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.     

Studies of DNA packaging into the heads of bacteriophage lambda

During the assembly of bacteriophage λ heads, a head-like, DNA-free structure called petite λ, is first constructed. Into this, λ DNA is then packaged. In this paper we examine early interactions between λ DNA and petite λ in a cell-free system. The two major findings of this paper are: (1) when seen through the electron microscope, an early petite λ-λ DNA complex appears with the circular petite λ having the DNA crossing through its center. These resemble a bead on a string or the Greek letter φ (hence they are called φ structures). The λ A protein is required in the formation of φ structures. Also, φ structures can be found in bacteria infected with phage λ. (2) The polyamine putrescine is required for phage head assembly. An earlier reported requirement for spermidine can be replaced by the addition of putrescine. Polyamine is required in the DNA packaging reaction after the packaging has begun.     

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.     

Nucleotide sequence of bacteriophage lambda DNA

The nucleotide sequence of the DNA of bacteriophage λ has been determined using the dideoxy chain termination method in conjunction with random cloning in M13 vectors. Various methods were studied for sequencing specific regions to complete the sequence, but all were much slower than the random approach. The DNA in its circular form contains 48,502 base-pairs. Open reading frames were identified and, where possible, ascribed to genes by comparing with the previously determined genetic map. The reading frames for 46 genes were clearly identified, though in about 20 the position of the protein initiation site could not be rigorously established. Probable positions for the kil, cIII and lom genes are suggested but remain uncertain. There are about 20 other unidentified reading frames that may code for proteins.The genome is fairly compact with comparatively little non-coding DNA. In many cases the translation terminators and initiators overlap, particularly in the sequence A-T-G-A where the TGA terminates one gene and the ATG initiates the next. Such structures seem to be characterized by a purine-rich sequence, rather than by a specific “Shine and Dalgarno” sequence, before the initiator. In the whole of the left arm the codon CTA, which is normally read by a minor leucine tRNA, is absent. The distribution of other rare codons in the genes of the left arm suggests that they may have a controlling function on the relative amounts of the proteins produced.     

New map of bacteriophage lambda DNA.

A map of bacteriophage lambda was constructed, including accurate positions for all 41 cut sites made by 12 different restriction enzymes. Over 100 fragments from single, multiple, and partial enzyme digestions were measured versus standards that were calibrated with respect to DNA molecules of known sequence. The data were subjected to least-squares analysis to assign map coordinates. In no case did a fragment size predicted from the map differ from the measurement of the fragment by more than +/- 5%. This low error rate was consistent in all size ranges of fragments. The total length of lambda was calculated as 49,133 nucleotide pairs. This probably is accurate to within 500 base pairs.     

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.     

Lysogenization by bacteriophage lambda

Summary An easy and sensitive way of measuring the proportion of E. coli cells which are lysogenized by lambda phage or lambda mutants has been devised. With this assay it was possible to analyse the lysogenic response as a function of the average phage input per cell. The results indicate that lysogenization of exponentially growing cells requires that the cells are infected by at least two phages able to replicate, or three or four phages unable to replicate.     

Large scale preparation of bacteriophage lambda by tangential flow ultrafiltration for isolation of lambda DNA

A preparative procedure for purifying bacteriophage lambda from large volumes of phage lysates by recirculating tangential flow ultrafiltration is described. Lambda DNA, isolated by deproteinization of the phage, is suitable for use in molecular biology.     

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.     

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.     

Transduction of bacteriophage lambda by bacteriophage T1.

When bacteriophage T1 was grown on bacteriophage lambda-lysogenic cells, phenotypically mixed particles were formed which had the serum sensitivity, host range, and density of T1 but which gave rise to lambda phage. T1 packaged lambda genomes more efficiently both when the length of the prophage was less than that of wild-type lambda and when the host cell was polylysogenic. Expression of the red genes of lambda or the recE system of Escherichia coli during T1 growth enhanced pickup of lambda by T1, whereas packaging was reduced in recB cells. If donors were singly lysogenic, the expression of transduced lambda genomes as a PFU required lambda-specified excisive recombination, whereas lambda genomes transduced from polylysogens required only lambda- or E. coli-specified general recombination to give a productive infection.