Virus DNA packaging: the strategy used by phage λ

Phage λ, like a number of other large DNA bacterio-phages and the herpesviruses, produces concatemeric DNA during DNA replication. The concatemeric DNA is processed to produce unit-length, virion DNA by cutting at specific sites along the concatemer. DNA cutting is coordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the λ DNA packaging process is the phage-encoded enzyme terminase, which is involved in (i) recognition of the concatemeric λ DNA; (ii) initiation of packaging, which includes the introduction of staggered nicks at cosN to generate the cohesive ends of virion DNA and the binding of the prohead; (iii) DNA packaging, possibly including the ATP-driven DNA translocation; and (iv) following translocation, the cutting of the terminal cosN lo complete DNA packaging. To one side of cosN is the site cosB, which plays a role in the initiation of packaging; along with ATP, cosB stimulates the efficiency and adds fidelity to the endo-nuclease activity of terminase in cutting cosN. cosB is essential for the formation of a post-cleavage complex with terminase, complex I, that binds the prohead, forming a ternary assembly, complex II. Terminase interacts with cosN through its large subunit, gpA, and the small terminase subunit, gpNul, interacts with cosB. Packaging follows complex II formation. cosN is flanked on the other side by the site cosQ, which is needed for termination, but not initiation, of DNA packaging. cosQ is required for cutting of the second cosN, i.e. the cosN at which termination occurs. DNA packaging in λ has aspects that differ from other λ DNA transactions. Unlike the site-specific recombination system of λ, for DNA packaging the initial site-specific protein assemblage gives way to a mobile, translocating complete, and unlike the DNA replication system of λ, the same protein machinery is used for both initiation and translocation during λ DNA packaging.     

DNA Packaging by λ-Like Bacteriophages: Mutations Broadening the Packaging Specificity of Terminase,the λ-Packaging Enzyme

The DNA-packaging specificities of phages λ and 21 depend on the specific DNA interactions of the small terminase subunits, which have support helix-turn-recognition helix-wing DNA-binding motifs. λ-Terminase with the recognition helix of 21 preferentially packages 21 DNA. This chimeric terminase''s ability to package λDNA is reduced ∼20-fold. Phage λ with the chimeric terminase is unable to form plaques, but pseudorevertants are readily obtained. Some pseudorevertants have trans-acting suppressors that change codons of the recognition helix. Some of these codons appear to remove an unfavorable base-pair contact; others appear to create a novel nonspecific DNA contact. Helper-packaging experiments show that these mutant terminases have lost the ability to discriminate between λ and 21 during DNA packaging. Two cis-acting suppressors affect cosB, the small subunit''s DNA-binding site. Each changes a cosB^λ-specific base pair to a cosB²¹-specific base pair. These cosB suppressors cause enhanced DNA packaging by 21-specific terminase and reduce packaging by λ-terminase. Both the cognate support helix and turn are required for strong packaging discrimination. The wing does not contribute to cosB specificity. Evolution of packaging specificity is discussed, including a model in which λ- and 21-packaging specificities diverged from a common ancestor phage with broad packaging specificity.VIRUSES must package viral chromosomes from nucleic acid pools that include host-cell nucleic acids, so specific recognition of the viral nucleic acid is essential during virion assembly. For large DNA viruses, including the tailed double-strand DNA (dsDNA) bacteriophages, the herpesviruses, and the adenoviruses, DNA-packaging proteins recognize specific sequences on the viral chromosomes (reviewed in Baines and Weller 2005 and Ostapchuk and Hearing 2005, respectively). For the dsDNA viruses that produce virion chromosomes by processing concatemeric DNA, a viral terminase enzyme functions in the recognition and cutting of concatemeric DNA and subsequently sponsors DNA translocation. λ-Terminase is a heterooligomer of large and small subunits, gpA and gpNu1, respectively. Cutting of concatemeric DNA is carried out by gpA''s endonuclease activity (Becker and Gold 1978; Davidson and Gold 1992; Hwang and Feiss 1996). Three DNA subsites, cosQ, cosN, and cosB, are contained in the ∼200-bp-long cos site and orchestrate DNA packaging through interactions with terminase (Figure 1A; reviewed in Feiss and Catalano 2005). gpA introduces staggered nicks in cosN to generate the 12-bp cohesive ends of mature λDNA molecules. Efficient and accurate nicking of cosN requires anchoring of gpA by gpNu1, which binds to the adjacent cosB subsite (Higgins and Becker 1994b; Hang et al. 2001).Open in a separate windowFigure 1.—The cos and terminase region of the λ-chromosome. (A) (Top) Map of cos and the terminase-encoding Nu1 and A genes. The black bar indicates the location of the winged helix-turn-helix DNA-binding motifs in the N-terminal domain of gpNu1. (Bottom) cos subsites: cosQ is required for termination of DNA packaging; cosN is the site where the large terminase subunit, gpA, introduces staggered nicks to generate the cohesive ends of virion DNA molecules; and cosB contains the gpNu1-binding sites R1, R2, and R3 along with the IHF-binding site I1. (B) (Top) Schematic of gpNu1 residues 1–42, including the support (blue) and recognition (red) α-helixes and the wing loop (magenta). β1 and β2 are short β-strands flanking the DNA-binding elements. (Bottom) Sequences are a comparison of residues of λ''s gpNu1 and phage 21''s gp1, with conserved resides indicated by vertical lines. Note that the recognition helixes of gpNu1 and gp1 differ by four residues, all likely solvent-exposed (Becker and Murialdo 1990; de Beer et al. 2002). (C) Three-dimensional structure of the winged helix-turn-helix-containing, N-terminal domain of gpNu1 (residues 1–68) (de Beer et al. 2002). Side groups of solvent-exposed residues of the recognition helix are displayed. Color coded as in B.λ''s cosB (cosB^λ) is a complex subsite containing three copies of a gpNu1-binding sequence, the R sequence, plus a site, I1, for the integration host factor (IHF), the Escherichia coli DNA-bending protein. The order of sites is cosN–R3–I1–R2–R1. The amino-terminal half of gpNu1 contains a winged helix-turn-helix DNA-binding motif (Figure 1, B and C; Gajiwala and Burley 2000) that interacts with the R sequences. Further, the amino-terminal domain of gpNu1 is a tight dimer (Figure 1C, de Beer et al. 2002). The IHF-induced bend at I1 creates a DNA hairpin in cosB that positions the major grooves of R3 and R2 to face inward, so that the helix-turn-helix motifs of dimeric gpNu1 can be docked into them. The wing loops are positioned to make minor groove contacts with R3 and R2. Thus it is proposed that gpA is positioned to nick cosN by assembly of a bent structure with dimeric gpNu1 bound to R3 and R2 (Becker and Murialdo 1990; de Beer et al. 2002). A variety of studies indicate that the positioning of gpNu1 at R3 is crucial and that the other interactions function to create and/or stabilize the R3–gpNu1 interaction (Cue and Feiss 1993a; Higgins and Becker 1994a; Hang et al. 2001).DNA packaging initiates when terminase binds and nicks a cos. Following cosN nicking and separation of the cohesive ends, terminase remains bound to the cosB-containing chromosome end (Becker et al. 1977; Yang et al. 1997). The DNA-bound terminase docks on the portal vertex of a prohead, the empty, immature virion head shell. Assembly of the ternary prohead–terminase–DNA complex activates gpA''s potent translocation ATPase, and the viral DNA is translocated into the prohead (Yang and Catalano 2003; Dhar and Feiss 2005). Translocation brings the next cos along the concatemer to the portal-docked terminase (Feiss and Widner 1982). The downstream cos is cleaved by terminase, completing packaging of the chromosome. Recognition of the downstream cos requires cosQ and cosN (Cue and Feiss 2001). Following DNA packaging, terminase undocks from the filled head. Attachment of a tail to the DNA-filled head completes virion assembly. The undocked terminase remains bound to and sponsors the packaging of the next chromosome along the concatemer.The interactions between the recognition helix of gpNu1 and an R sequence are typical for helix-turn-helix proteins, as shown by genetic studies of chimeras between λ and its relative, phage 21, as follows: λ and 21 have similarly organized cos sites; the cosB of 21 also has the R3–I1–R2–R1 structure. Nevertheless, the two phages have distinct packaging specificities. Base-pair differences in the R sequences account for packaging specificity (Becker and Murialdo 1990; Smith and Feiss 1993). cosN and cosQ are interchangeable between λ and 21 (Feiss et al. 1981). The consensus R sequences are 5′-CGTTTCCtTTCT-3′ for cosB^λ and 5′-CaTGTCGGncCT-3′ for cosB²¹, where capitalized residues are conserved in all three R sequences of both phages; underlined and capitalized are two residues conserved in all three R sequences of both phages, but which differ between cosB^λ and cosB²¹ (Becker and Murialdo 1990). These two conserved but phage-specific base pairs are likely to be of major importance for specificity. Similarly, the recognition helixes of the helix-turn-helix motifs of the small subunits of λ (gpNu1) and 21 (gp1) terminases differ in four amino acid residues that account for packaging specificity (Figure 1; Becker and Murialdo 1990).In earlier work (de Beer et al. 2002), we showed that modifying λ-terminase by replacing the gpNu1 recognition helix with that of 21''s gp1 created a terminase (gpNu1^hy1 terminase) that was specific for the cosB of phage 21 (designated cosB²¹). That is, λ cosB²¹ Nu1^hy1 was viable, but λ cosB^λ Nu1^hy1 was inviable due to the specificity mismatch between cosB^λ and the cosB²¹-specific recognition helix of the chimeric small terminase subunit, gpNu1^hy1. The Nu1^hy1 terminase packages cosB²¹ chromosomes ∼10-fold more efficiently than it does cosB^λ chromosomes. This 10-fold discrimination between cosB²¹ and cosB^λ chromosomes is much weaker than the >10⁴-fold discrimination shown by wild-type λ and 21 terminases (de Beer et al. 2002). Because of the modest discrimination of Nu1^hy1 terminase, the yield of λ cosB^λ Nu1^hy1 is only slightly below the yield required for plaque formation. Lysates of λ cosB^λ Nu1^hy1 contain plaque-forming pseudorevertants at a level expected for single mutations. A number of these pseudorevertants were sequenced and found to contain mutations in cosB^λ or in the Nu1^hy1 gene. Here we report on in vivo packaging studies on the effects of these Nu1^hy1 and cosB^λ suppressor mutations on packaging specificity.     

Mutations That Extend the Specificity of the Endonuclease Activity of λ Terminase

Terminase, an enzyme encoded by the Nu1 and A genes of bacteriophage lambda, is crucial for packaging concatemeric DNA into virions. cosN, a 22-bp segment, is the site on the virus chromosome where terminase introduces staggered nicks to cut the concatemer to generate unit-length virion chromosomes. Although cosN is rotationally symmetric, mutations in cosN have asymmetric effects. The cosN G₂C mutation (a G-to-C change at position 2) in the left half of cosN reduces the phage yield 10-fold, whereas the symmetric mutation cosN C₁₁G, in the right half of cosN, does not affect the burst size. The reduction in phage yield caused by cosN G₂C is correlated with a defect in cos cleavage. Three suppressors of the cosN G₂C mutation, A-E₅₁₅G, A-N₅₀₉K, and A-R₅₀₄C, have been isolated that restore the yield of λ cosN G₂C to the wild-type level. The suppressors are missense mutations that alter amino acids located near an ATPase domain of gpA. λ A-E₅₁₅G, A-N₅₀₉K, and A-R₅₀₄C phages, which are cosN⁺, also had wild-type burst sizes. In vitro cos cleavage experiments on cosN G₂C C₁₁G DNA showed that the rate of cleavage for A-E₅₁₅G terminase is three- to fourfold higher than for wild-type terminase. The A-E₅₁₅G mutation changes residue 515 of gpA from glutamic acid to glycine. Uncharged polar and hydrophobic residues at position 515 suppressed the growth defect of λ cosN G₂C C₁₁G. In contrast, basic (K, R) and acidic (E, D) residues at position 515 failed to suppress the growth defect of λ cosN G₂C C₁₁G. In a λ cosN⁺ background, all amino acids tested at position 515 were functional. These results suggest that A-E₅₁₅G plays an indirect role in extending the specificity of the endonuclease activity of λ terminase.     

Packaging of coliphage lambda DNA. I. The role of the cohesive end site and the gene A protein

The cohesive ends of the DNA of bacteriophage λ particles are normally formed by the action of a nuclease on the cohesive end sites (cos) of concatemeric λ DNA (reviewed by Hohn et al., 1977). The nuclease also cuts the cos site of an integrated prophage, and DNA located to the right is preferentially packaged into phage particles. This process occurs with approximately the same efficiency and rate in a single lysogen as in a tandem polylysogen. Thus, the rate of cos cutting does not increase when the number of cos sites per molecule increases, an hypothesis that has been proposed to explain why cohesive ends are not formed in circular monomers of λ DNA. We propose instead that the interaction of Ter with cos is influenced by the configuration of the DNA outside of cos during packaging, and that this configuration is different for circular monomers than for other forms of λ DNA. A model that gives rise to such a difference is described.We also found that missense mutations in the λ A gene changed the efficiency of packaging of phage relative to host DNA. This was not the case for missense mutations in several phage genes required for capsid formation. Thus, the product of gene A plays a role in determining packaging specificity, as expected if it is or is part of the nuclease that cuts λ DNA at cos.     

Bacteriophage lambda derivatives carrying two copies of the cohesive end site

A spontaneously arising tandem duplication derivative of bacteriophage lambda has been isolated, which carries two copies of the site where the cohesive ends are formed (designated cos). Its structure has been determined by electron microscopy of DNA heteroduplexes. These heteroduplexes reveal that the duplication is usually, but not always, carried on the left end of the chromosome. A second duplication phage having two copies of cos, constructed by Feiss &; Campbell (1974), has also been studied by electron microscopy and is found to have a similar property.Unlike most tandem duplication derivatives of phage λ, the mutant studied here is not stable during growth in the absence of generalized recombination, but segregates both the triplication and the parental phage. This verifies that both cos sites are functional. The triplication does not arise as a result of end-to-end aggregation of phage chromosomes or site-specific recombination catalyzed by the chromosome maturation system at cos. It must therefore result from the cutting of mature ι chromosomes from concatemeric replication intermediates. The pattern of cutting observed shows that the λ cohesive ends are not created by a free nuclease acting on unpackaged DNA. The cutting appears to be influenced by the amount of DNA previously packaged into a phage head. A model for λ packaging is presented which explains the results.The duplication phage of Feiss &; Campbell (1974) carries a novel addition containing self-complementary sequences.     

Processing of bacteriophage lambda DNA during its assembly into heads

DNA purified from bacteriophage λ added to a cell-free extract derived from induced λ lysogens can be packaged into infectious phage particles (Kaiser & Masuda, 1973). In this paper the structure of the DNA which is the substrate for in vitro packaging and head assembly is described. The active precursor is a multichromosomal polymer that contains covalently closed cohesive end sites. Neither circular or linear DNA monomers nor polymers with unsealed cohesive ends are packaged efficiently into heads. The unit length monomer is packaged when it is either contained in the interior of a polymer (both of its ends are in cos sites) or when it has a free left end and a cos site on its right. The monomer unit with a free right end is not a substrate for packaging.A procedure is given for the purification of λ DNA fragments that contain either the left or the right cohesive end. The fragments are produced by digesting λ DNA with the site-specific Escherichia coli R₁ endonuclease; the left and right ends are separated by sedimentation through a sucrose gradient. These fragments are used to construct small polymers that have a unit length λ monomer with (1) a free left end and a closed right end, (2) a free right end and a closed left end, or (3) both ends closed in cos sites.     

Packaging of coliphage lambda DNA. II. The role of the gene D protein

The gene D protein (pD) of coliphage λ is normally an essential component of the virus capsid. It acts during packaging of concatemeric λ DNA into the phage prohead and is necessary for cutting the concatemers at the cohesive end site (cos). In this report we show that cos cutting and phage production occur without pD in λ deletion mutants whose DNA content is less than 82% that of λ wild type. D-independence appears to result directly from DNA loss rather than from inactivation (or activation) of a phage gene. (1) In cells mixedly infected with undeleted λ and a deletion mutant, particles of the deletion mutant alone are efficiently produced in the absence of pD; and (2) D-independence cannot be attributed to loss of a specific segment of the phage genome. pD-deficient phage resemble pD-containing phage in head size and DNA ends; they differ in their extreme sensitivity to EDTA, greater density, and ability to accept pD.pD appears to act by stabilizing the head against disruption by overfilling with DNA rather than by changing the capacity of the head for DNA. This is shown by the observation that the amount of DNA packaged by a “headful” mechanism, normally in excess of the wild-type chromosome size, is not reduced in the absence of pD. In fact, pD is required for packaging headfuls of DNA. This implies that a mechanism exists for preventing the entry of excess DNA into the head during packaging of concatemers formed by deletion mutants, and we suggest that this is accomplished by binding of cos sites to the head.The above results show that pD is not an essential component of the nuclease that cuts λ concatemers at cos during packaging, and they imply that 82% of a wild-type chromosome length can enter the prohead in the absence of pD. Yet, pD is needed for the formation of cohesive ends after infection with undeleted phage. We propose two models to account for these observations. In the first, cos cutting is assumed to occur early during packaging. The absence of pD leads to release of packaged DNA and the loss of cohesive ends by end-joining. In the second, cos cutting is assumed to occur as a terminal event in packaging. pD promotes cos cutting indirectly through its effect on head stability. We favor the second model because it better explains the asymmetry observed in the packaging of the chromosomes of cos duplication mutants (Emmons, 1974).     

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3′ extended DNAs, with the following sequence: 5′-GTTAGGCTT-3′ 3′-CAATCCGAA-5′. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3′ extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5′ extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.     

Mutations in Nu1, the gene encoding the small subunit of bacteriophage lambda terminase, suppress the postcleavage DNA packaging defect of cosB mutations.

The linear double-stranded DNA molecules in lambda virions are generated by nicking of concatemeric intracellular DNA by terminase, the lambda DNA packaging enzyme. Staggered nicks are introduced at cosN to generate the cohesive ends of virion DNA. After nicking, the cohesive ends are separated by terminase; terminase bound to the left end of the DNA to be packaged then binds the empty protein shell, i.e., the prohead, and translocation of DNA into the prohead occurs. cosB, a site adjacent to cosN, is a terminase binding site. cosB facilitates the rate and fidelity of the cosN cleavage reaction by serving as an anchoring point for gpNu1, the small subunit of terminase. cosB is also crucial for the formation of a stable terminase-DNA complex, called complex I, formed after cosN cleavage. The role of complex I is to bind the prohead. Mutations in cosB affect both cosB functions, causing mild defects in cosN cleavage and severe packaging defects. The lethal cosB R3- R2- R1- mutation contains a transition mutation in each of the three gpNu1 binding sites of cosB. Pseudorevertants of lambda cosB R3- R2- R1- DNA contain suppressor mutations affecting gpNu1. Results of experiments that show that two such suppressors, Nu1ms1 and Nu1ms3, do not suppress the mild cosN cleavage defect caused by the cosB R3- R2- R1- mutation but strongly suppress the DNA packaging defect are presented. It is proposed that the suppressing terminases, unlike the wild-type enzyme, are able to assemble a stable complex I with cosB R3- R2- R1- DNA. Observations on the adenosine triphosphatase activities and protease susceptibilities of gpNu1 of the Nu1ms1 and Nu1ms3 terminases indicate that the conformation of gpNu1 is altered in the suppressing terminases.     

Coupling with Packaging Explains Apparent Nonreciprocality of Chi-Stimulated Recombination of Bacteriophage Lambda by Reca and Recbc Functions

Chi (χ, 5'-GCTGGTGG) is a recombinator in RecA- and RecBC-mediated recombination in Escherichia coli. In vegetative recombination between two bacteriophage lambda strains, one with and the other without Chi (a⁺χ⁺b^- x a^-χ^ob⁺), the χ-containing recombinant (a^-χ⁺b ^-) is less abundant than the non-χ-containing recombinant (a⁺χ^ob⁺). Previously this was taken was evidence for nonreciprocality of χ-stimulated exchange. This inequality, however, is now seen to result from an event at cos (λ's packaging origin) that both activates Chi and initiates DNA packaging. An event at rightward cos leads to activation of leftward χ on the same chromosome for an exchange to its left. From the resulting circulating dimer (—cos-a⁺-χ^o-b ⁺-cos-a ^--χ⁺-b-—), the cos that activated χ is more likely to be used for rightward packaging initiation than is the cos from the other parent. Consistent with this coupling model is "biased packaging" in λ carrying two cos sites per monomer genome. When their maturation is dependent on dimerization by χ-stimulated exchange, the phage particles result more often from packaging from the cos that activates χ than from packaging from the other cos. Since Chi activation and packaging can be uncoupled, we infer that some early and reversible step in packaging activates χ. A strong candidate for this step is a double-strand break at cos that provides an oriented entry site for a recombinase.     

Transduction of a Plasmid Carrying the Cohesive End Region from Lactococcus lactis Bacteriophage ΦLC3

Plasmids carrying the cohesive end region from temperate lactococcal bacteriophage ΦLC3 could be packaged in vivo by ΦLC3 and transduced into its host strain, Lactococcus lactis subsp. cremoris NCDO 1201. The transduction frequencies were between 10^-4 and 10^-3 transducing particles per PFU, depending on the size of the phage DNA insert. This transduction system is limited to only certain lactococcal strains. The ΦLC3 cohesive site region (cos) appears to play an important role in plasmid transduction.     

Ionic switch controls the DNA state in phage λ

We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid by changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. These results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.     

Genetic Variation of Lactobacillus delbrueckii subsp. lactis Bacteriophages Isolated from Cheese Processing Plants in Finland

The genomes of four Lactobacillus delbrueckii subsp. lactis bacteriophages were characterized by restriction endonuclease mapping, Southern hybridization, and heteroduplex analysis. The phages were isolated from different cheese processing plants in Finland between 1950 and 1972. All four phages had a small isometric head and a long noncontractile tail. Two different types of genome (double-stranded DNA) organization existed among the different phages, the pac type and the cos type, corresponding to alternative types of phage DNA packaging. Three phages belonged to the pac type, and a fourth was a cos-type phage. The pac-type phages were genetically closely related. In the genomes of the pac-type phages, three putative insertion/deletions (0.7 to 0.8 kb, 1.0 kb, and 1.5 kb) and one other region (0.9 kb) containing clustered base substitutions were discovered and localized. At the phenotype level, three main differences were observed among the pac-type phages. These concerned two minor structural proteins and the efficiency of phage DNA packaging. The genomes of the pac-type phages showed only weak homology with that of the cos-type phage. Phage-related DNA, probably a defective prophage, was located in the chromosome of the host strain sensitive to the cos-type phage. This DNA exhibited homology under stringent conditions to the pac-type phages.     

Sequence analysis of bacteriophage T4 DNA packaging/terminase genes 16 and 17 reveals a common ATPase center in the large subunit of viral terminases

Phage DNA packaging is believed to be driven by a rotary device coupled to an ATPase ‘motor’. Recent evidence suggests that the phage DNA packaging motor is one of the strongest force-generating molecular motors reported to date. However, the ATPase center that is responsible for generating this force is unknown. In order to identify the DNA translocating ATPase, the sequences of the packaging/terminase genes of coliphages T4 and RB49 and vibriophages KVP40 and KVP20 have been analyzed. Alignment of the terminase polypeptide sequences revealed a number of functional signatures in the terminase genes 16 and 17. Most importantly, the data provide compelling evidence for an ATPase catalytic center in the N-terminal half of the large terminase subunit gp17. An analogous ATPase domain consisting of conserved functional signatures is also identified in the large terminase subunit of other bacteriophages and herpesviruses. Interestingly, the putative terminase ATPase domain exhibits some of the common features found in the ATPase domain of DEAD box helicases. Residues that would be critical for ATPase catalysis and its coupling to DNA packaging are identified. Com binatorial mutagenesis shows that the predicted threonine residues in the putative ATPase coupling motif are indeed critical for function.     

Molecular Mechanism of Heat Shock-Provoked Disassembly of the Coliphage λ Replication Complex

We have found previously that, in contrast to the free O initiator protein of λ phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the λO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43°C resulted in the decay of the λO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of λ plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from λ DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroEL/S and ClpP/ClpX proteins. In contrast to λcro⁺, in λcro⁻ plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the λcrots plasmid-harboring cells, a Cro repressor-independent control of λ DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to λ DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the λcro⁻ plasmid replication control.     

Single-Molecule and FRET Fluorescence Correlation Spectroscopy Analyses of Phage DNA Packaging: Colocalization of Packaged Phage T4 DNA Ends within the Capsid

Linear DNAs of any sequence can be packaged into empty viral procapsids by the phage T4 terminase with high efficiency in vitro. Packaging substrates of 5 kbp and 50 kbp, terminated by energy transfer dye pairs, were constructed from plasmid and λ phage DNAs. Nuclease and fluorescence correlation spectroscopy (FCS) assays showed that ∼ 20% of the substrate DNA was packaged and that the DNA dye ends of the packaged DNA were protected from nuclease digestion. Upon packaging, both 5-kbp and  50-kbp DNAs produced comparable fluorescence resonance energy transfer (FRET) between Cy5 and Cy5.5 double-dye terminated DNAs. Single-molecule FRET (sm-FRET) and photobleaching analysis shows that FRET is intramolecular rather than intermolecular upon packaging of most procapsids and demonstrates that single-molecule detection allows mechanistic analysis of packaging in vitro. FRET-FCS and sm-FRET measurements are comparable and show that both the 5-kbp and the  50-kbp packaged DNA ends are held within 8-9 nm of each other, within the dimensions of the long axis of the procapsid portal. The calculated distribution of FRET distances is relatively narrow for both FRET-FCS and sm-FRET, suggesting that the two packaged DNA ends are held at the same fixed distance relative to each other in most capsids. Because one DNA end is known to be positioned for ejection through the portal, it can be inferred that both DNAs ends are held in proximity to the portal entrance and ejection channel. The analysis suggests that a DNA loop, rather than a DNA end, is translocated by the packaging motor to fill the procapsid.     

Genomic Analysis of Pseudomonas putida Phage tf with Localized Single-Strand DNA Interruptions

The complete sequence of the 46,267 bp genome of the lytic bacteriophage tf specific to Pseudomonas putida PpG1 has been determined. The phage genome has two sets of convergently transcribed genes and 186 bp long direct terminal repeats. The overall genomic architecture of the tf phage is similar to that of the previously described Pseudomonas aeruginosa phages PaP3, LUZ24 and phiMR299-2, and 39 out of the 72 products of predicted tf open reading frames have orthologs in these phages. Accordingly, tf was classified as belonging to the LUZ24-like bacteriophage group. However, taking into account very low homology levels between tf DNA and that of the other phages, tf should be considered as an evolutionary divergent member of the group. Two distinguishing features not reported for other members of the group were found in the tf genome. Firstly, a unique end structure – a blunt right end and a 4-nucleotide 3′-protruding left end – was observed. Secondly, 14 single-chain interruptions (nicks) were found in the top strand of the tf DNA. All nicks were mapped within a consensus sequence 5′-TACT/RTGMC-3′. Two nicks were analyzed in detail and were shown to be present in more than 90% of the phage population. Although localized nicks were previously found only in the DNA of T5-like and phiKMV-like phages, it seems increasingly likely that this enigmatic structural feature is common to various other bacteriophages.