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.     

Model for DNA packaging into bacteriophage T4 heads.

The mechanism of DNA packaging into bacteriophage T4 heads in vivo was investigated by glucosylation of hydroxymethylcytosine residues in a conditionally glucose-deficient host. Cytoplasmic DNA associated with partially packaged ts49 heads can be fully glucosylated, whereas DNA already packaged into these heads is shown to be resistant to glucosylation. After temperature shift and completion of arrested packaging into the reversible temperature-sensitive ts49 head, the structure of the DNA in the mature ts49 phage was investigated by restriction enzyme digestion, autoradiography, and other techniques. Such mature DNA appears to be fully glucosylated along part of its length and nonglucosylated on the remainder. Its structure suggests that the DNA is run into the head linearly and unidirectionally from one mature end and that there is little sequence specificity in that portion of the T4 DNA which first enters the capsid. This technique should be useful in investigation of the three-dimensional structure of first- and last-packaged DNA within the head; preliminary studies including autoradiography of osmotically shocked phage suggest that the DNA which first enters the head is deposited toward the center of the capsid and that the end of the DNA which first enters the head exits first upon injection. In conjunction with studies of the structure of condensed DNA, the positions and functions of T4 capsid proteins in DNA packaging, and the order of T4 packaging functions [Earnshaw and Harrison, Nature (London) 268:598-602, 1977; Hsiao and Black, Proc. Natl. Acad. Sci. U.S.A. 74:3652-3656, 1977; Müller-Salamin et al., J. Virol. 24:121-134, 1977; Richards et al., J. Mol. Biol. 78:255-259, 1973], the features described above suggest the following model: the first DNA end is fixed to the proximal apex of the head at p20 and the DNA is then pumped into the head enzymatically by proteins (p20 + p17) which induce torsion in the DNA molecule.     

Identification of sequences necessary for packaging DNA into lambda phage heads

Several species of DNA molecules are packaged into lambda phage heads if they carry the region around the cohesive end site of lambda phage (cos lambda). The minimal functional sequence around cos lambda needed for packaging was examined by cloning in pBR322. The results showed that the minimal region contained 85 bp around cos lambda; 45 bp of the left arm of lambda phage and 40 bp of the right arm. A 75-bp region located to the right of the minimal region seems to enhance packaging. A 223-bp fragment containing these regions can be used as a portable element for plasmid DNA packaging into lambda phage heads. Plasmid ppBest 322, a derivative of pBR322 carrying this portable packager and both amp and tet genes, was constructed. This plasmid is useful for cloning of large DNA fragments.     

Cloning of mini-mu bacteriophage in cosmids: in vivo packaging into phage lambda heads

A technique has been developed that permits the packaging of mini-Mu-carrying cosmids into phage lambda heads. This procedure has several advantages over packaging into Mu helper capsids: the amounts of DNA to be packaged can be increased, the packaging efficiency is improved, and the stability of transducing lysates is high.     

DNA packaging steps in bacteriophage lambda head assembly

Petit λ is an empty spherical shell of protein which appears wherever λ grows. If phage DNA and petit λ are added to a cell-free extract of induced lysogenic bacteria, then phage particles are formed that contain the DNA and protein from the petit λ. Petit λ is transformed, without dissociation, into a phage head by addition of DNA and more phage proteins.The products of ten genes, nine phage and one host, are required for λ head assembly. Among these, the products of four phage genes, E, B, C, and Nu3 and of the host gene groE are involved in the synthesis of petit λ, consequently these proteins are dispensable for head assembly in extracts to which petit λ has been added. The products of genes A and D allow DNA to combine with petit λ to form a head that has normal morphology. In an extract, DNA can react with A product and petit λ to become partially DNAase-resistant, as if an unstable DNA-filled intermediate were formed. ATP and spermidine are needed at this stage. This intermediate is subsequently stabilized by addition of D product. The data suggest a pathway for head assembly.     

Assembly of bacteriophage lambda terminase into a viral DNA maturation and packaging machine

Terminase enzymes are common to complex double-stranded DNA viruses and function to package viral DNA into the capsid. We recently demonstrated that the bacteriophage lambda terminase gpA and gpNu1 proteins assemble into a stable heterotrimer with a molar ratio gpA1/gpNu1(2). This terminase protomer possesses DNA maturation and packaging activities that are dependent on the E. coli integration host factor protein (IHF). Here, we show that the protomer further assembles into a homogeneous tetramer of protomers of composition (gpA1/gpNu1(2))4. Electron microscopy shows that the tetramer forms a ring structure large enough to encircle duplex DNA. In contrast to the heterotrimer, the ring tetramer can mature and package viral DNA in the absence of IHF. We propose that IHF induced bending of viral DNA facilitates the assembly of four terminase protomers into a ring tetramer that represents the catalytically competent DNA maturation and packaging complex in vivo. This work provides, for the first time, insight into the functional assembly state of a viral DNA packaging motor.     

Formation of oligomeric structures from plasmid DNA carrying cos lambda that is packaged into bacteriophage lambda heads.

Plasmids that carry cos lambda, the region necessary for lambda phage packaging and that are as small as four kilobases in size can be packaged into lambda phage heads in head-to-tail tandem oligomeric structures. Multimeric oligomers as large as undecamers have been detected. Oligomer formation depends upon the products of red and gam of lambda, and the general recombination occurs between different plasmids that share homologous DNA regions. The packaging efficiency of plasmids depends on its copy number in cells and its genome size. Upon injection into a cell, the DNA establishes itself as a plasmid in a tandem structure. When such a plasmid in a high oligomeric structure is used as the source of packaging DNA, the packaging efficiency of the plasmids is elevated. The oligomers are stable in recA cells, whereas they drift toward lower oligomers in recA+ cells.     

Maturation of the head of bacteriophage T4. III. DNA packaging into preformed heads

An estimate was made of the amount of DNA packaged into gene 49-defective heads when P49 is activated by a temperature shift. The uptake of DNA into preformed heads following activation of P49 was studied using bromo-deoxyuridine as a label. The rate of inactivation by visible light of the phage matured in the presence of BrdU as well as their buoyant density in CsCl, indicate that over half of the particles package, on the average, at least 25% of the DNA complement following P49 activation. This is a minimum estimate, since the BrdU-labeled DNA has to compete with unlabeled DNA. Analysis on alkaline sucrose gradients of the size of the DNA extracted from phage matured in the presence of BrdU following irradiation reveals that extended irradiation at 313 nm breaks the DNA close to half of its original size. These experiments clearly show that up to half of the DNA can be packaged into the preformed heads made at high temperature following activation of the product of gene 49 (P49), strongly supporting the pathway for phage head maturation described by Laemmli &; Favre (1973).The so-called τ-particles, which accumulate in 24-defective cells, can serve as precursors of the mature phage (Bijlenga et al., 1973). We have measured the uptake of BrdU-labeled DNA into τ-particles during their maturation. We find that a very large proportion of DNA made after activation of P24 is apparently incorporated into preformed τ-particles as these particles are converted into mature heads. This indicates that the τ-particles contain very little or no DNA prior to P24 activation and supports the pathway described by Laemmli &; Favre (1973).     

Injection of DNA into liposomes by bacteriophage lambda

Small unilamellar vesicles (75-100 nm diameter) and large liposomes (greater than 1 micron in diameter) were prepared containing the lamB protein, an outer membrane protein of Escherichia coli and Shigella which serves as the receptor for bacteriophage lambda. Bacteriophage were observed to bind to these liposomes and vesicles by their tails and in most cases the heads of the bound bacteriophage appeared empty or partially empty of DNA. The lambda DNA was usually only partially ejected from the bacteriophage head when small unilamellar liposomes were used, presumably because the vesicles are too small to contain all the DNA. The partially ejected DNA was not susceptible to DNase unless the vesicle bilayer was first disrupted suggesting that DNA injection of phage DNA into the vesicle had occurred. After disruption of these vesicles on electron microscope grids, the bacteriophage are seen to have partially empty heads and a small mass of DNA associated with their tails. Using larger liposomes prepared by the fusion of lamB bearing vesicles with polyethylene glycol and n-hexyl bromide, the heads of most of the bound bacteriophage appeared to be completely empty of DNA. Disruption of these preparations on electron microscope grids revealed circular arrays of empty-headed bacteriophage surrounding DNA which had apparently been contained within the intact liposomes. These results indicate that high molecular weight DNA can be entrapped within liposomes with high efficiency by ejection from bacteriophage lambda. The possible use of these DNA-containing liposomes to facilitate gene transfer in eukaryotic cells is discussed.     

Packaging of DNA into bacteriophage heads: A model

A model is suggested for the geometry of DNA entry into a bacteriophage head. It accounts for recent observations indicating absence of a unique, ordered sequence of windings in the packaged DNA.     

Polarized packaging of bacteriophage lambda chromosomes.

Packaging of chromosomes during lytic growth of cohesive end-site (cos site) duplication strains of phage lambda is strikingly asymmetric; the duplication segment is generally at the left chromosome end (Emmons, 1974). In the present study, the packaging of non-replicating cos duplication chromosomes is shown to be similarly asymmetric. It is, therefore, likely that the packaging process itself is polarized, in an A to R direction. This conclusion is based on the study of packaging of repressed prophage chromosomes of dilysogenic strains of Escherichia coli by a heteroimmune helper. In these strains one of the two prophages contains a cos duplication (see Fig. 2). The frequency with which helper-packaged chromosomes carry the cos duplication segment agrees well with expectations derived from lytically grown phage.Haploid segregants are produced from the cos duplication strain at a lower level (35%) during lytic growth than during packaging of repressed prophage chromosomes (50%). This is expected if chromosomes are packaged processively (in sequence) during lytic growth.Packaging of repressed cos triplication chromosomes by a heteroimmune helper also yields a distribution of haploid and duplication chromosomes that agrees with expectations from lytically grown cos duplication phage and the assumption that the initial cutting of a cos site to initiate a packaging sequence is made at random.Polarized, processive packaging and random initial cutting are elements of a model of lambda chromosome packaging proposed by Emmons (1974), for which our experiments provide support.     

Defining cosQ, the site required for termination of bacteriophage lambda DNA packaging

Bacteriophage lambda is a double-stranded DNA virus that processes concatemeric DNA into virion chromosomes by cutting at specific recognition sites termed cos. A cos is composed of three subsites: cosN, the nicking site; cosB, required for packaging initiation; and cosQ, required for termination of chromosome packaging. During packaging termination, nicking of the bottom strand of cosN depends on cosQ, suggesting that cosQ is needed to deliver terminase to the bottom strand of cosN to carry out nicking. In the present work, saturation mutagenesis showed that a 7-bp segment comprises cosQ. A proposal that cosQ function requires an optimal sequence match between cosQ and cosNR, the right cosN half-site, was tested by constructing double cosQ mutants; the behavior of the double mutants was inconsistent with the proposal. Substitutions in the 17-bp region between cosQ and cosN resulted in no major defects in chromosome packaging. Insertional mutagenesis indicated that proper spacing between cosQ and cosN is required. The lethality of integral helical insertions eliminated a model in which DNA looping enables cosQ to deliver a gpA protomer for nicking at cosN. The 7 bp of cosQ coincide exactly with the recognition sequence for the Escherichia coli restriction endonuclease, EcoO109I.     

Self-association properties of the bacteriophage lambda terminase holoenzyme: implications for the DNA packaging motor

Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of viral DNA into a protective capsid. Bacteriophage lambda terminase holoenzyme is a hetero-oligomer composed of the A and Nu1 lambda gene products; however, the self-association properties of the holoenzyme have not been investigated systematically. Here, we report the results of sedimentation velocity, sedimentation equilibrium, and gel-filtration experiments studying the self-association properties of the holoenzyme. We find that purified, recombinant lambda terminase forms a homogeneous, heterotrimeric structure, consisting of one gpA molecule associated with two gpNu1 molecules (114.2 kDa). We further show that lambda terminase adopts a heterogeneous mixture of higher-order structures, with an average molecular mass of 528(+/-34) kDa. Both the heterotrimer and the higher-order species possess site-specific cos cleavage activity, as well as DNA packaging activity; however, the heterotrimer is dependent upon Escherichia coli integration host factor (IHF) for these activities. Furthermore, the ATPase activity of the higher-order species is approximately 1000-fold greater than that of the heterotrimer. These data suggest that IHF bending of the duplex at the cos site in viral DNA promotes the assembly of the heterotrimer into a biologically active, higher-order packaging motor. We propose that a single, higher-order hetero-oligomer of gpA and gpNu1 functions throughout lambda development.     

Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease

The five EcoRI² restriction sites in bacteriophage lambda DNA have been mapped at 0.445, 0.543, 0.656, 0.810, and 0.931 fractional lengths from the left end of the DNA molecule. These positions were determined electron-microscopically by single-site cleavage of hydrogen-bonded circular λ DNA molecules and by cleavage of various DNA heteroduplexes between λ DNA and DNA from well defined λ mutants. The DNA lengths of the EcoRI fragments are in agreement with their electrophoretic mobility on agarose gels but are not in agreement with their mobilities on polyacrylamide gels. These positions are different from those previously published by Allet et al. (1973). Partial cleavage of pure λ DNA by addition of small amounts of EcoRI endonuclease does not lead to random cleavage between molecules. Also, the first site cleaved is not randomly distributed among the five sites within a molecule. The site nearest the right end is cleaved first about ten times more frequently than either of the two center sites.     

Location of DNA ends in P2, 186, P4 and lambda bacteriophage heads

When mature phage particles were suspended in a solution containing formaldehyde (0.07 m-Na⁺, pH 9.0, 10% HCHO for 10 min at 23 °C) and the mixture then spread for electron microscopy in the presence of 50% formamide and cytochrome c, the phage lysed and a high proportion of the DNA molecules were seen to be attached to phage tails. The phage tails were found to be attached at only one end of each DNA molecule and denaturation mapping showed that this end was unique for each of the phages P2, 186, P4 and λ. It is argued that in these mature phage particles one specific end of the DNA molecule is present at the head-tail attachment site.     

Fluorescence measurement of the kinetics of DNA injection by bacteriophage lambda into liposomes

Bacteriophage lambda attaches to Gram-negative bacteria using the outer membrane protein LamB as its receptor. Subsequently, DNA is injected by the bacteriophage into the host cell for replication and expression. The mechanism of DNA injection, however, is poorly understood. In order to begin to characterize DNA injection, a quantitative kinetic assay to detect injection into reconstituted LamB liposomes is described. The technique involves monitoring the increase in fluorescence of liposome-encapsulated ethidium bromide, which occurs as DNA enters the aqueous compartment of the vesicles. The data indicate that injection is several times faster than indicated by earlier studies and is complete within 1 min. Such assays which allow direct observation of this process are necessary first steps toward a mechanistic understanding.     

Models of bacteriophage DNA packaging motors

An ATP-dependent motor drives a DNA genome into a bacteriophage capsid during morphogenesis of double-stranded DNA bacteriophages both in vivo and in vitro. The DNA molecule enters the capsid through a channel in the center of a symmetric protein ring called a connector. Mechanisms in two classes have been proposed for this motor: (1) An ATP-driven rotating connector pulls a DNA molecule via serial power strokes. (2) The connector rectifies DNA motion that is either thermal, biased thermal, or oscillating electrical field-induced (motor-ratchet hypothesis). Mechanisms in the first class have previously been proposed to explain the detailed structure of DNA packaging motors. The present study demonstrates that the motor-ratchet hypothesis also explains the current data, including data in the following categories: biochemical genetics, energetics, structure, and packaging dynamics.