Initial cos cleavage of bacteriophage lambda concatemers requires proheads and gpFI in vivo

The development of bacteriophage lambda and double-stranded DNA viruses in general involves the convergence of two separate pathways: DNA replication and head assembly. Clearly, packaging will proceed only if an empty capsid shell, the prohead, is present to receive the DNA, but genetic evidence suggests that proheads play another role in the packaging process. For example, lambda phages with an amber mutation in any head gene or in FI, the gene encoding the accessory packaging protein gpFI, are able to produce normal amounts of DNA concatemers but they are not cut, or matured, into unit length chromosomes for packaging. Similar observations have been made for herpes simplex 1 virus. In the case of lambda, a negative model proposes that in the amber phages, unassembled capsid components are inhibitory to maturation, and a positive model suggests that assembled proheads are required for cutting. We tested the negative model by using a deletion mutant devoid of all prohead genes and FI in an in vivo cos cleavage assay; in this deleted phage, the cohesive ends were not cut. When lambda proheads and gpFI were provided in vivo via a second prophage, cutting was restored, and gpFI was required, results that support the positive model. Phage 21 is a sister phage of lambda, and although its capsid proteins share approximately 60% residue identity with lambda's, phage 21 proheads did not restore cutting, even when provided with the accessory protein gpFI. Models for the role of proheads and gpFI in cos cutting are discussed.     

Bacteriophage lambda DNA packaging. The product of the FI gene promotes the incorporation of the prohead to the DNA-terminase complex

Lambda DNA packaging in vitro can be examined in stages. In a first step, lambda DNA interacts with terminase to form a DNA-enzyme complex, called complex I. Upon addition of proheads, in a second step, a ternary complex, complex II, containing DNA, terminase and the prohead is formed. Finally, upon addition of the rest of the morphogenetic components, complete phages are assembled. We have investigated the effect of the FI gene product (gpFI) in these reactions and found that a stimulation in phage yield is observed when gpFI is included early in the reaction, at the time when DNA, terminase and proheads interact to form complex II. Measurements of complex II formation revealed that gpFI stimulated the rate of formation of this intermediate. gpFI was further shown to stimulate the addition of proheads to preformed complexes I to give complex II, but the protein did not stimulate complex I formation.     

RNA-mediated specificity of DNA packaging into hybrid lambda/phi 29 proheads.

A small RNA (pRNA, 174 nt) is known to be essential for DNA packaging in bacteriophage phi 29. However, in an in vitro DNA packaging system based on hybrid lambda/phi 29 proheads (made up of head proteins from phage lambda and connectors from phage phi 29), the specificity of DNA packaging is lost, and different RNA molecules fulfil the requirements for DNA packaging, albeit with less efficiency than phi 29 pRNA. Competition assays with RNAs from different sources have shown that phi 29 connectors bind preferentially pRNA. An increase in the efficiency of phi 29 DNA packaging into hybrid proheads induced by phi 29 pRNA is observed because, when phi 29 pRNA is incubated with hybrid proheads, phi 29 DNA is packaged more efficiently than other DNAs of similar length. Furthermore, when hybrid proheads carrying phi 29 pRNA are incubated with a mixture of DNAs from different sources, phi 29 DNA is selectively packaged, thus indicating that phi 29 pRNA determines the specificity of DNA packaging.     

Isolation and characterization of mutations in the bacteriophage lambda terminase genes.

The terminase enzyme of bacteriophage lambda is a hetero-oligomeric protein which catalyzes the site-specific endonucleolytic cleavage of lambda DNA and its packaging into phage proheads; it is composed of the products of the lambda Nul and A genes. We have developed a simple method to select mutations in the terminase genes carried on a high-copy-number plasmid, based on the ability of wild-type terminase to kill recA strains of Escherichia coli. Sixty-three different spontaneous mutations and 13 linker insertion mutations were isolated by this method and analyzed. Extracts of cells transformed by mutant plasmids displayed variable degrees of reduction in the activity of one or both terminase subunits as assayed by in vitro lambda DNA packaging. A method of genetically mapping plasmid-borne mutations in the A gene by measuring their ability to rescue various lambda Aam phages showed that the A mutations were fairly evenly distributed across the gene. Mutant A genes were also subcloned into overproducing plasmid constructs, and it was determined that more than half of them directed the synthesis of normal amounts of full-length A protein. Three of the A gene mutants displayed dramatically reduced in vitro packaging activity only when immature (uncut) lambda DNA was used as the substrate; therefore, these mutations may lie in the endonuclease domain of terminase. Interestingly, the putative endonuclease mutations mapped in two distinct locations in the A gene separated by a least 400 bp.     

Analysis of a mutation affecting the specificity domain for prohead binding of the bacteriophage lambda terminase.

Genetic studies have identified a specificity domain for prohead binding in the C-terminal 32 amino acids of gpA, the large subunit of bacteriophage lambda terminase (S. Frackman, D. A. Siegele, and M. Feiss, J. Mol. Biol. 180:283-300, 1984). In the present work, an amber mutation, Aam42, in the fifth-to-last codon of the A gene was found to be lethal in nonsuppressing hosts. The mutation, expected to generate gpA lacking the last five amino acids, caused the production of a terminase that cut cos efficiently both in vivo and in vitro but was defective in DNA packaging. lambda Aam42 lysates contained unused proheads, consistent with a defect in prohead binding. Aam42 terminase was more strongly dependent than wild-type terminase on gpFI, the catalyst of prohead binding. Like wild-type terminase, Aam42 terminase did not cut cos in vivo when prohead assembly was blocked by a mutation in one of the genes encoding the prohead.     

DNA packaging of bacteriophage T4 proheads in vitro. Evidence that prohead expansion is not coupled to DNA packaging

We developed a system for DNA packaging of isolated bacteriophage T4 proheads in vitro and studied the role of prohead expansion in DNA packaging. Biologically active proheads have been purified from a number of packaging-deficient mutant extracts. The cleaved mature prohead is the active structural precursor for the DNA packaging reaction. Packaging of proheads requires ATP, Mg2+ and spermidine, and is stimulated by polyethylene glycol and dextran. Predominantly expanded proheads (ELPs) are produced at 37 degrees C and predominantly unexpanded proheads (ESPs) are produced at 20 degrees C. Both the expanded and unexpanded proheads are active in DNA packaging in vitro. This is based on the observations that (1) both ESPs and ELPs purified by chromatography on DEAE-Sephacel showed DNA packaging activity; (2) apparently homogeneous ELPs prepared by treatment with sodium dodecyl sulfate (which dissociates ESPs) retained significant biological activity; (3) specific precipitation of ELPs with anti-hoc immunoglobulin G resulted in loss of DNA packaging activity; and (4) ESPs upon expansion in vitro to ELPs retained packaging activity. Therefore, contrary to the models that couple DNA packaging to head expansion, in T4 the expansion and packaging appear to be independent, since the already expanded DNA-free proheads can be packaged in vitro. We therefore propose that the unexpanded to expanded prohead transition has evolved to stabilize the capsid and to reorganize the prohead shell functionally from a core-interacting to a DNA-interacting inner surface.     

The maturation of coliphage lambda DNA in the absence of its packaging

In vivo, λ DNA cannot be cleaved at cos (matured) if proheads are not present; in vitro, however, cos cleavage readily takes place in the absence of proheads. In order to investigate this paradox, we have constructed plasmids that synthesize λ terminase in vivo upon induction. The plasmids also contain cos at the normal position, about 190 bp upstream of λ gene Nul. One of the plasmids, pFM3, produces levels of terminase comparable to those found after phage induction. If cells carrying pFM3 are thermoinduced, almost 100% of the intracellular plasmid DNA has a double-strand interruption at or near cos.

Since the only λ genes that pFM3 carries are Nul, A, W and B, this in vivo cleavage is occurring in the absence of proheads. Previous failure to observe 2 maturation with phages carrying prohead mutations may be due to exonucleolytic degradation of the unprotected DNA ends, a different DNA topology or compartmentalization, or terminase inhibition in the absence of prohead by the product of another λ gene that maps to the right of gene B.     

In vitro packaging of DNA of the Bacillus subtilis bacteriophage SPP1

In vitro packaging of bacteriophage SPP1 DNA into procapsids is described and the requirements of this process were determined. Combination of proheads with an extract supplying terminase, DNA and phage tails yielded up to 10(7 )viable phages per milliliter of in vitro reaction under optimized conditions. The presence of neutral polymers and polyamines had a concentration and type dependent effect in the packaging reaction. The terminase donor extract lost rapidly activity at 30 degrees C in contrast to the stability of the prohead donor extract. Maturation to infective virions was observed using both procapsids assembled in SPP1 infected cells and procapsid-like structures assembled in Escherichia coli that overexpressed the SPP1 prohead gene clusters. Neither a majority of aberrant capsid-related structures present in the latter material nor procapsids lacking the portal protein inhibited DNA packaging. Addition of purified portal protein reduced DNA packaging activity in vitro only at concentrations 20-fold higher than those found in the SPP1 infected cell. The SPP1 DNA packaged in vitro originated exclusively from the terminase donor extract. This packaging selectivity was not observed in vivo during mixed infections. The data are compatible with a model for processive headful DNA packaging in which terminase and DNA co-produced in the same cell are tightly associated and can effectively discriminate the portal vertex of DNA packaging-proficient proheads from aberrant structures, from portal-less procapsids, and from isolated portal protein.     

Role of the amino-terminal domain of bacteriophage phi 29 connector in DNA binding and packaging.

The connector of bacteriophage phi 29 is required for prohead assembly, binds DNA, and drives DNA packaging into viral proheads. Limited proteolysis of the connector protein with endoproteinase Glu-C from Staphylococcus aureus V8 and chymotrypsin showed that a domain of the NH2-terminal region is involved in DNA binding and in the subsequent packaging into preformed proheads, but not in prohead assembly. Mutants in specific amino acids of the NH2-terminal domain, obtained by directed mutagenesis techniques, showed that the Ala1-Arg2-Lys3-Arg4 region of the connector is absolutely necessary for DNA packaging into the proheads as well as for efficient DNA binding.     

Evidence that a phage T4 DNA packaging enzyme is a processed form of the major capsid gene product

A phage T4 DNA packaging enzyme appears to arise as a processed form of the major T4 capsid structural protein gp23. The enzyme activity and antigen are missing from all head gene mutants that block the morphogenetic proteolytic processing reactions of the head proteins in vivo. The enzyme antigen can be formed in vitro by T4 (gp21) specific processing of gp23 containing extracts. Enzyme antigen is found in active processed proheads but not in full heads. The enzyme and the major capsid protein show immunological cross-reactivity, produce common peptides upon proteolysis, and share an assembly-conformation-dependent ATP binding site. The packaging enzyme and the mature capsid protein (gp23*) both appear to arise from processing of gp23, the former as a minor product of a specific gp23 structure in the prohead, acting in DNA packaging as a DNA-dependent ATPase, and a headful-dependent terminase.     

Improved in vitro packaging of coliphage lambda DNA: a one-strain system free from endogenous phage

In previous systems for in vitro packaging of lambda DNA, phages are produced from the packaging components as well as from added DNA. We have developed a new genetic strategy for in vitro packaging that bypasses this endogenous phage problem. Our system employs a single bacterial strain whose lambda prophage codes for all of the packaging proteins but is deleted for cos, the packaging origin. Crude extracts of the single lysogen: (i) are virtually free from endogenous phages, (ii) package added lambda DNA efficiently and (iii) are easy to prepare. Using the cos- in vitro packaging system we show that packaging of lambda linear monomers is a second-order reaction, but that packaging from concatemers prepared by annealing or ligation is first order. We conclude that in our cos- system, linear monomers are a poor substrate for in vitro packaging but that packaging from concatemers works well.     

EcoK restriction during in vitro packaging of coliphage lambda DNA

The K restriction system of Escherichia coli works in vitro [Meselson and Yuan, Nature 217 (1968) 1110-1114]. E. coli C lacks the K restriction system. I show that in vitro packaging in standard E. coli K-12-derived systems effects a loss of plaque-former output from K-unmodified lambda DNA relative to K-modified lambda DNA when compared with packaging in the E. coli C-derived system of Rosenberg et al. [Gene 38 (1985) 165-175]. I conclude that the EcoK restriction system is active in standard in vitro packaging systems. EcoK restriction during in vitro packaging could specifically depress recovery of some lambda and cosmid clones of eukaryotic DNA or any other DNA not modified for EcoK restriction.     

Structure and inherent properties of the bacteriophage lambda head shell. IV. Small-head mutants

Missense mutants of bacteriophage lambda that produce small proheads were found among prophage mutants defective in the major head protein gpE. Measurements of the sedimentation coefficient and molecular weight of the small proheads showed that they have the T = 4 structure composed of 240 molecules of gpE instead of the wild-type T = 7 structure composed of 420 molecules of gpE. When the phage mutants were grown in groE mutants of Escherichia coli, they produced small unprocessed proheads, which contained a smaller number (about 60) of the core protein (gpNu3) molecules than normal unprocessed proheads, which contain about 180 molecules of gpNu3. This shows that the major head protein determines the size of not only the shell but also the core of unprocessed proheads. These mutants by themselves produce very few mature small-headed phage particles, partly because the lambda DNA molecule, whose cos sites are separated at a distance of 48,500 bases, is too long to be packaged into the small proheads. However, the small proheads can package shorter DNA in vivo and in vitro at somewhat reduced efficiency, if the length or a multiple of the length between the cos sites of the DNA is 13,000 to 19,000 bases.     

The Nu1 subunit of bacteriophage lambda terminase

The maturation and packaging of bacteriophage lambda DNA are catalyzed by the phage terminase enzyme. Terminase is composed of two protein subunits, gpNu1 and gpA. The holoenzyme is multifunctional in vitro; it binds to and cleaves lambda DNA at the cos site (where cos represents cohesive-end site), packages DNA into lambda proheads, and is also a DNA-dependent ATPase. The genes of the two subunits have been cloned separately into powerful expression vectors which allow for very high levels of protein overproduction. The gpNu1 protein has been purified to homogeneity and has a monomeric molecular weight of 21,200, in close agreement with the Mr of 20,444 expected from its amino acid sequence. Both gel filtration and sedimentation velocity centrifugation indicate that the native gpNu1 protein exists as a Mr greater than 500,000 aggregate. The sequence of the first 20 amino acids and the overall composition both match those predicted by the nucleotide sequence of the Nu1 gene. Purified gpNu1 is able to complement gpA-containing extracts in both lambda DNA packaging and cos cleavage assays. The Nu1 gene amino acid sequence predicts DNA binding by the protein, and gpNu1 does show specific binding to lambda DNA by filter binding assays. Also, as predicted from its sequence, gpNu1 exhibits ATPase activity; but in contrast to the holoenzyme, this activity is DNA-independent.     

The last duplex base-pair of the phage lambda chromosome. Involvement in packaging, ejection and routing of lambda DNA.

cosN is the site at which the bacteriophage lambda DNA packaging enzyme, terminase, introduces staggered nicks to generate the cohesive ends of mature lambda chromosomes. Genetic and molecular studies show that cosN is recognized specifically by terminase and that effects of cosN mutations on lambda DNA packaging and cosN cleavage are well correlated. Mutations affecting a particular base-pair of cosN are unusual in being lethal in spite of causing only a moderate defect in cosN cleavage and DNA packaging. The particular base-pair is the rightmost duplex base-pair in mature chromosomes, at position 48,502 in the numbering system of Daniels et al; herein called position - 1. A G.C to T.A transversion mutation at position - 1, called cosN - 1T, reduces the particle yield of lambda fivefold, and the particles formed are not infectious. lambda cosN - 1T particles have wild-type morphology, and contain chromosomes that have normal cohesive ends. The chromosomes of lambda cosN - 1T particles, like the chromosomes of lambda + particles, are associated with the tail. lambda cosN - 1T particles, in spite of being normal structurally, are defective in injection of DNA into a host cell. Only approximately 25% of lambda cosN - 1T particles are able to eject DNA from the capsid in contrast to 100% for lambda +. Furthermore, for the 25% that do eject, there is a further injection defect because the ejected lambda cosN - 1T chromosomes fail to cyclize, in contrast to the efficient cyclization found for wild-type chromosomes following injection. The cosN - 1T mutation has no effect on Ca2+ mediated transformation by lambda DNA, indicating that the effect of the mutation on DNA fate is specific to the process of DNA injection. Models in which specific DNA : protein interactions necessary for DNA injection, and involving the rightmost base-pair of the lambda chromosome, are considered.     

Morphogenesis of bacteriophage phi 29 of Bacillus subtilis: oriented and quantized in vitro packaging of DNA protein gp3.

The assembly of phage phi 29 occurs by a single pathway, and the DNA protein (DNA-gp3) of "packaging intermediates" can be obtained after DNase I interruption of in vitro complementation. A broad spectrum of DNA molecules of variable length was isolated from DNase I-treated proheads. Restriction endonuclease EcoRI digestion and electrophoretic analysis of these DNA molecules suggested that DNA-gp3 packaging was oriented with respect to the physical map and was a complex process. Proteinase K-treated exogenous DNA was not packaged. When exogenous DNA-gp3 was predigested with the restriction endonucleases BstEII. EcoRI, HpaI, and HpaII, the left-end fragments, ranging in size from 8 to 0.9 megadaltons, were selectively and efficiently packaged. During in vivo and in vitro assembly, DNA-gp3 is packaged into proheads, the "core-scaffolding" protein gp7 exits from the particles, and the DNA-filled heads assume the angular morphology of phage phi 29. The packaging of a 4.1-megadalton DNA-gp3 left-end fragment (one third of the genome) resulted in the exit of gp7 and the transition to angularity.     

Role of RNA in bacteriophage phi 29 DNA packaging

A novel bacteriophage phi 29 RNA of 174 nucleotides is essential for the in vitro packaging of the DNA-terminal protein complex into proheads. The RNA, bound to the prohead portal vertex (connector), participates in assembly and function of the DNA translocating ATPase and in recognition of the DNA left-end during the course of the packaging reaction. The RNA is present in related phages and varies widely in primary sequence, but its secondary structure, as deduced by phylogenetic analysis, is both highly conserved and unique among small RNAs.     

DNA binding and bending to initiate packaging of phage lambda DNA

Physical and genetic studies verify that the DNA binding domain of protein gpNu1 (which initiates packaging of phage lambda DNA) is a winged helix-turn-helix (w HTH) and that gpNu1 dimers bind sites that are brought close through DNA bending.