Characterization of a bacteriophage related to R-type pyocins.

A bacteriophage with a contractile tail which shows very similar features to R-type pyocins was isolated and characterized. This phage, named PS17,was purified by DEAE-cellulose chromatography and CsCl density gradient centrifugation. It was a DNA-containing phage, and the density of the purified particles in CsCl was found to be 1.468. DNA from this phage had a density of 1.720 in CsCl, indicating its guanine plus cytosine content to be 61.2%. The head was polyhedral, 69 nm in diameter, and the tail was 150 nm in length. This phage was neutralized by antiserum preparations against five R-type pyocins, and the antiserum against this phage was active in neutralizing R-type pyocins. The properties of this phage, PS17, were compared with another similar phage, PS3, which was previously reported.     

The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage

Pseudomonas aeruginosa produces three types of bacteriocins: R-, F- and S-type pyocins. The S-type pyocin is a colicin-like protein, whereas the R-type pyocin resembles a contractile but non-flexible tail structure of bacteriophage, and the F-type a flexible but non-contractile one. As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage. In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages. The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with P2 phage and the F-type from lambda phage. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins. A systematic polymerase chain reaction (PCR) analysis of P. aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity.     

The pyocins of Pseudomonas aeruginosa

Pyocins are produced by more than 90% of Pseudomonas aeruginosa strains and each strain may synthesise several pyocins. The pyocin genes are located on the P. aeruginosa chromosome and their activities are inducible by mutagenic agents such as mitomycin C. Three types of pyocins are described. (i). R-type pyocins resemble non-flexible and contractile tails of bacteriophages. They provoke a depolarisation of the cytoplasmic membrane in relation with pore formation. (ii). F-type pyocins also resemble phage tails, but with a flexible and non-contractile rod-like structure. (iii). S-type pyocins are colicin-like, protease-sensitive proteins. They are constituted of two components. The large component carries the killing activity (DNase activity for pyocins S1, S2, S3, AP41; tRNase for pyocin S4; channel-forming activity for pyocin S5). It interacts with the small component (immunity protein). The synthesis of pyocins starts when a mutagen increases the expression of the recA gene and activates the RecA protein, which cleaves the repressor PrtR, liberating the expression of the protein activator gene prtN. R and F-pyocins are derived from an ancestral gene, with similarities to the P2 phage family and the lambda phage family, respectively. The killing domains of S1, S2, AP41 pyocins show a close evolutionary relationship with E2 group colicins, S4 pyocin with colicin E5, and S5 pyocin with colicins Ia, and Ib.     

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.     

Partial characterization of coliphage WPK and a comparison with coliphage T3.

Coliphage WPK was originally isolated from sewage in Kiel, Germany, because its plaque diameter continued to expand for days. Electron microscopy revealed an isometric capsid with dimensions of 54 nm between opposite apices, and a short, noncontractile tail 16 nm long, placing phage WPK into morphogroup C1. The nucleic acid of phage WPK was linear double stranded DNA. The host ranges of phages WPK and T3 were identical. Of ten E. coli strains tested for host range, two were resistant and of eighteen other Enterobacteriaceae only four were susceptible. Seven gram-negative species which are not members of the Enterobacteriaceae were refractory. However, there were differences in plaque morphology and plaque expansion between the two phages. Phage T3 plaques expanded for at least seven days on E. coli B only, while phage WPK plaques expanded for at least seven days on four strains of E. coli. The buoyant density of WPK, determined by isopycnic density gradient centrifugation in CsCl, was 1,508 g/ml which was significantly different than that of T3 at 1.493 g/ml (P less than 0.05). Phage-encoded proteins were examined for each phage using [35S]methionine incorporation, SDS-PAGE, and autoradiography. Of thirty proteins identified in phage WPK and twenty-eight in phage T3, only fourteen were of the same size in both. We concluded that phage WPK was distinct, but related to T3.     

Characteristics of φT, the Temperate Bacteriophage Carried by Bacillus megaterium 899a

Phage T was the only phage observed in lysates of Bacillus megaterium 899a induced with mitomycin C, 0.35 mug/ml. The phage adsorbed slowly to its host in nutrient agar, giving rise to plaques of varying sizes and turbidity. Only clear plaques were observed when the phage and host cells were preincubated in an adsorption buffer and plated under optimum conditions. Plaque turbidity was caused by either the addition of 0.5 x 10(-2) to 1.0 x 10(-2) M CaCl(2) to the phage assay medium, or by raising the incubation temperature to 34 C. Phage T purified on a CsCl gradient had a density of 1.48 g/ml in CsCl and the extracted phage DNA had a buoyant density in CsCl of 1.6975 g/ml, equivalent to 38.2% guanine plus cytosine. The phage was rapidly inactivated at 75 C and was unstable in the presence of chloroform at 4 C, but it was stable in buffer stored in ice. When stage I sporulating cells were induced with mitomycin C, phage were carried into spores which when germinated lyse with the release of phi T. The burst size on induction of early-log vegetative cells was 52, whereas the burst size of induced T(0) sporulating cells, diluted in fresh medium, was 47 for a sporulating strain and 140 for an asporogenous mutant. A typical phage T had a long, noncontracting tail 240 nm long, 9 to 11 nm wide, with a repeating disk unit along the tail, 4 nm in size center to center. The tail ended in a small disk (15 nm wide) which is presumably for attachment to the host. The hexagonal head measures 68 by 57 nm and is composed of donut-shaped units 9 nm in diameter.     

MP13, a generalized transducing bacteriophage for Bacillus megaterium.

The first generalized transducing bacteriophage reported for Bacillus megaterium has been characterized. Optimum conditions for lysate production and transduction procedures were established so that transducing frequencies of 8 x 10(-6) and higher are now possible. The phage, MP13, has a head diameter of 97 nm and a contractile tail (202 by 17 nm) and adsorbs to the periphery of the cell. MP13 was inactivated rapidly at 60 degrees C, but not at 55 degrees C, and was sensitive to toluene, ether, and chloroform. When centrifuged in a neutral CsCl gradient, two bands were observed, a major band of 1.490 g cm-3 and a minor band of 1.482 g cm-3 buoyant density. The major band contained only infective particles, whereas the minor band contained both infective and transducing particles. Phage DNA was resistant to several restriction endonucleases, but yielded 9 fragments with MboI, more than 34 with HindIII, and 7 with BstEII. The molecular weights for the fragments from MboI-BstEII double digests total 97 x 10(9).     

Properties of Thermoactinomyces vulgaris phage Ta1 and its extracted DNA

The virulent phage Ta1 was obtained in good yields from infected cultures of Thermoactinomyces vulgaris 1227. The purified phage was found to sediment with a single band, the sedimentation constant being (519 +/- 14)S, and to exhibit a typical nucleoprotein behaviour in UV-spectrophotometric and CD experiments. The Ta1 phage consists of a hexagonal head about 0.056 micrometers in diameter and a very short tail. It is morphologically similar to the temperate Salmonella phage P22. The nucleic acid extracted from the phage was found to be a double-stranded linear DNA with a G+C content of 42 mole-% as deduced both from its melting temperature and buoyant density in CsCl. Analytical sedimentation revealed a high degree of molecular homogeneity of Ta1 Dna. the sedimentation constant of this DNA amounts to (35.9 +/- 0.3)S, corresponding to a DNA molecular weight of about 29 millions daltons. The biological activity of Ta1 DNA was indicated by its ability to infect the mycelium of the components T. vulgaris strain 1227 and to give rise to mature phages.     

Mechanical Transmission, Purification and Properties of an Isolate of Maize Stripe Virus from Venezuela

A Venezuelan isolate of maize stripe virus (MStpV) was successfully transmitted mechanically and by the leafhopper Peregrinus maidis from field infected plants to sweet cv. Iochief. After purification of maize infected with MStpV, fine spiral filamentous particles about 4 nm in diameter and with variable lengths were consistently associated with a nucleoprotein band present in CsCl or Cs₂SO₄ isopycnic gradients. Purified preparations exhibited a typical nucleoprotein absorption spectrum with a maximum at 260–263 nm and a minimum at 240–243 nm and a 260–280 ratio of 1.38. The density of the nucleoprotein in CsCl gradients was estimated at 1.29 g/ml. The sedimentation coefficient was calculated at 62 S. The nucleoprotein consisted of 5 % single stranded RNA and a capsid protein of molecular weight 33.500 daltons. Large quantities of non-capsid proteins were isolated from infected tissue with a molecular weight of 17.500 and 16.500 daltons. Peregrinus maidis, injected with purified MStpV preparation failed to transmit the disease to healthy plants. However, they were infectious when injected with clarified infected plant sap. Antisera against capsid and non-capsid proteins from MStpV-Florida strain reacted positively with the Venezuelan antigens.     

Fractionation of nucleic acids from Penicillium chrysogenum and associated ribonucleic acid viruses by selective exclusion and retention in agarose gels

Double-stranded nucleic acids from a strain of Penicillium chrysogenum containing RNA viruses were isolated by agarose-gel filtration, and separated into DNA and double-stranded RNA fractions by agarose-gel chromatography in 2.5m-NaCl. The DNA fraction contained less than 1% alkali-labile polynucleotides, and sedimented homogeneously at 8-10S in alkaline sucrose gradients. In CsCl gradients it tended to band in the density region of 1.66-1.72g/ml. It had a ;melting' temperature (T(m)) of 75 degrees C in 0.015m-NaCl-0.0015m-trisodium citrate, corresponding to 51.5mol% of G+C. The double-stranded RNA fraction did not contain detectable DNA. It could not band in CsCl up to a density of 1.78g/ml, and mainly consisted of a 14-15S RNA species with a T(m) of 88.5 degrees C in the above solvent, and a G+C content of 49.3 mol%.     

Isolation and characterization of a new bacteriophage, Cp-1, infecting Streptococcus pneumoniae.

Several pneumococcal phages showing a morphology completely different from those of all other previously found pneumococcal bacteriophages have been isolated. Bacteriophage Cp-1, one of the phages isolated, showed an irregular hexagonal structure and a short tail of 20 nm. The virion density was 1.46 g/cm3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of nine polypeptides. The polypeptide showing a molecular weight of 39,000 accounted for more than the 90% of the total protein. The nucleic acid of Cp-1 was linear, double-stranded DNA with a mean length of 6.3 microns and a guanine-plus-cytosine content of 41%; its buoyant density was 1.699 and 1.422 g/cm3 in CsCl and CS2SO4, respectively. Its sedimentation coefficient (S20,w) was 19S. Cp-1 DNA showed a remarkable resistance to a large number of restriction endonucleases. A total of 12 fragments, ranging in molecular weight from 1.3 X 10(6) to 0.09 X 10(6), were produced by AluI, two fragments (molecular weight, 5.5 X 10(6) and 0.9 X 10(6)) were generated by HindIII, and two fragments (molecular weight, 6.0 X 10(6) and 5.7 X 10(6)) were produced by HaeIII. The easy visualization of th plaques produced by Cp-1, the small size of Cp-1 DNA (12 X 10(6) daltons), and other biological and physiochemical properties make this phage potentially useful for genetic studies.     

Isolation and characterization of Caulobacter crecentus bacteriophage phi Cd1.

A DNA-containing bacteriophage, phiCd1, was isolated from sewage and shown to infect both stalked and swarmer cells of Caulobacter crescentus strain CB13B1a. phiCd1 is a small, icosohedral bacteriophage, 60 nm in diameter, which possesses a short, noncontractile tail, 10 to 12 nm in length. The bacteriophage particle is composed of at least eight structural proteins. phiCd1 nucleic acid exists as a linear duplex of DNA as judged by: (i) thermal denaturation (Tm), (ii) CsCl density gradient centrifugation, and (iii) chemical analysis of its base composition. The DNA is 61% guanosine plus cytosine, has a buoyant density in CsCl of 1.721 +/- 0.001 g/cm3, and denatures sharply at 78.5 C in 0.1 SSC (standard saline citrate) buffer. The S20, w value for the DNA is 34.3 +/- 0.1S as compared with T7 DNA, indicating a molecular weight of about 29 x 10(6).     

Physical properties of phage 299

Summary Phage 299, on equilibrium sedimentation in CsCl, gives 3 major bands whose relative proportions depend on growth conditions. One band is whole heads without tails, the other two are infectious phage of differing degrees of disarray and stability. Electron micrographs show that the infectious phage has a head about 60 nm across, probably icosahedral in shape, and a straight tail of approximately 140 nm in length. The tail assemblies appear defective and incomplete. Sedimentation in a sucrose gradient of the DNA extracted from phage 299 is monodisperse with a molecular weight of 20.6±1.5×10⁶ daltons based on comparison with λ and 186 phage DNA’s. The DNA has a base composition of 51.7% guanine and cytosine as determined by bouyant density in CsCl. A comparison of its denaturation behavior by analysis of the hyperchromic shift at 260 nm with that of phage P2 suggests a considerable number of common characteristics, and an absence of a low guanine and cytosine portion on the part of 299 which amounts to approximately 10% of the total DNA.     

Properties of "diplophage": a lipid-containing bacteriophage.

We describe the purification and properties of Dp-1, a bacteriophage isolated from Diplococcus pneumoniae. The phage was sensitive to the organic solvents deoxycholate and Sarkosyl, and its infectivity was reduced by treatment with phospholipase C. Electron microscopy indicated the presence of a double-layered coat around the phage particles. Purified phage preparations contained lipid amounting to about 8.5% of the dry weight of the phage, and thin-layer chromatography resolved the lipids into four components. The phage had a buoyant density in CsCl of 1.47 g/cm3, and a sedimentation constant in 0.1 M NaCl of 313S. Analysis in acrylamide gel electrophoresis indicated the presence of three major proteins. Dp-1 DNA shows a density of 1.681 g/cm3. Neutralizing antisera against the phage have a low potency (K less than 120/min).     

Incomplete and Aberrant Particles of Actinophage MSP2 from Lysates of Streptomyces venezuelae

Lysates of actinophage MSP2, propagated on Streptomyces venezuelae S13, contain at least 10(11) PFU/ml. During purification by centrifugation methods and by adsorption chromatography, a number of types of aberrant and incomplete phage particles were seen by electron microscopy. Infectious MSP2 had a buoyant density in CsCl of 1.52 g/cm(3) and an absorbance at 260 nm relative to that at 280 nm (A(260)/A(280)) of 1.53. Empty capsids banded at 1.276 g/cm(3) and partially filled capsids banded at 1.351 g/cm(3), and A(260)/A(280) ratios were 0.77 and 1.24, respectively. Two kinds of light capsids found in CsCl fractions of 1.278 g/cm(3) probably include the 1.276 component. Some capsids were joined by tail-like structures. Ghosts and polyheads also were present. Aberrant particles observed by electron microscopy included two-tailed actinophage, phage with abnormal tail positions, and large-headed phage.     

Isolation and Characterization of a Bacteriophage for Thiobacillus novellus

A DNA-containing bacteriophage for Thiobacillus novellus has been isolated from sewage and purified by ammonium sulfate precipitation, differential centrifugation, and cesium chloride equilibrium centrifugation. The buoyant density of this phage in CsCl is 1.51 g/cm(3). Electron microscopy studies have revealed a polyhedral head about 60 nm in diameter and a tail surrounded by a number of fine filaments. It has an adsorption rate constant of 1.1 x 10(-9) ml/min, a latent period of 45 min, and an average burst size of 150. The mole guanine and cytosine content in its DNA has been estimated to be 57 to 58%. Five structural proteins with molecular weights of 62,000, 42,500, 30,500, 17,750, and 13,500 have been detected by polyacrylamide gel electrophoresis techniques.     

Characterization of the bacteriophage B2 of Lactobacillus plantarum ATCC 8014

Bacteriophage B2 of Lactobacillus plantarum ATCC 8014, isolated in 1971, belonged to Bradley's group B. Electron microscopy revealed an isometric head (110 nm) and a long non-contractile and flexible tail (500 nm) containing about 75 regularly aligned lateral striations. Burst size was 12-14 phages per infectious centre. The latent period for phage development was 75 min and the rise period approximately 90 min. The phage particle contained 5 major proteins. The buoyant density of the phage in CsCl was measured as 1.575 g/cm3. B2 genome was a linear double-stranded DNA molecule of 37 +/- 1% guanosine-cytosine. Its size was 73 kilobase pairs (kbp). Restriction analysis of the genome showed that 4 restriction enzymes (Xba I, Sac I, Bgl II and Sma I) gave single site cuts in the DNA, while Ava I and Sal I formed 2 and 5 cuts, respectively.     

Characterization and classification of virus particles associated with hepatitis A. I. Size, density, and sedimentation.

Virus-like particles were purified from stools of patients in an epidemic of hepatitis A in Germany. When reference MS-1 chimpanzee pre-inoculation and convalescent sera were used, the close serological relationship of the purified particles to well-known isolates of hepatitis A could be established. On the other hand, the physicochemical characteristics of the particles were determined in parallel to the characteristics of a marker parvovirus (LuIII) and a marker picornavirus (poliovirus type 2). It could be shown that the majority of the hepatitis A-associated particles band at 1.34 g/ml in CsCl and, like poliovirus, sediment at about 160S. In addition, a distinct hepatitis A antigen was observed, which banded at 1.305 g/ml and sedimented between 50 and 90S. A further component accumulated in the density range of between 1.38 and 1.44 g/ml. However, it seemed to be rather labile. Upon reisolation from CsCl and sedimentation in sucrose, it resolved into a 160S, a 90 to 100S, and a 50S form. The size of the 160S particles (27 to 29 nm) could be readily distinguished from that of the parvovirus (22 to 24 nm). It is concluded, therefore, that hepatitis A-associated virus particles are more likely to be classified with the picornaviruses than with the parvoviruses.     

Stalked Bacteria: Properties of Deoxriybonucleic Acid Bacteriophage φCbK

The Caulobacter crescentus bacteriophage phiCbK was studied with respect to the physical and chemical properties of both the phage and its deoxyribonucleic acid (DNA). Electron micrographs reveal the phage to be among the largest DNA bacteriophages reported, with head dimensions of 64 by 195 nm and a flexible tail 275 nm in length. The phage is composed of 57% DNA. This DNA is double-stranded as indicated by (i) the sharp increase in extinction coefficient over a narrow range of temperature increase, (ii) an increase in density in CsCl upon thermal denaturation, and (iii) the equivalence of guanine and cytosine as well as adenine and thymine, determined by chemical analysis. phiCbK DNA cosediments with Escherichia coli phage T2 DNA and has therefore been assigned an S(20,w) value of 63.5S. The size of the phage and its DNA and the percentage of DNA indicate that the phiCbK phage head is relatively loosely packed. The properties of the DNA from bacteriophage phiCbK are similar to those of host C. crescentus DNA with respect to buoyant density, thermal transition point, and guanine plus cytosine content.     

Physiological, morphological, and physicochemical characterization of a novel Escherichia coli bacteriophage, phage MM

A double-stranded DNA containing, T even-like, Escherichia coli bacteriophage, called MM, has been isolated from the local sewage and purified by polyethylene glycol precipitation followed by banding on a cesium chloride three-step gradient. It yields a burst size of 75 particles per infected cell, and has an adsorption coefficient of 3.3 x 10(-10) cm3/min and a latent period of 45 min. Electron microscopy of phage MM reveals an isometric icosahedral head, 92 nm long and 81 nm wide, and a 112-nm-long contractile tail with six pairs of 40-nm-long fibers attached to its baseplate. Phage MM appears similar to E. coli phage T4 or Salmonella phage O1. The density of phage MM in cesium chloride is 1.515 g/ml, and its total mass is 144 MDa. Gel electrophoresis of purified MM capsids displays two major capsid proteins in approximately equimolar amounts and with apparent molecular masses of 38 and 15 kDa. Similarly, purified MM tails yield two major polypeptides with apparent molecular masses of 55 and 16 kDa, most likely representing the major tail sheath and tail tube polypeptides. Its double-stranded DNA has a G-C content of 50%, a length of 131 kilobases (kb), and a mass of 89 MDa.