Packaging of genomes in bacteriophages: a comparison of ssRNA bacteriophages and dsDNA bacteriophages.

In complex DNA bacteriophages like lambda, T4, T7, P22, P2, the DNA is packaged into a preformed precursor particle which sometimes has a smaller size and often a shape different from that of the phage head. This packaging mechanism is different from the one suggested for the RNA phages, according to which RNA nucleates the shell formation. The different mechanisms could be understood by comparing the genomes to be packaged: single stranded fII RNA has a very compact structure with high helix content. It might easily form quasispherical structures in solution (as seen in the electron microscope by Thach & Thach (1973)) around which the capsid could assemble. Double stranded phage DNA, on the other hand, is a rigid molecule which occupies a large volume in solution and has to be concentrated 15-fold during packaging into the preformed capsid, and the change in the capsid structure observed hereby might provide the necessary DNA condensation energy.     

Recombination of uracil-containing lambda bacteriophages.

Controlled incorporation of uracil into the deoxyribonucleic acid (DNA) of lambda bacteriophages was achieved by growth on dut ung thy mutants of Escherichia coli. The frequency of substitution of uracil for thymine, estimated by alkaline sucrose sedimentation of phage DNA treated in vitro with uracil DNA glycosylase, ranged from 0.17 to 1.9%. The corresponding ratio between the plating efficiencies on wild-type (Ung+) and glycosylase-deficient (Ung-) bacteria ranged from 0.70 to 0.05. If a single-hit dependence of plating efficiency on uracil content is assumed, the probability that any given uracil residue is lethal is approximately 1% (about one-fifth the probability for a pyrimidine dimer). The effect of uracil on recombination was studied in experiments with lambda tandem duplication phages (ethylenediaminetetraacetic acid [EDTA] sensitive), which are converted to single-copy phages (EDTA resistant) by general recombination. For repressed infections (of homoimmune lysogens), recombination was measured by a two-stage assay (DNA extraction, transfection of spheroplasts, and EDTA treatment). The frequencies observed for uracil-containing phages (2 to 4%) were 5 to 10 times higher than control values. However, comparisons with ultraviolet irradiated phages indicated that uracil residues promoted recombination less than 1/100 as efficiently as ultraviolet-induced lesions. Recombination of uracil-containing phages during repressed infections was negligible in recA and partially reduced in recB bacteria. Recombination was very low in ung cells, suggesting that excision repair was responsible for the stimulation. Interestingly, uracil-stimulated recombination was elevated about twofold in xth bacteria.     

Lysogenic pneumococci and their bacteriophages.

About half of pneumococci recovered from pediatric patients and one-third of isolates from adult patients yielded bacteriophages active against one or more of four noncapsulated indicator strains of pneumococcus. Strains of capsular types most frequently causing pediatric infections were associated with lysogeny. Classical restriction-modification phenomena have been demonstrated in vivo with some of the temperate phages, and correlation of restriction with the presence of one or the other of the two known pneumococcal restriction endonucleases has been established. The temperate phages differ serologically and in several other characteristics from virulent pneumococcal phages previously described. All pneumococcal phages so far studied can be classified into a minimum of three serological groups.     

Sporulation-converting bacteriophages for Bacillus pumilus.

Thirty-three sporulation-converting bacteriophages for Bacillus pumilus NRS576 were assigned to two apparently unrelated groups on the basis of morphology and antiserum neutralization. Bacterial sporulation mutants responded similarly (conversion or nonconversion) to representatives of both phage groups. Evidence is presented indicating that PMB1 and related phages specify a restriction and/or modification system.     

Characterization of Streptococcus bovis bacteriophages.

About 25 Streptococcus bovis bacteriophages were isolated from abattoir wastes, bovine rumen fluid, and lysogenic strains of S. bovis. Eight phages were selected and characterized by morphology, stability, rate of adsorption, single-step growth curve, serum neutralization, and antigenic relationship. Two distinct morphological phage types were found, one of which has not been previously reported for group D streptococci.     

Inducible bacteriophages from ruminal bacteria.

The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria.     

Characterization of some pneumococcal bacteriophages.

The growth of pneumococcal phages at high cell and phage densities is enhanced strongly by the substitution of potassium for sodium in the medium. Initial titers of 2 X 10(10) to 4 X 10(10) PFU/ml are readily obtained, and concentrated stocks are stable in a storage buffer described here. The mechanism of the cation effect is obscure. Phages omega3 and omega8 each have linear double-stranded DNA of 33 X 10(6) daltons per particle, with an apparent guanine plus cytosine content of 47 to 49 mol%, as determined by buoyancy and melting temperature, but with an unusual absorbance spectrum. Efficiency of plating is high if sufficient time is allowed for a relatively slow adsorption, which differs several-fold in rate between the two phages. Morphologically, these and other pneumococcal phages are similar to coliphage lambda but with a longer tail and tail fiber. Upon UV inactivation, omega3 and omega8 have D37 values of 33 and 55 J/m2, respectively, and each shows multiplicity reactivation. A total of 13 ts mutants have been isolated from the two phages, representing only two complementation groups; complementation and recombination occur between omega3 and omega8 mutants. Both phages provoke high-titer antisera with extensive cross-reactivity against a number of newly isolated pneumococcal phages.     

Genomic polymorphism in the T-even bacteriophages.

We have compared the genomes of 49 bacteriophages related to T4. PCR analysis of six chromosomal regions reveals two types of local sequence variation. In four loci, we found only two alternative configurations in all the genomes that could be analyzed. In contrast, two highly polymorphic loci exhibit variations in the number, the order and the identity of the sequences present. In phage T4, both highly polymorphic loci encode internal proteins (IPs) that are encapsidated in the phage particle and injected with the viral DNA. Among the various T4-related phages, 10 different ORFs have been identified in the IP loci; their amino acid sequences have the characteristics of internal proteins. At the beginning of each of these coding sequences is a highly conserved 11 amino acid leader motif. In addition, both 5' and 3' to most of these ORFs, there is a approximately 70 bp sequence that contains a T4 early promoter sequence with an overlapping inversely repeated sequence. The homologies within these flanking sequences may mediate the recombinational shuffling of the IP sequences within the locus. A role for the new IP-like sequences in determining the phage host range is proposed since such a role has been previously demonstrated for the IP1 gene of T4.     

Exopolysaccharide depolymerases induced by Rhizobium bacteriophages.

Enzymes induced by two Rhizobium trifolii bacteriophages caused depolymerization of exopolysaccharides from most R. trifolii and R. leguminosarum strains tested, but did not, in general, attack the exopolysaccharides of R. meliloti, the slow-growing rhizobia, or Agrobacterium. Ca2+ and (or) Mg2+ were required for enzyme activity. In all strains tested, depolymerization of exopolysaccharide occurred when there was successful phage infection, but depolymerization also occurred with exopolysaccharides from nonsusceptible strains.     

Characteristics of bacteriophages for Micromonospora purpurea.

Chemical and physical stabilities of bacteriophages ?UW 21 and ?UW 51 infecting Micromonospora purpurea ATCC 15835 were examined. Both phages were stable over the pH range of 5 to 8 and to heating at temperatures up to 50 degrees C and especially stable in buffer containing magnesium ion. Exposure to 1 M Ca(NO3)2 inactivated both phages, and phage ?UW 51 was also susceptible to 1 M CaCl2, 0.1 M tris(hydroxymethyl)aminomethane, and 0.3% H2O2. Phage plating efficiency was highest on the cultures at logarithmic phase and sometimes much influenced by host growth. Phage ?UW 51 has a latent period of 2 h at 34 degrees C and a burst size between 35 and 40. The latent period for phage ?UW 21 is about 12 h, and the burst size is smaller than 30.     

Characteristics of bacteriophages for Micromonospora purpurea.

Chemical and physical stabilities of bacteriophages øUW 21 and øUW 51 infecting Micromonospora purpurea ATCC 15835 were examined. Both phages were stable over the pH range of 5 to 8 and to heating at temperatures up to 50 degrees C and especially stable in buffer containing magnesium ion. Exposure to 1 M Ca(NO3)2 inactivated both phages, and phage øUW 51 was also susceptible to 1 M CaCl2, 0.1 M tris(hydroxymethyl)aminomethane, and 0.3% H2O2. Phage plating efficiency was highest on the cultures at logarithmic phase and sometimes much influenced by host growth. Phage øUW 51 has a latent period of 2 h at 34 degrees C and a burst size between 35 and 40. The latent period for phage øUW 21 is about 12 h, and the burst size is smaller than 30.     

Escherichia coli capsule bacteriophages. V. Lysozyme 29.

In addition to the spike-associated host capsule depolymerase, infection by Escherichia coli capsule bacteriophage no. 29 also induces the synthesis of a large bacteriolytic enzyme which has been purified to homogeneity. On incubation of isolated host murein sacculi with this enzyme, no amino groups but reducing sugar groups were liberated, and muraminitol, but no glucosaminitol, was found in the degraded sacculi after subsequent reduction with NaBH4. The bacteriolytic enzyme is thus another lysozyme (mucopeptide N-acetylmuramylhydrolase; EC 3.2.1.17). Electron optical visualization of negatively stained lysozyme specimens showed oblong particles of roughly 4.5 to 5.5 nm in diameter and 15 to 19 nm in length. Although the material tended to dissociate, a crude estimate of its molecular weight (270,000 plus or minus 30,000) could be obtained from these dimensions, from its sedimentation equilibrium, and from its behavior in gel chromatography. After disintegration of homogeneous lysozyme 29 by heating in solution with sodium dodecyl sulfate and dithiothreitol, polypeptides of one size only (about 46,000 dalton, probably six copies per molecule) were found in sodium dodecyl sulfate-polyacrylamide electrophoresis. The amino acid analysis of the enzyme accounted for more than 90% of its dry weight. One percent or less of the bacteriolytic activity in phage 29 lysates was found to be associated with the intact or disrupted virus particles, and a polypeptide of 46,000 daltons was not detected in the virions. These results strongly suggest that, in contrast to the host capsule depolymerase also induced by the same phage, and in spite of its comparatively large size, "lysozyme 29" does not constitute an integral part also of the homologous bacteriophage particles.