: 2819. pmid:2140565
View Article
PubMed/NCBI
Google Scholar

[212] View Article

[213] PubMed/NCBI

[214] Google Scholar

[ref58] 58. Feiss M, Geyer H, Klingberg F, Moreno N, Forystek A, Maluf NK, et al. (2015) Novel DNA packaging recognition in the unusual bacteriophage N15. Virology 482: 260–268. pmid:25956737
View Article
PubMed/NCBI
Google Scholar

[216] View Article

[217] PubMed/NCBI

[218] Google Scholar

[ref59] 59. Frackman S, Siegele DA, Feiss M (1985) The terminase of bacteriophage lambda. Functional domains for cosB binding and multimer assembly. J Mol Biol 183: 225. pmid:2989542
View Article
PubMed/NCBI
Google Scholar

[220] View Article

[221] PubMed/NCBI

[222] Google Scholar

[ref60] 60. Xu SY, Feiss M (1991) Structure of the bacteriophage lambda cohesive end site. Genetic analysis of the site (cosN) at which nicks are introduced by terminase. Journal of Molecular Biology 220: 281. pmid:1830343
View Article
PubMed/NCBI
Google Scholar

[224] View Article

[225] PubMed/NCBI

[226] Google Scholar

[ref61] 61. Feiss M, Fisher R, Siegele DA, Widner W (1981) Bacteriophage lambda and 21 packaging specificities. In: DuBow MS, editor. Bacteriophage Assembly. New York: Alan R. Liss Inc. pp. 213.

[ref62] 62. Cue D, Feiss M (1992) Genetic analysis of mutations affecting terminase, the bacteriophage lambda DNA packaging enzyme, that suppress mutations in cosB, the terminase binding site. J Mol Biol 228: 72–87. pmid:1447796
View Article
PubMed/NCBI
Google Scholar

[229] View Article

[230] PubMed/NCBI

[231] Google Scholar

[ref63] 63. Murray K, Murray NE, Bertani G (1975) Base changes in the recognition site for ter functions in lambdoid phage DNA. Nature 254: 262. pmid:1113894
View Article
PubMed/NCBI
Google Scholar

[233] View Article

[234] PubMed/NCBI

[235] Google Scholar

[ref64] 64. Feiss M, Reynolds E, Schrock M, Sippy J (2010) DNA packaging by lambda-like bacteriophages: mutations broadening the packaging specificity of terminase, the lambda-packaging enzyme. Genetics 184: 43. pmid:19841094
View Article
PubMed/NCBI
Google Scholar

[237] View Article

[238] PubMed/NCBI

[239] Google Scholar

[ref65] 65. Little JW (2010) Evolution of complex gene regulatory circuits by addition of refinements. Curr Biol 20: R724–734. pmid:20833317
View Article
PubMed/NCBI
Google Scholar

[241] View Article

[242] PubMed/NCBI

[243] Google Scholar

[ref66] 66. Hendrix RW, Lawrence JG, Hatfull GF, Casjens S (2000) The origins and ongoing evolution of viruses (Review). Trends Microbiol 8: 504. pmid:11121760
View Article
PubMed/NCBI
Google Scholar

[245] View Article

[246] PubMed/NCBI

[247] Google Scholar

[ref67] 67. Arber W, Enquist L, Hohn B, Murray NE, Murray K (1983) Experimental methods for use with lambda. In: Hendrix RW, Roberts JW, Stahl FW, Weisberg RA, editors. Lambda II. Cold Spring Harbor: Cold Spring Harbor Press. pp. 433–466.

[ref68] 68. Sternberg N, Austin S (1983) Isolation and characterization of P1 minireplicons. J Bacteriol 153: 800. pmid:6296053
View Article
PubMed/NCBI
Google Scholar

[250] View Article

[251] PubMed/NCBI

[252] Google Scholar

[ref69] 69. Pal SK, Chattoraj DK (1988) P1 plasmid replication: Initiator sequestration is inadequate to explain control by initiator-binding sites. J Bacteriol 172: 2819.
View Article
Google Scholar

[254] View Article

[255] Google Scholar

[ref70] 70. Granston AE, Alessi DM, Eades LJ, Friedman DI (1988) A point mutation in the Nul gene of bacteriophage lambda facilitates phage growth in Escherichia coli with himA and gyrB mutations. Mol Gen Genet 212: 149–156. pmid:2836702
View Article
PubMed/NCBI
Google Scholar

[257] View Article

[258] PubMed/NCBI

[259] Google Scholar

[ref71] 71. Feiss M, Siegele DA (1979) Packaging of the bacteriophage lambda chromosome: dependence of cos cleavage on chromosome length. Virology 92: 190. pmid:419690
View Article
PubMed/NCBI
Google Scholar

[261] View Article

[262] PubMed/NCBI

[263] Google Scholar

Figures

Abstract

Introduction

Results

Genetics of a cohesive end mismatch: Phages with mismatched cohesive ends are viable; the mismatches can persist to post-DNA replication

Strategy for studying DNA packaging specificity.

Genetics of mismatched cohesive ends.

Quantitating effects of a cohesive end mismatch on infectious phage production.

N15’s Packaging Specificity

cosN specificity.

Termination specificity.

Packaging specificities of λ, 21 and N15.

Discussion

Functional asymmetry of the cohesive ends of λ-like phages

Genetic consequences of the λ versus N15 cohesive end mismatch

Packaging specificity in the N15-like phages

cosB architecture and function

Genetic consequences of the asymmetric, shared packaging specificities of λ and N15

DNA recognition by TerS

Materials and Methods

Media

Bacteria, phages and plasmids

Microbiological methods

Helper phages

Dilysogens

MF611 (Tables 3 and 6).

MF3510 (Table 5).

Helper phages.

Helper packaging protocol.

Supporting Information

S1 Table. Dilysogens used in Helper Packaging Experiments.

Acknowledgments

Author Contributions

References