Bacteriophage phiNS11: a lipid-containing phage of acidophilic thermophilic bacteria. IV. Sedimentation coefficient, diffusion coefficient, partial specific volume, and particle weight of the phage.

The particle weight (molecular weight) of phiNS11 was determined from the sedimentation coefficient, diffusion coefficient, and partial specific volume of the phage. The sedimentation coefficient of the phage (S(0)20, W) is 416 +/- 2.7S. The diffusion coefficient D(0)20, W), which was determined by quasielastic light scattering measurement, is (0.57 +/- 0.03) x 10(-7) cm2/s. The partial specific volume was determined by the mechanical oscillation technique to be 0.747 +/- 0.007 cm3/g. Based on these values, the particle weight of the phage was calculated to be (70.3 +/- 4.3) x 10(6) daltons, which agrees well with the particle weight (69--72 x 10(6) daltons) estimated from the molecular weight of phage DNA and the content of DNA. The Stokes radius of the phage particle was calculated to be 37.7 +/- 2 nm and hydration of the phage was estimated to be 1.18 cm3/g of dry phage. From the particle weight and the chemical composition of the phage, we estimated that one phage particle contains one double-stranded DNA molecule, 16,000 residues of fatty acid, 72 protein I molecules, 920 protein II, 42 protein III, 48 protein IV, 290 protein V molecules, and 3,700 molecules of polyamines.     

Purification and Some Properties of Bacteriophage ST-1

Bacteriophage ST-1 is shown to be a small, isometric, single-stranded deoxyribonucleic acid (SS-DNA) virus with a diameter of about 260 nm. Standard methods for growth, assay, preparation of high-titer lysates, and purification of the phage are suggested. ST-1 infects K-12 and not C strains of Escherichia coli and requires a divalent cation to adsorb to susceptible bacteria. Adsorption also requires an activation of the particle brought on by incubation at 37 C. The latent and eclipse periods are essentially identical (9 to 11 min) in ST-1 infections, with an average burst size about 250 phages per cell. Multiple densities of ST-1 infectivity are observed during purification in CsCl gradients. The virus recovered from different densities has the same sedimentation coefficient and, therefore, all phage containing fractions are pooled during purification. The purified ST-1 particle has a sedimentation coefficient of 121S relative to phiX-174 (114S) in a sucrose gradient and a molecular weight of 6.8 x 10(6) (as estimated from its relative sedimentation). The nucleic acid is assumed to be SS-DNA on the basis of (i) the specific incorporation of (3)H-thymine, (ii) the dependence of its UV absorption on temperature, and (iii) its reaction with formaldehyde. ST-1 SS-DNA sediments at 24.4S relative to phiX-174 SS-DNA (23.8S).     

Characterization of Inducible Bacteriophages in Bacillus licheniformis

Two morphologically distinct and physically separable defective phages have been found in Bacillus licheniformis NRS 243 after induction by mitomycin C. One of them (PBLB) is similar to the defective phage PBSX of B. subtilis, which has a density of 1.373 g/cm(3) in CsCl and a sedimentation coefficient of 160S. PBLB incorporates into its head mainly bacterial deoxyribonucleic acid (DNA) which has a sedimentation coefficient of 22S and a buoyant density in CsCl of 1.706 g/cm(3). The other phage (PBLA) has a morphology similar to the temperate phage phi105 of B. subtilis; the head diameter is about 66 nm, and it possesses a long and noncontractile tail. PBLA has a density of 1.484 g/cm(3) in CsCl and the phage-specific DNA, which is exclusively synthesized after induction by mitomycin C, has a density of 1.701 g/cm(3). PBLA DNA is double-stranded and has a sedimentation coefficient of 36S, corresponding to a molecular weight of 34 x 10(6) to 35 x 10(6) daltons. The phage DNA has one interruption per single strand, giving single-stranded segments with molecular weights of 13 x 10(6) and 4 x 10(6) daltons. Common sequences between the two phage DNA species and with their host DNA have been demonstrated by DNA-DNA hybridization studies. Both phage particles kill sensitive bacteria. However, all attempts thus far to find an indicator strain to support plaque formation have been unsuccessful.     

Factors Affecting Infection of Protoplasts with Deoxyribonucleic Acid of Actinophage PK-66

To establish a method for transmission of genetic materials in the genus Streptomyces, the conditions of infection of protoplasts of S. kanamyceticus by actinophage PK-66 deoxyribonucleic acid (DNA) were studied. The protoplasts of Streptomyces were prepared by treatments with lysozyme and trypsin. The infectivity of the phage DNA was enhanced by the presence of NaCl in the medium. The optimal concentration of the protoplasts for infection with DNA was 7 x 10(7) to 4 x 10(8)/ml. A proportional relationship was found between the infectivity and the DNA concentration within a certain range. The maximal production of mature phage was achieved after 19 hr of incubation. The number of phage propagated in the infection mixture reached 10(4) plaque-forming units per ml under the appropriate conditions. The phage DNA infected not only protoplasts prepared from S. kanamyceticus but also those prepared from S. violaceoniger and S. acidomyceticus, which were resistant to intact phage PK-66.     

Isolation and Characterization of a New Generalized Transducing Bacteriophage Different from Pl in Escherichia coli

A new generalized transducing bacteriophage in the Escherichia coli system was isolated and characterized. This phage, designated D108, makes clear plaques on E. coli K-10, K-12, K-12(P1kc), K-12(D6), B/r, C, and 15 T(-), and Shigella dysenteriae. The plaque of phage D108 is larger in size than that of phage P1kc. Electron-microscopic observation revealed that phages D108 and P1kc are morphologically different from each other, suggesting that phage D108 belongs to a phage group different from phage P1. The fact that all of the 10 markers tested were transduced by phage D108 indicates that this phage is a generalized transducing phage in the E. coli system. The transduction frequency by phage D108 of chromosomal markers and of a drug resistance factor (R factor) ranged from 2 x 10(-6) to 3 x 10(-8) and 3 x 10(-9) to 6 x 10(-10) per phage, respectively. The cotransduction frequency of the thr and leu markers was 2.8% for phage P1kc and 1.5% for phage D108. The CM and TC markers (chloramphenicol-resistant and tetracycline-resistant markers, respectively) of the R factor were not cotransduced by phage D108, but the markers were generally cotransduced by phage P1kc. The results suggest that the transducing particle of phage D108 contains a smaller amount of host deoxyribonucleic acid than does phage P1kc.     

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.     

Characterization of bacteriophage gh-1 for Pseudomonas putida

Lee, Lucy F. (Michigan State University, East Lansing), and J. A. Boezi. Characterization of bacteriophage gh-1 for Pseudomonas putida. J. Bacteriol. 92:1821-1827. 1966.-Bacteriophage gh-1 of Pseudomonas putida A.3.12 was isolated and purified by differential centrifugation and diethylaminoethyl (DEAE) cellulose chromatography. An electron micrograph of the phage stained with uranyl acetate revealed a regular hexagonal outline about 50 mmu across with a short wedge-shaped tail attached at one corner of the head. The phage formed 10% as many plaques on P. putida C1S as on P. putida A.3.12, the organism used in the isolation procedure. No plaques were formed on P. fluorescens (ATCC 9712) or P. aeruginosa. The latent period of the infectious cycle was 21 min, and the average burst size was 103. The nucleic acid component of gh-1 is double-stranded deoxyribonucleic acid (DNA), with a base composition of 57.0% guanine plus cytosine (G + C) as determined by chemical analysis. The per cent G + C of P. putida A.3.12 DNA measured in a similar manner was 63.7%. The buoyant density of phage gh-1 measured by cesium chloride equilibrium centrifugation was 1.45 g/cm(3), whereas that of gh-1 DNA, heat-denatured gh-1 DNA, and P. putida A.3.12 DNA was 1.716, 1.730, and 1.722 g/cm(3), respectively. The per cent G + C of gh-1 DNA and P. putida A.3.12 DNA calculated from the buoyant densities was 57.1 and 63.3%, respectively. The sedimentation coefficients, S(50) (20,w), of gh-1 and the phenol-extracted gh-1 DNA, measured by the boundary sedimentation velocity method, were 460 and 18.9, respectively. The molecular weight of phenol-extracted gh-1 DNA, calculated by use of the equation of Burgi and Hershey, is 6 x 10(6).     

Characterization of two Salmonella newport bacteriophages

Salmonella newport phages 16--19 and 7--11 have very long heads and are members of two rare and so far little-known phage groups. Both produce various morphological aberrations. Preparations of phage 7--11 contain numerous polyheads and about 0.4% short heads belonging to nine size classes. In addition, one giant phage particle was observed. The head of phage 7--11 seems to be an icosahedron which became elongated by adding successive rows of subunits. Phages 16--19 and 7--11 have buoyant densities in CsCl of 1.43 and 1.48 g/mL and particle weights of 103 and 204 x 10(6) respectively. Both viruses contain double-stranded DNA, internal proteins, and sugars. Phage 16--19 contains 46.5% DNA of 35 x 10(6) molecular weight, and glucose. Phage 7--11 contains 47.5% DNA of 108 x 10(6) molecular weight, and mannose. Base compositions of phage and S. newport DNAs were determined from buoyant densities, melting point, and acid hydrolysis. Phage 16--19 contains 5.4% 5-methylcytosine.     

Characterization of two inducible bacteriophages, alpha 1 and alpha 2, isolated from Clostridium botulinum type A 190L and their deoxyribonucleic acids

Two inducible bacteriophages, alpha 1 and alpha 2, isolated from Clostridium botulinum type A strain 190L and their deoxyribonucleic acids (DNAs) were purified and characterized. Phage alpha 1, which is unable to form plaques on any strain of C. botulinum, was produced in large quantities after treatment with mitomycin C (MC), whereas phage alpha 2, which was induced in much lower quantities than phage alpha 1, propagated in cultures of type A strain Hall. The phage DNAs were exclusively synthesized after induction with MC. Alpha 1 and alpha 2 DNAs had sedimentation coefficients of 34.0 and 30.6 S, corresponding to molecular weights of 31.9 x 10(6) and 23.5 x 10(6), respectively. The buoyant density in CsC1 was 1.682 g/cm3 for alpha 1 DNA and 1.680 g/cm3 for alpha 2 DNA. Based on thermal denaturation characteristics, the genomes of both phages were shown to be double-stranded DNAs. Agarose gel electrophoretic profiles of the phage DNAs digested with restriction endonuclease EcoRI revealed nine fragments for alpha 1 DNA and six fragments for alpha 2 DNA. The molecular weights of the phage DNAs as determined by restriction enzyme analysis were 30.55 x 10(6) for alpha 1 DNA and 25.83 x 10(6) for alpha 2 DNA. Nontoxigenic mutants obtained from strain 190L could, like the toxigenic parent strain, produce the two phages after treatment with MC. Lysogenic conversion to toxigenicity by phage alpha 2 was not observed with the nontoxigenic mutants. It seems likely that there is no relationship between either phage genome and the toxigenicity of C. botulinum type A.     

Structure of Bacillus subtilis bacteriophage phi 29 and the length of phi 29 deoxyribonucleic acid

Anderson, D. L. (University of Minnesota, Minneapolis), D. D. Hickman, and B. E. Reilly. Structure of Bacillus subtilis bacteriophage phi29 and the length of phi29 deoxyribonucleic acid. J. Bacteriol. 91:2081-2089. 1966-Bacillus subtilis bacteriophage phi29 were negatively stained with phosphotungstic acid. The head of phi29 has a hexagonal outline with a flattened base, and is about 315 A wide and 415 A in length. The virus has an intricate tail about 325 A in length. Twelve spindle-shaped appendages are attached to the lower of two collars which comprise the proximal portion of the tail. The distal 130 A of the tail axis has a diameter of about 60 A and is larger in diameter than the axis of the upper portion of the tail. Comparison of electron microscopic counts of phi29 with plaque-forming units indicated that about 50% of the microscopic entities were infective. Phenol-extracted phi29 deoxyribonucleic acid (DNA) molecules were prepared for electron microscopy by the cytochrome c film technique of Kleinschmidt et al. Measurement of contour lengths of DNA molecules from three preparations gave skewed distributions of lengths with observed modal class values ranging from 5.7 to 5.9 mu. Assuming that phi29 DNA is a double helix in the B form, the corresponding molecular weights would be 10.9 x 10(6) to 11.3 x 10(6) daltons. The largest DNA molecules would have a volume of 1.9 x 10(7) A(3) which is about 25% greater than the estimated 1.4 x 10(7) A(3) internal volume of the phage head.     

Isolation of a Polyvalent Bacteriophage for Escherichia coli, Klebsiella pneumoniae, and Aerobacter aerogenes

A lytic bacteriophage isolated from sewage was found to attack strains of Aerobacter aerogenes, Escherichia coli, and Klebsiella pneumoniae, but not members of the genera Salmonella, Proteus, and Serratia. The phage, designated phimp, contained deoxyribonucleic acid with a 50% guanine plus cytosine ratio and a molecular weight of 23.1 x 10(6) daltons. Single-step growth experiments of phimp plated at 37 C on A. aerogenes A2 gave a mean latent period of 20 min, an average burst size of 103 plaque-forming units/infected cell, and an average adsorption rate constant of 3 x 10(-10) ml/min. Electron microscopy of phimp revealed a phage with a flexible tail (165 nm long and 6 nm wide). The phage head had a hexagonal outline (62 nm in diameter).     

Transfection of Escherichia coli Spheroplasts I. General Facilitation of Double-Stranded Deoxyribonucleic Acid Infectivity by Protamine Sulfate

The addition of 25 mug of protamine sulfate per ml to lysozyme-ethylenediamine-tetraacetic acid spheroplasts of Escherichia coli stimulates transfection not only for T1 phage deoxyribonucleic acid (DNA; Hotz and Mauser, 1969) but also for the following phage DNA species: lambda, 10,000-fold to an efficiency of 10(-3) infective centers per DNA molecule; phiX174 replicative form, 300-fold to an efficiency of 5 x 10(-2); fd replicative form, 300-fold to 10(-6); T7, 300-fold to 3 x 10(-7). Three native phage DNA species were not infective at all in the absence of protamine sulfate but were infective in the presence of protamine sulfate with the following efficiencies: T4, 10(-5); T5, 3 x 10(-6); and P22, 3 x 10(-9). The effect of protamine sulfate is specific for double-stranded DNA. The application of infectivity assays to the study of phage DNA replication, recombination, prophage integration, prophage excision, and interspecies transfection are discussed.     

Transfection of Lysostaphin-treated Cells of Staphylococcus aureus

After treatment with 1 unit of lysostaphin per ml for 3 min, two strains of Staphylococcus aureus, 233 and PS 44A HJD, were transfected with phenol-extracted deoxyribonucleic acid (DNA) from the staphylococcal bacteriophages, 53 and 44A HJD, respectively. The number of transfected cells was low in both systems, approximately two in 10(7) enzyme-treated cells. There was a saturation effect at high concentrations of DNA; optimal results were obtained at concentrations between 10 to 25 mug/ml. Growth curves and fluctuation tests indicated that cells of strain 44A HJD infected with phage, then converted to protoplasts by a 10-min treatment with lysostaphin, produce only one phage particle and lose their ability to lyse spontaneously in hypertonic media.     

Transduction of R Factors by a Proteus mirabilis Bacteriophage

A transducing phage, designated phim, was isolated from a lysogenic strain of Proteus mirabilis and was characterized with respect to its physical and genetic properties. The phage contains double-stranded deoxyribonucleic acid (DNA) with an S(20,w) degrees of 29 which corresponds to a molecular weight of 24 x 10(6) daltons. The base composition of phim DNA was estimated to be 40% guanine plus cytosine on the basis of the buoyant density of the DNA. phim carries out generalized transduction of chromosomal genes in P. mirabilis at a frequency of 5 x 10(-8) to 2 x 10(-6) per adsorbed phage. To obtain R-factor transduction, it was necessary to have a resident R factor in the recipient cells. In these experiments, different combinations of genetically distinguishable R factors were used in the donor and recipient cells. The frequencies of R-factor transduction were 10(-9) to 2 x 10(-8). The transduction of R factors using an R(-) recipient could not be detected. Transductant R factors were usually recombinant between donor and resident R factors. All of the transduced R factors were transferable by conjugation. A plausible explanation for the requirement for a resident R factor in the recipient cells is that phim transduces only a portion of the R-factor genome and therefore requires a resident R factor for genetic recombination. The reason for the low frequencies of R-factor transduction is not known, but some possible interpretations have been discussed.     

Bacteriophage T4 Head Morphogenesis III. Some Novel Properties of Gene 13-Defective Heads

The nature of phage precursors in gene 13-defective infected cells was studied by electron microscopy and pulse-chase isotopic labeling experiments. Our results suggest that both stable (20%) and fragile (70%) filled-head precursors accumulated in the absence of gene 13 product. Upon extraction, the fragile heads were found to lose most of their deoxyribonucleic acid and appeared unfilled with an average density of 1.34 g/cm(3) and a sedimentation coefficient of 300S. These unfilled heads differed from empty gene 13-defective heads which did not have any associated deoxyribonucleic acid and banded at an average density of 1.31 g/cm(3). Furthermore, it was found that a tsN38 (temperature-sensitive mutant in gene 13)- infected culture maintained at 41.5 C for increasing times led to a decrease in specific infectivity of 1,000S phagelike particles. Electron microscopy of these particles revealed that the decreased infectivity was due to an improper union of head and tails.     

Control of the Replication Complex of Bacteriophage P22

A replication complex for the vegetative synthesis of the deoxyribonucleic acid (DNA) of the temperate phage P22 previously has been described. This complex is an association of parental phage DNA, most of the newly synthesized phage DNA made during pulses with (3)H-thymidine, and other cell constituents, and has a sedimentation rate in neutral sucrose gradients of at least 1,000S. The complex is one of the intermediates, intermediate I, in the synthesis and maturation of phage P22 DNA after infection or induction. Evidence supporting the replicative nature of intermediate I is presented. Phage replication is repressed in lysogenic bacteria. On superinfection of P22 lysogens with nonvirulent phage, little association of the input phage DNA with a rapidly sedimenting fraction is demonstrable. However, after induction with ultraviolet light, the superinfecting parental phage DNA quickly acquires the rapid sedimentation rate characteristic of intermediate I; phage DNA synthesis follows; and progeny phages are produced. Infection with a virulent mutant of P22 produces progeny phages in lysogens. Its DNA associates with intermediate I. In mixed infection with the virulent phage, replication of nonvirulent phage P22 is still repressed, even though the virulent replicates normally. The nonvirulent input DNA does not associate with intermediate I. The repressor of the lysogenic cell prevents replication by interfering with the physical association of template material with intermediate I. A phage function is required for association of phage template with the replication machinery.     

A Bacteriophage of Bacillus subtilis Which Forms Plaques Only at Temperatures Above 50 C I. Physical and Chemical Characteristics of TSP-1

Bacteriophage TSP-1 was isolated from soil in a search for phage which would form plaques on Bacillus subtilis W168 at 53 C. It forms clear plaques only at temperatures from 50 to 55 C. Approximately 95% of the free phage adsorb after 2 min at 53 C. The lytic cycle is between 55 and 60 min long with a burst size of about 55 particles per infected bacterium. The phage was shown to contain double-stranded deoxyribonucleic acid with a base composition of 44.7% guanine plus cytosine. This deoxyribonucleic acid does not contain a base analogue for thymine and has a molecular weight estimated at 56 x 10(6) daltons.     

Skeletal structure of clathrin triskelion in solution: experimental and theoretical approaches

The physicochemical properties of the clathrin triskelion were determined by dynamic and static light-scattering and sedimentation analyses in Tris and triethanolamine (TEA) buffers of about pH 8, in which the clathrin triskelion has been found to be in different conformational states by electron microscopy [Heuser, J., & Kirchhausen, T. (1985) J. Ultrastruct. Res. 92, 1-27]. Dynamic light-scattering measurements provided diffusion coefficients (D0(20,w)) of 1.22 x 10(-7) and 1.23 x 10(-7) cm2/s, and ultracentrifugal analysis gave sedimentation coefficients (S0(20,w)) of 8.39 and 8.32 S in Tris and TEA buffer, respectively. The average Stokes radius of the protein was determined to be 175 A from its diffusion and sedimentation coefficients and its molecular weight. Static light-scattering analysis provided molecular weights of 6.58 x 10(5) and 6.41 x 10(5) and radii of gyration of 311 and 301 A in the respective buffers. These results indicate that the clathrin triskelion has a similar conformation in the two buffers. For clarification of the skeletal structure of the clathrin triskelion in solution, the physicochemical parameters were calculated by using two models in which the clathrin arms are bent at various angles in a plane, on the basis of the Bloomfield approximation and a formula derived to estimate the radius of gyration of proteins consisting of various structural units. Values for the Stokes radius, diffusion and sedimentation coefficients, and radius of gyration in the ranges of 178-170 A, (1.20-1.26) x 10(-7) cm2/s, 8.26-8.66 S, and 316-266 A, respectively, were obtained with these models with the arms bent in the range of 0-60 degrees.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of spermidine on the RNA-A protein complex isolated from the RNA bacteriophage MS2.

The polyamine spermidine has recently been reported to be a substantial component of the RNA phage particle. Its effect on the isolated RNA-A protein complex of the phage MS2 is investigated here. This complex infects intact Escherichia coli cells via F-pili, as does the whole phage. It is shown that the infectivity of the complex on intact E. coli cells was enhanced by incubation with spermidine. Optimal stimulation (20-fold) of the complex infectivity was achieved by incubation with 3 x 10(-4) M spermidine for 20 to 30 min at 37 degrees C. This gave a more compact structure to the complex, as could be seen by its faster sedimentation in sucrose gradients. Although spermidine and Mg2+ are known to partially replace one another in several systems, no enhancement of the infectivity of the complex, but only its considerably faster sedimentation in sucrose gradients, occurred after incubation with 3 x 10(-4) M Mg2+. Only if the Mg2+ concentration was raised by more than one order of magnitude could increased infectivity of the complex be observed. At concentrations of spermidine and Mg2+ that maximally stimulated the infectivity of the complex on intact E. coli cells, no increase in infectivity of phenol-extracted RNA to E. coli spheroplasts was detected. From these in vitro results, the role of the polyamine spermidine in the RNA phage particle for the infecting, RNA-A protein complex molecules in phage infection is discussed.     

Intracellular forms of lambda deoxyribonucleic acid in Escherichia coli infected with clear or virulent mutants of bacteriophage lambda

Weissbach, Arthur (National Institutes of Health, Bethesda, Md.), Allan Lipton, and Arnold Lisio. Intracellular forms of lambda deoxyribonucleic acid in Escherichia coli infected with clear or virulent mutants of bacteriophage lambda. J. Bacteriol. 91:1489-1493. 1966.-Infection of either the sensitive or lysogenic strain of Escherichia coli K-112S by lambda(+) leads to the formation of a new phage deoxyribonucleic acid (DNA) species having the properties of a twisted circular DNA duplex. This new phage DNA species is also seen in cells infected with clear or virulent mutants of lambda which cannot lysogenize, or do so at a low frequency. The sedimentation rate of circular lambda DNA duplex at various pH values and its lability were examined.