Genetic Map of Bacteriophage α

Temperature-sensitive mutants of phage alpha were obtained by means of various mutagens and assigned to 25 complementation groups. Temperature-sensitive mutants belonging to 21 complementation groups and a mutant giving turbid plaques were used to perform two- and three-factor crosses. Seventeen of the cistrons and the turbid mutant were shown to belong to the same linear linkage group, which showed no signs of circularity. The remaining four unlinked cistrons showed peculiarities in their recombination properties. Genes which are known to be expressed earlier apear to be grouped together in a terminal segment of the linkage group.     

How Bacteriophage χ Attacks Motile Bacteria

Bacteriophage chi attaches to the filament of a bacterial flagellum by means of a tail fiber, but the ultimate receptor site for the phage is located at the base of the bacterial flagellum. Here, the phage injects its deoxyribonucleic acid into the bacterium, leaving the empty phage attached at the base. It is suggested that chi slides along the filament of the flagellum to the base, owing to the movement of the flagellum. The role of motility would thus be to provide for rapid adsorption of the phage by guiding the phage to the adsorption sites at the bases of the flagella. Bacteria whose motility has been strongly inhibited by cold or anaerobic conditions still adsorb chi at the filaments and bases of flagella if a high multiplicity is used. This indicates that direct collisions with the bases may also be possible. Bacteria must be flagellated in order for chi to attach, but only a short flagellum, perhaps only the flagellar base, is necessary.     

Purification and Chemistry of Bacteriophage ?

From a stock of varkappa phage grown on Salmonella, a host-range mutant which attacks Escherichia coli was isolated. As in the case of Salmonella, only motile strains of E. coli are sensitive to varkappa. The phage shows an eclipse period of 35 min and a minimal latent period of 52 min. The adsorption rate constant is 3 x 10(-9) ml/min. Adsorption shows a marked dependence on temperature. Bacteriophage varkappa was purified by differential centrifugation and CsCl density gradient centrifugation. It contains deoxyribonucleic acid (DNA) which is double-stranded. The DNA has a molecular weight of 42 million and a guanine plus cytosine content of 57%. Of 68 molecules of DNA inspected, 7 were circular. The phage particle weight is about 90 million.     

Evidence that Bacillus subtilis Bacteriophage SPO2 is Temperate and Heteroimmune to Bacteriophage φ105

Some characteristics of Bacillus subtilis phage SPO2 which show that it is a temperate phage are presented. Wild-type SPO2 forms turbid plaques, similar to those of other temperate phages. SPO2 lysogenic strains which are resistant to SPO2 can be isolated; these strains remain stable lysogens despite the fact that they can no longer adsorb SPO2. SPO2 lysogenic strains can be grown for many generations in SPO2 antiserum and remain lysogenic. Phage SPO2 plates on phi105 lysogens and phage phi105 plates on SPO2 lysogens; this indicates that SPO2 and phi105 are heteroimmune. Phage phi105 plates on an SPO2-resistant strain; this indicates that SPO2 and phi105 adsorb to different receptor sites on the bacterial surface.     

Genetic Recombination in Bacteriophage φX174

Genetic recombination in bacteriophage X174 usually takes place early in the infection process and involves two parental replicative form (double-stranded) DNA molecules. The host recA protein is required; none of the nine known X174 cistron products is essential. The products of a single recombination event are nonreciprocal and asymmetric. Typically, only one of the parental genotypes and one recombinant genotype are recovered from a single cell. An alternative, less efficient recombination mechanism which requires an active X174 cistron A protein is observed in the absence of the host recA gene product.     

Deoxyribonucleic Acid Replication in λ Bacteriophage Mutants

After ultraviolet light induction of Escherichia coli K-12 strain W3350(λ), several structural intermediate forms of phage deoxyribonucleic acid (DNA) are synthesized. The early defective lysogens of λ, sus O₈, sus P₃, and T₁₁, were found to synthesize none of the DNA structural intermediates. A lysogen believed to be defective in all known phage activities, λsus N₇, was found to be able to synthesize an early phage DNA intermediate. The lysogen λsus Q₂₁, defective in late phage functions, is able to synthesize the early phage DNA intermediate and a concatenated molecule of greater molecular weight than the mature λ DNA.     

Genetic Heterozygosity in Unreplicated Bacteriophage λ Recombinants

Bacteriophage crosses using density-labeled parents have been carried out under conditions restricting DNA synthesis. The parental material and genetic contributions to progeny manifesting recombination within a genetic interval sufficiently short to exhibit high negative interference have been examined. The unreplicated products of recombination isolated as phage particles appear to contain long continuous heteroduplex regions which are heterozygous for the closely linked markers. Recombination between closely linked markers seems to be the consequence of the removal of base-pair mismatches that are present within the heteroduplex regions. This localized reduction of heterozygosity within the heteroduplex regions that join the parental components of recombinant DNA molecules can account for high negative interference.     

Bacteriophage therapy--cooked goose or phoenix rising?

Recent animal and human trials of bacteriophage therapy have demonstrated its potential to alleviate bacterial diseases, both in internal and in external applications. The regulatory requirements are becoming clearer as more examples are presented. A core of GLP (Good Laboratory Practice) studies will be needed to validate safety and clinical trials to validate efficacy. GMP (Good Manufacturing Practice) production requirements and quality issues will mean that comparable costs to the production of conventional antibiotics should be anticipated. The definition of the 'active substance' will be central to the success of bacteriophage therapy to ensure that the variety and evolutionary potential of bacteriophages are exploited.     

Bacteriophage Lysozyme Synthesis in Escherichia coli K-12(λ) Infected with rII Mutant of Bacteriophage T4

When Escherichia coli K-12(λ) was infected with a T4rII bacteriophage, synthesis of lysozyme appeared at the normal time but stopped 15 min after infection. The lysozyme produced was 1% of the normal level.     

RNA Polymerase Activity Associated with Bacteriophage φ6

The Pseudomonas phaseolicola bacteriophage phi6 incorporated labeled UTP into an acid-insoluble precipitate. Incorporation was dependent on the presence of manganese acetate, ATP, GTP, CTP, and a short heat treatment of the phage; the reaction was stimulated by NH(4)Cl. The substitution of (14)C-ATP, -CTP or -GTP for UTP, together with the appropriate unlabeled ribonucleoside triphosphates, disclosed that CMP was incorporated to the greatest extent followed by GMP, UMP, and AMP. Radioactive RNAs formed by the reaction were resistant to RNases A and T(1) in high salt but susceptible to these nucleases in low salt. The labeled RNA co-sedimented and co-electrophoresed with phi6 double-stranded (ds) RNA. However, the distribution of the radioactivity into the three ds-RNA components varied depending on the (14)C-ribonucleoside triphosphate used in the reaction. The incorporation of UMP was primarily into the two smaller ds-RNA segments, GMP primarily into the large ds-RNA segment, and CMP and AMP were about equally distributed into all three ds-RNA segments.     

R17 Bacteriophage Replicase: Association with the Inhibition of Qβ, fd Bacteriophage and β-Galactosidase Production

Superinfection of Escherichia coli with amber mutants of R17 phage which produce R17 replicase inhibits production of the RNA phage Qbeta, the DNA phage fd, and the host enzyme beta-galactosidase. Inhibition required R17 replicase production and was related to the amount of replicase produced.     

Gene Regulation in N Mutants of Bacteriophage λ

Mutants (N(-)nin) of bacteriophage lambda in which the N gene product is not required for growth on wild-type Escherichia coli do not plate on recA bacterial mutants. Secondary mutants, selected for growth on recA, lie within the immunity region to the right of gene cI and appear identical to the cro mutants of Eisen et al. In an N(+) phage, a cro mutation causes enhanced and prolonged production of lambda exonuclease. N(-)cro phages make no detectable exonuclease, but show an increased rate of specific excision from lysogens and are excluded by P2 prophage. These properties, together with the ability to plate on recA, suggest that N(-)cro phages express genes to the left of N at a rate that is very low but higher than that for N(-)cro(+) phages. N(-)nin phages can integrate at the normal site on the bacterial chromosome, but specific excision from lysogens is immeasurably low.     

Mapability of Very Close Markers of Bacteriophage λ

Recombinant frequency was compared with nucleotide distance in crosses involving markers in either the P_RM or the cy region of phage λ. For each pair of markers, we performed reciprocal four-factor crosses of the following types: (I) A⁺m₁ ⁺m₂^-B^- x A^-m₁ ^-m₂⁺B⁺; and (II) A⁺m₁ ^-m₂⁺B^- x A^-m₁ ⁺m₂^-B⁺. In crosses of type I, the frequency of A⁺m₁ ⁺m₂⁺B⁺ recombinants among total (selected) A⁺B⁺ progeny was directly proportional to nucleotide distance between m₁ and m₂ in the range from 3 to 160 nucleotides. When less than three nucleotides separated m₁ and m₂, the measured yields of m₁⁺m₂⁺ recombinants were significantly depressed.

We also found that the frequency of A⁺m₁ ⁺m₂⁺B⁺ recombinants among total A⁺B⁺ progeny was significantly lower (about 10-fold on the average) in crosses of type II than in the corresponding crosses of type I. Since mismatch correction should yield A⁺m₁ ⁺m₂⁺B⁺ recombinants with approximately equal frequencies in type I and II crosses, we suggest: (1) that most m₁⁺m₂⁺ recombinants produced in type I crosses must arise from the formation of heteroduplex structures with a discontinuity (in the source of genetic information) between sites m₁ and m₂, and (2) that mismatch correction is not a major pathway for production of recombinants for close markers in normal λ infection.

     

Antigenic Properties of Bacteriophage φ29 Structural Proteins

Serological methods and electron microscopy were used to study the structural proteins of the small Bacillus subtilis bacteriophage phi29. This virus has a large number of fibers attached at both ends of its prolate head. A complex neck assembly is comprised of 12 symmetrically arranged appendages as the outer component. Head fibers, neck appendages, and the head surface bind anti-phi29 antibodies. Immune sera absorbed with defective lysates of suppressor-sensitive (sus) mutants have been used to determine the genetic control of neck appendages production. Studies on the serum-blocking power of lysates defective in different tail components showed that appendages contain the main serum-blocking protein. This finding suggests an essential role of the neck appendages in phage adsorption or DNA injection.     

Synthesis of Bacteriophage φ29 Proteins in Bacillus subtilis

Seventeen bacteriophage phi29 proteins were detected in ultraviolet light-irradiated Bacillus subtilis by autoradiography of polyacrylamide slab gels. The appearance of phi29 proteins occurred either before or concomitantly with viral DNA replication. Viral proteins detected early in the infectious cycle consisted of nine polypeptides ranging from 5,200 daltons to 54,000 daltons. Two of the early proteins were identified as, respectively, the major capsid protein and the protein comprising the filaments which extend from the head of the virus. Late phi29 proteins were composed of eight polypeptides ranging from 14,000 daltons to 95,000 daltons. Only three late proteins were noncapsid proteins. Among the early proteins, six were synthesized at diminishing rates late in the infectious cycle. One of the early proteins (protein 12) lacked histidine, whereas two (proteins 10 and 15) lacked tryptophan. Among the 17 proteins detected, 10 were viral noncapsid proteins. The amount of viral genetic information required to code for the 17 proteins detected in these experiments (81% of the potential genetic information of phi29 DNA) compares favorably with the genetic information detected as mRNA in a previous report, 85% of the potential information on the phi29 chromosome.     

Genetic Analysis of the Bacteriophage λ Attl Nucleoprotein Complex

Site-specific recombination in bacteriophage λ involves interactions among proteins required for integration and excision of DNA molecules. We have analyzed the elements required to form an in vivo nucleoprotein complex of integrase (Int) and integration host factor (IHF). Interaction of Int with the core (the site of strand exchange) is stabilized by the flanking arm region of attL. IHF, in addition to Int, is required for efficient Int-core binding. We used the in vivo attL binding assay to characterize several Int variants for their abilities to form stable attL complexes. Substitution of Int active site tyrosine 342 by phenylalanine had no effect on the ability of the protein to form attL complexes. Three other amino acids that are completely conserved in the integrase family of recombinases (arginine 212, histidine 308, and arginine 311) were separately substituted by glutamine, leucine, and histidine, respectively. In each case, the mutant protein was altered in its ability to form attL complexes while retaining its ability to bind to the λ arm-type sites. We propose that, in addition to their role in catalysis, this triad of amino acids helps the Int protein to interact with the λ core sites.     

On the Nature of CIS-Acting Regulatory Proteins and Genetic Organization in Bacteriophage: The Example of Gene Q of Bacteriophage λ

We note the existence of a "partially cis-acting" regulatory protein of bacteriophage λ: the product of the phage Q gene. We suggest that there may be a complete spectrum from "all cis" to "all trans" for such regulatory proteins. This behavior might arise because a DNA-binding protein either acts at a nearby (cis) site soon after synthesis or becomes "lost" for its trans activity on another genome through nonspecific interactions with DNA. Our proposed explanation provides one evolutionary basis for the linkage of genes for regulatory proteins and the sites at which such proteins act; it also suggests a possible rationale for the "metabolic instability" of certain regulatory proteins.     

Properties of Caulobacter Ribonucleic Acid Bacteriophage φCb5

The ribonucleic acid (RNA) bacteriophage phiCb5, which specifically infects only one form of the dimorphic stalked bacterium Caulobacter crescentus, has been obtained in high yield. Since the phage is extremely salt-sensitive, a purification procedure was devised which avoided contact with solutions of high ionic strength. Phage phiCb5 was studied with respect to the physical and chemical properties of both the phage and its RNA. In an electron microscope, the phage particles appear as small polyhedra, 23 nm in diameter. The phage is similar to the Escherichia coli RNA phages in that it (i) sediments at an S(20, w) of 70.6S, (ii) is composed of a single molecule of single-stranded RNA and a protein coat, (iii) contains two structural proteins, and (iv) apparently contains the genetic capacity to code for a coat protein subunit, a maturation-like protein, and an RNA polymerase. Phage phiCb5 differs from the E. coli RNA phages in (i) host specificity, (ii) salt sensitivity, and (iii) the presence of histidine, but not methionine, in the coat protein.     

Evidence for a New Endonuclease Synthesized by λ Bacteriophage

Infection of nonlysogenic Escherichia coli CR34(S) (Thy(-)) with bacteriophage lambda C(I)857 resulted in the formation of twisted circular double-stranded phage deoxyribonucleic acid (DNA; species I). When such infected bacteria were incubated in the absence of thymine, there was a significant decrease in the amount of species I DNA after 60 min of incubation. A similar loss of species I lambda DNA during incubation in a thymine-deficient medium was also observed after infection of the endonuclease I-deficient strain, E. coli 1100(S) (Thy(-)). This destruction of twisted, circular lambda DNA in thymine-deprived cells did not occur in the presence of chloramphenicol nor in lysogenic E. coli CR34 carrying a noninducible lambda prophage. It is therefore concluded that the endonuclease which attacks this circular configuration of lambda DNA is newly synthesized after infection and is directed by the phage chromosome.