Viable Phage Particles Research Articles

A mutation downstream from the signal peptidase cleavage site affects cleavage but not membrane insertion of phage coat protein.

Morphogenesis of filamentous phage includes synthesis of the phage major coat protein in precursor form, its insertion into the host cell plasma membrane, its cleavage to the mature form of the protein, and its assembly there into virions. The M13 mutant am8H1R6 encodes a coat protein in which leucine replaces glutamic acid as residue 2 of the mature protein [Boeke, J. D., Russel, M. & Model, P. (1980) J. Mol. Biol. 144, 103-116]. The coat protein precursor produced by this variant is a poor substrate for the Escherichia coli signal peptidase both in vivo and in vitro. This pre-coat protein, which is eventually processed and assembled into viable phage particles, is associated with the membrane fraction of the infected cell. We conclude that the domain recognized by the signal peptidase extends beyond the signal peptide itself. Furthermore, membrane association and signal peptide cleavage can be separated temporally under conditions that permit membrane insertion, cleavage, and phage assembly.

In Vitro Packaging of Damaged Bacteriophage T7 DNA

Experiments using in vitro packaging to monitor the biological activity of DNA recovered after in vitro repair, replication, and recombination reactions are described. These results suggest that the in vitro systems mimic the in vivo situation sufficiently well to allow generation (or restoration) of DNA molecules which can be encapsulated to form fully viable T7 phage particles. The in vitro packaging system has proved to be a convenient and relatively sensitive means for determining the amount of biological damage present in T7 DNA and for examining the response of various DNA metabolic systems to that damage.

Mutations in Coliphage P1 Affecting Host Cell Lysis

A total of 103 amber mutants of coliphage P1 were tested for lysis of nonpermissive cells. Of these, 83 caused cell lysis at the normal lysis time and have defects in particle morphogenesis. Five amber mutants, with mutations in the same gene (gene 2), caused premature lysis and may have a defect in a lysis regulator. Fifteen amber mutants were unable to cause cell lysis. Artificially lysed cells infected with five of these mutants produced viable phage particles, and phage particles were seen in thin sections of unlysed, infected cells. However, phage production by these mutants was not continued after the normal lysis time. We conclude that the defect of these five mutants is in a lysis function. The five mutations were found to be in the same gene (designated gene 17). The remaining 10 amber mutants, whose mutations were found to be in the same gene (gene 10), were also unable to cause cell lysis. They differed from those in gene 17 in that no viable phage particles were produced from artificially lysed cells, and no phage particles were seen in thin sections of unlysed, infected cells. We conclude that the gene 10 mutants cannot synthesize late proteins, and it is possible that gene 10 may code for a regulator of late gene expression for P1.

Incorporation of 5-substituted uracil derivatives into nucleic acids. The isolation and gamma-radiation-sensitivity of bacteriophage T3 containing the thymine analogue 5-vinyluracil.

Bacteriophage T3 was produced in a form that contained 32% of its normal DNA thymine residues replaced with 5-vinyluracil residues by infecting a thymine-requiring strain of Escherichia coli with phage T3 in a medium containing 5-vinyluracil. When 2'-deoxy-5-vinyluridine was added to the medium instead, no incorporation was observed into the phage DNA, and the presence of the deoxyribonucleoside severely decreased the number of viable phage particles produced. The analogue-containing phage, although initially viable, rapidly lost viability when stored, but it was no more sensitive than was normal phage T3 to the effect of gamma-radiation.

The isolation of structural genes from libraries of eucaryotic DNA

We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of β-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked β-globin genes.

In Vitro Packaging of UV Radiation-Damaged DNA from Bacteriophage T7

When DNA from bacteriophage T7 is irradiated with UV light, the efficiency with which this DNA can be packaged in vitro to form viable phage particles is reduced. A comparison between irradiated DNA packaged in vitro and irradiated intact phage particles shows almost identical survival as a function of UV dose when Escherichia coli wild type or polA or uvrA mutants are used as the host. Although uvrA mutants perform less host cell reactivation, the polA strains are identical with wild type in their ability to support the growth of irradiated T7 phage or irradiated T7 DNA packaged in vitro into complete phage. An examination of in vitro repair performed by extracts of T7-infected E.coli suggests that T7 DNA polymerase may substitute for E. coli DNA polymerase I in the resynthesis step of excision repair. Also tested was the ability of a similar in vitro repair system that used extracts from uninfected cells to restore biological activity of irradiated DNA. When T7 DNA damaged by UV irradiation was treated with an endonuclease from Micrococcus luteus that is specific for pyrimidine dimers and then was incubated with an extract of uninfected E. coli capable of removing pyrimidine dimers and restoring the DNA of its original (whole genome size) molecular weight, this DNA showed a higher packaging efficiency than untreated DNA, thus demonstrating that the in vitro repair system partially restored the biological activity of UV-damaged DNA.

Normal expression of the viral gene N interferes with growth of bacteriophage λ in Escherichia coli 15T-

Growth of λ and of some lambdoid phages is considerably inhibited on strain 3057 derived from E. coli 15T-. Mutants of λ which overcome this inhibition map in gene N. Some of these λhty mutants are temperature sensitive for growth on E. coli K12. Thus plating of λ on strain 3057 allows one to isolate temperature sensitive N mutants. The λhty mutants produce less than normal N activity as judged by their low efficiency of plating on a nus- host and by the extended latent period of some of them on normal hosts. The inability of strain 3057 to propagate λ can be at least partially reversed by addition of thymidine to the medium and the growth difference between λhty and λ in 3057 increases with decreasing thymidine concentration. The amount of DNA produced by λ in 3057 at low thymidine concentration is lower than that produced by λhty under the same conditions. Only a small percentage of the DNA produced by λ in 3057 is packaged into viable phage particles. This suggests that λ not only produces less DNA in 3057 than λhty but that an important part of the λ DNA in 3057 is in a form which can not be packaged or which is noninfective for other reasons. A hypothesis is discussed that hty mutations enable λ to grow on E. coli 15T- at low thymidine concentration because they lead to reduction in the number of single strand nicks in the λ DNA by reducing the intracellular endonuclease activity. Under permissive conditions conditional lethal N mutants are favored for growth on 3057 over λN+ which confirms the idea that N activity or the activity of a gene under N control interferes with λ growth in 3057 at low thymidine concentration.

The production of undegraded cytosine-containing DNA by bacteriophage T4 in the absence of dCTPase and endonucleases II and IV, and its effects on T4-directed protein synthesis

We have studied the in vivo effects of T4 endonucleases II and IV on the cytosine-containing T4 DNA made after infection of Escherichia coli B with dCTPase amber mutants of bacteriophage T4. Both nucleases are specific for cytosine-containing DNA; endonuclease II is a nickase, and endonuclease IV is specific for single-stranded stretches of DNA. The small amount of DNA made by dCTPase amber mutants is rapidly degraded; in contrast, the (dCTPase, endonuclease II, endonuclease IV) mutant makes normal amounts of DNA. Although this cytosine-containing T4 DNA is actually larger than that made by T4D + , as determined by both neutral and alkaline density-gradient sedimentation, no viable phage particles are produced. The relative importance of the two nucleases in the degradation process differs from that observed previously for host DNA degradation. Degradation of cytosine-containing T4 DNA is largely prevented by abolishing endonuclease IV alone but occurs normally in the absence of endonuclease II, whereas the reverse is true for host DNA degradation. Possible reasons for this disparity are discussed. To determine the reason for the non-viability of the (dcTPase, endonuclease II, endonuclease IV) triple mutant, we have studied the in vivo patterns of protein synthesis. Using polyacrylamide gel electrophoresis, we find that the synthesis of all late proteins we can analyze is almost totally abolished when cytosine replaces hydroxymethylcytosine in the progeny DNA, even though DNA degradation has been prevented by the nuclease mutations. In fact, substantially more late-protein synthesis is seen with T4 mutants totally defective in DNA synthesis than when the progeny DNA contains cytosine rather than hydroxymethyl-cytosine. Only with gene 45 or 55 mutants, both of which affect RNA polymerase alterations, is the block of late-protein synthesis more stringent. In contrast, the timing of shutoff of early-enzyme synthesis is almost normal, although there is some overproduction of most early enzymes. Those proteins synthesized by T4D + throughout the infection cycle are produced in similar manner and amounts by our triple mutant, except that the gene 32 protein is grossly overproduced, as it is after infection with certain other mutants that make aberrant unencapsulated DNA.

In vitro packaging of covalently closed circular monomers of bacteriophage DNA.

Extracts from coliphage P2 or P4-infected cells can package exogenous P2 or P4 DNA into viable phage particles. This in vitro system was used to determine the structure of the DNA substrate for the packaging reaction. Results obtained with covalently joined linear concatemers, covalently closed circles, and mature phage DNA as the substrates indicate that DNA packaging in the phages P2 and P4 does not involve the cutting of mature phage DNA from a concatemeric precursor. Instead, the substrate of the in vitro DNA packaging reaction appears to be either a covalently closed circular monomer of the phage DNA or the mature phage DNA itself. Sedimentation analysis of P2 phage DNA synthesized in cells infected with a P2 head mutant showed that covalently closed circular DNA molecules accumulate in such cells. Together, the in vitro and in vivo results suggest that covalently closed monomer circles are the substrates of the P2 and P4 DNA packaging reactions.

Polyacrylamide gel electrophoresis of intact bacteriophage T4D particles

A method for the electrophoresis of intact bacteriophage T4D particles through polyacrylamide gels has been developed. It was found that phage particles will migrate through dilute polyacrylamide gels (less than 2.1%) in the presence of a low concentration of MgCl2. As few as 5 x 10(9) phage particles can be seen directly as a light-scattering band during the course of electrophoresis. The band can also be detected by scanning gels at 260 to 265 nm or by eluting viable phage particles from gel slices. A new mutant (eph1) has been identified on the basis of its decreased electrophoretic mobility compared with that of the wild type; mutant particles migrated 14% slower than the wild type particles at pH 8.3 and 35% slower at pH 5.0. The isoelectric points of both the wild type and eph1 mutant were found to be between pH 4.0 and 5.0. Particles of T4 with different head lengths were also studied. Petite particles (heads 20% shorter than normal) migrated at the same rate as normal-size particles. Giant particles, heterogenous with respect to head length (two to nine times normal), migrated faster than normal-size particles as a diffuse band. This diffuseness was due to separation within the band of particles having mobilities ranging from 8 to 35% faster than those of normal-size particles. These observations extend the useful range of polyacrylamide gel electrophoresis to include much larger particles than have previously been studied, including most viruses.

Intercellular transfer by phage receptor site lipopolysaccharide.

TREATMENT of Escherichia coli cells with lysozyme and EDTA partially removes the outer layer of the cell wall containing lipopolysaccharide (LPS), leaving osmotically unstable spheroplasts1. These can be infected with phage nucleic acid2 and can produce viable phage particles. Removal of LPS-containing phage receptor sites3–5, however, leaves spheroplasts resistant to infection by intact phages1. We now show that LPS, obtained from phage-sensitive cells by aqueous phenol extraction, can provide functional phage receptor sites to spheroplasts prepared from cells lacking receptor sites.

The inability of T-even phages to produce heat-stable density mutants and its bearing on chromosome maturation.

Heat stable (st) mutants have been isolated from phages T5 (Rubenstein, 1968), T1, T3, T7 (Ritchie & Malcolm, 1970) and λ (Parkinson & Huskey, 1971). In addition to their increased resistance to heat inactivation the st mutant phage particles also have a lower buoyant density and, with the possible exception of T1, contain less DNA than their respective wild-types. All these phages have DNA molecules with non-permuted base sequences (Olligs, 1967; Thomas & MacHattie, 1967) which have been suggested to arise from intracellular concatenated DNA forms (found with T5, T7 and λ) by a sequence specific cutting mechanism which recognizes sites marking the ends of mature DNA molecules (Thomas, Kelly & Rhoades, 1968). Therefore the deletion of non-essential DNA sequences would produce viable phage particles with less DNA than wild-type; this would reduce the buoyant density and apparently also increases the heat stability.

Conversion of T4 gene 46 mutant deoxyribonucleic acid into nonviable bacteriophage particles.

The T4 amber mutant N130 in gene 46 produces, upon infection of a nonpermissive host, about 10 times more phage equivalent of deoxyribonucleic acid (DNA) than viable phage particles. Intracellular DNA is shorter than its mature phage counterpart and is converted into nonviable, light phage particles (287S) with about half of the normal DNA content, incapable either of adsorbing to or killing host bacteria. This DNA is highly unstable and breaks down, upon extraction, into subunits one-tenth the normal length of T4 DNA. Electron micrographs showed that the nonviable particles consist of normal-sized capsids of various degrees of fullness. Abnormal tail structures (naked cores with the sheaths missing) were also observed. Under conditions of arrested DNA synthesis, late messenger ribonucleic acid is produced, but some species are rare or missing, resulting in uneven transcription of the late genes. Our findings indicate that continuous DNA replication is necessary for normal transcription in T4.

Three cistrons in bacteriophage Qβ

Amber mutants of bacteriophage Qβ were isolated and classified into three groups by complementation tests. Physiological studies showed that the three cistrons of Qβ could be related to the three known genes of the f2 group of RNA phages. Gene I mutants do not produce either phage RNA polymerase or phage specific RNA in Su − hosts. Gene II mutants in Su − hosts produce defective phage particles which are lighter in density than viable phage particles. Gene III mutants do not grow on either Su-2 or Su-3 suppressor strains; they hyperproduce double-stranded RNA and viral RNA polymerase on these hosts. On Su − hosts they appeared to have a polar effect on the synthesis of phage RNA polymerase. None of the Qβ mutants lysed nonpermissive hosts.

Effect of nalidixic acid on deoxyribonucleic acid synthesis in bacteriophage SPO1-infected Bacillus subtilis.

The effect of nalidixic acid on deoxyribonucleic acid (DNA) synthesis in Bacillus subtilis cells infected with bacteriophage SPO1 was studied. Nalidixic acid had little inhibitory effect on SPO1 DNA synthesis at concentrations that drastically inhibited B. subtilis DNA synthesis. Inhibition of DNA synthesis, appropriate to the concentration used, was imposed within 1 min after addition of nalidixic acid, suggesting that it acts directly on DNA synthesis in both infected and uninfected cells. The SPO1 DNA synthesized in the presence of high concentrations of nalidixic acid had a density characteristic of normal SPO1 DNA and was packaged into viable progeny phage particles, but its rate of synthesis was reduced and bacterial lysis was delayed.

The action of 5-azacytidine on bacteria infected with bacteriophage T4

1. 1. 5-Azacytidine, in concentrations higher than 0.5 μg/ml, strongly inhibits the production of viable phage particles by bacteria infected with bacteriophage T4. The inhibitory action can be abolished completely by cytidine and mitigated slightly by deoxycytidine. The inhibition becomes irreversible about 3 min after the addition of 5-azacytidine. When added later than 12 min after infection, 5-azacytidine does not interfere with phage maturation. 2. 2. Synthesis of RNA is not affected by 5-azacytidine but synthesis of DNA is strongly inhibited. The inhibition of DNA synthesis cannot be reversed by deoxy-cytidine, but is reversed by cytidine. In un-infected cells 5-azacytidine does not inhibit the synthesis of DNA. 3. 3. When 5-azacytidine is added immediately after infection, total protein synthesis is inhibited only in the late period of phage development; the synthesis of late phage-specific proteins is inhibited, whereas the early enzymes are slightly over-produced. 4. 4. The frequency of genetic recombination is not affected by 5-azacytidine.

Assembly of phage lambda in vitro.

Edgar and Wood [1] have shown recently that it is possible to assemble, in vitro, the parts of phage T4 which accumulate in mutant infected cells to form infectious, viable phage particles. This complementation in vitro can also be demonstrated for phage lambda, as is shown below. One would expect lambda to be less complex in structure than T4, since the chromosome of lambda has only one fourth of the DNA content of T4 and presumably contains fewer genes. Indeed, it is found that the physiological functions of the genes of lambda, as revealed by in vitro assemblage, divide into two groups only-head donors and tail donors, in the terminology of Edgar and Wood [1]. Evidence is presented which suggests that the heads and tails of lambda can unite to form viable phage in the absence of other factors.

The ability of single phage particles to form plaques and to multiply in liquid cultures.

SUMMARY: The results of testing a bacteriophage to a strain of clover nodule bacteria using young (1 day) and old (5 days) bacterial cultures both fit to the hypothesis that phage multiplication can be initiated by single phage particles. As the same phage preparations gave more plaques on solid media and higher proportions of liquid cultures in which phage multiplication could be detected, with young than with old bacterial cultures, the fit to the hypothesis is not an evidence that every single phage particle will multiply. It may be so when young bacterial cultures are used, although there is no positive evidence for it. With older bacterial cultures definitely only a proportion of viable phage particles succeed in starting phage multiplication, the proportion decreasing with the increasing age of bacterial cultures used for testing.