Homologous Phage Research Articles

High frequency transduction of kanamycin resistance in Proteus mirabilis.

Summary: Properties of a transducing system with a phage able to transduce a kanamycin resistance marker of the T compatibility group R factor R394 at high frequency are described. The phage was detected in Proteus mirabilis strain pm5006 transduced to kanamycin resistance by phage 34 grown on P. mirabilis strain pm13 carrying the R factor. The pm5006 transductants, specially selected at the lowest multiplicities of infection (m.o.i.) of the high frequency transducing (h.f.t.) phage, are normal lysogens. They liberate h.f.t. phage spontaneously and high-titre phage may be obtained by u.v. induction. The phage is serologically identical to phage 34 and is defective in that, at low m.o.i., transduction frequency is increased by the simultaneous presence of homologous non-transducing phage. Ultraviolet irradiation of the h.f.t. lysate produces an exponential fall in transduction frequency. It is concluded that the defective phage transduces by lysogenization. Phage present in h.f.t. lysates can also transduce various chromosomal markers of pm5006 at low frequencies. These low-frequency transductants are usually not kanamycin resistant. Transductants do not transfer the kanamycin resistance marker conjugally and produce kanamycin-sensitive normal lysogenic segregants at a high rate. Strain pm5006 is cryptically lysogenic for a phage which is morphologically and serologically identical to phage 34 and many of the particular features of this transduction system are explicable in terms of complementation or recombination between genes of the phages involved.

Isolation, Characterization, and Classification of Three Bacteriophage Isolates for the Genus Levinea

Bacteriophages for the genus Levinea were isolated from sewage samples. On the basis of plaque morphology and host specificity, three different phage isolates were obtained and purified by standard techniques. Cross-neutralization experiments with homologous and heterologous phage antisera showed no serological relatedness among the three phages and, therefore, they were placed into three serological groups. Electron micrographs showed the isolates to have different and distinctive morphological types, one of which was unique in that it possessed an elongated "bullet-shaped" capsid. Nucleic acid types for the three phages were determined by the acridine orange-fluorescent staining technique to be double-stranded deoxyribonucleic acid. Based on serological grouping, the three phages were classified and designated DM-11, DM-21, and DM-31. The value of the phage isolates in defining the species of the genus Levinea is discussed.

Relations between Phage Sensitivity and Virulence in Pseudomonas morsprunorum

SUMMARY The phage reactions and pathogenicity of English isolates of Pseudomonas morsprunorum were compared with those of isolates from other countries. Nonindigenous cherry strains resembled English cherry strains pathologically, but not all were sensitive to A7, the type-determining phage for English cherry strains. Plum strains resistant to A7 adsorbed the phage as readily as cherry strains, indicating that phage receptor materials are not involved in host specificity. Cherry strain mutants resistant to specific and/or non-specific phages showed marked attenuation of virulence, although retaining the essential pathological characters of cherry strains. The resistance of all but one of the mutants was due to failure to adsorb the homologous phage, showing that the wall structure had changed and suggesting that wall components may function as general virulence determinants in P. morsprunorum.

Spackle and immunity functions of bacteriophage T4.

Cells of Escherichia coli B infected with the immunity-negative (imm2) mutant of bacteriophage T4 are able to develop a substantial level of immunity to superinfecting phage ghosts if the ghost challenge is made late in infection. This background immunity is not seen in infections with phage carrying the spackle (s) mutation in addition to the imm2 lesion. The level of immunity in s(-) infections is intermediate between that of imm(-) and wild-type infections under standard assay conditions. With respect to genetic exclusion of superinfecting phage, cells infected with imm(-) phage are completely deficient, whereas infections with the s(-) phage are only partially deficient compared to wild-type infections. Whereas s(-)-infected cells are unable to resist lysis from without by a high multiplicity of infection (MOI) of superinfecting phage, cells infected with imm(-) phage show less than wild-type levels of resistance and the majority of cells remaining intact are unable to incorporate leucine or form infective centers. Under conditions of superinfection by low MOI of homologous phage, imm(-)-infected cells are lysis inhibited, whereas s(-)-infected cells do not show this property. Superinfecting phage inject their DNA into imm(-)-infected cells with the same efficiency as seen in wild-type infections, but this efficiency is reduced when the cells are first infected with s(-) phage. The s function of T4 appears not only to affect the host cell wall as previously postulated by Emrich, but may also affect the junctures of cell wall and membrane with consequences similar to those of the imm function.

Temperate Bacteriophages for Streptomyces coelicolor A3(2) Isolated from Soil

SUMMARY: Three phages, isolated from soil samples collected in different localities, produced turbid plaques with a characteristic morphology on Streptomyces coelicolor A3(2). Surviving growth from the plaques was lysogenic: when isolated and purified it was resistant to lysis by the homologous phage and released phage at a low frequency during growth. The three phages were homoimmune. The chromosomal attachment site of one of the prophages, VP5, was mapped in a position between hisE and cysD - that is, in a region devoid of standard markers - on the circular linkage map of the host, by crossing defective lysogens with sensitive strains. The host range of VP5 was restricted; only certain strains belonging to the S. coelicolor (S. violaceoruber) group were attacked. VP5 gave rise to clear-plaque mutants, incapable of lysogenizing the host, at a spontaneous frequency of 10−4, which was increased by u.v. irradiation and by hydroxylamine mutagenesis; the clear-plaque mutants were incapable of lysing strains carrying the wild-type prophage. Electron microscopy showed that VP5 belongs to group B of Bradley’s morphological classification.

Effects of Homologous Bacteriophage on Growth of Pseudomonas fragi WY in Milk1

Pseudomonas fragi strain WY and its homologous bacteriophage were added in varying concentrations to sterile skim milk which was stored at 7 C for 72 hr. When the initial concentration of the bacterial host was 100,000/ml, addition of as few as 10 plaque-forming units per ml of bacteriophage resulted in significantly lower counts in treated skim milk than in the controls which contained no phage. There was no significant effect, however, when the phage input was 1 in 10 ml and the bacterial count was 1,000 or 100,00/ml. No differences in bacterial counts occurred even when the phage concentration was 1,000/ml if the initial bacterial concentration was only 1,000/ml.

Effects of homologous bacteriophage on growth of Pseudomonas fragi WY in milk.

Pseudomonas fragi strain WY and its homologous bacteriophage were added in varying concentrations to sterile skim milk which was stored at 7 C for 72 hr. When the initial concentration of the bacterial host was 100,000/ml, addition of as few as 10 plaque-forming units per ml of bacteriophage resulted in significantly lower counts in treated skim milk than in the controls which contained no phage. There was no significant effect, however, when the phage input was 1 in 10 ml and the bacterial count was 1,000 or 100,00/ml. No differences in bacterial counts occurred even when the phage concentration was 1,000/ml if the initial bacterial concentration was only 1,000/ml.

Characterization of Temperate Actinophage φC31 Isolated from Streptomyces coelicolor A3(2)

Actinophage phiC31 isolated from Streptomyces coelicolor A3(2), the only strain among actinomycetes for which a genetic map had been constructed, appears to be a typical temperate phage. After phiC31 infection, true lysogenic cultures arose which liberated phage and were immune to infection with homologous phage after repeated single-colony isolations and treatment with phage-specific antiserum. Clear-plaque (c) mutants were derived from phiC31 phage which failed to lysogenize sensitive cultures. Actinophage phiC31 has a temperature-sensitive stage of reproduction. A phage which reproduces with the same effectiveness at high (37 C) and low (28 C) temperatures has also been obtained. Heat-inducible (ct) mutants were isolated from this phage which were able to lysogenize sensitive cultures at 28 C but failed to do so at 37 C. Properties of ct mutants suggest that ct mutations involve a gene controlling maintenance of the lysogenic state in actinomycetes and synthesizing repressor, which may become heat-sensitive as a result of mutation.

Superinfection in bacteriophage S13 and determination of the number of bacteriophage particles which can function in an infected cell.

Bacteriophage S13 shows exclusion of superinfecting homologous phage, but the exclusion is only partial. The superinfecting phage can form infectious replicative form deoxyribonucleic acid (RF), can direct protein synthesis, and can form progeny particles even at a superinfection time as late as 60 min after the first infection. Exclusion is also only partial for the closely related phage phiX174. Seven min after the first infection, the exclusion mechanism begins to operate, requiring continuous phage-specified protein synthesis. The gene A protein (required for synthesis of progeny RF) appears to be involved in the exclusion mechanism. In superinfection experiments, it was found that at least 40 phage particles per cell can replicate and can carry out protein synthesis, though the number of sites for binding of RF to the membrane is only about 15 per cell. The results suggest that attachment of RF to a binding site is not required for protein synthesis. Evidence is presented that non-attached parental RF can serve as a template for single-stranded deoxyribonucleic acid synthesis.

Ultrastructure du bactériophage agissant dans certaines conditions comme une staphylococcine

The lysogenization of Staphylococcus aureus, strain BSS, by staphylophage [Formula: see text] of serological group A may be accompanied by the loss of its capacity to synthesize δ haemolysin. The high multiplicity of infection of [Formula: see text] phage changes the tinctorial affinity of BSS cell walls and lyses the same strain lysogenic for the homologous phage; this lysis is similar to that of the staphylococcin. In negative coloration by PTA, this bacteriophage shows one elongated head (750 Å × 420 Å) and a long tail (2650 Å). Electron microscopy demonstrated also the existence of structural subunits of the tail and the hexagonal end plate. By its morphology and dimensions, the [Formula: see text] phage is similar to 594n and b594n phages studied by Bradley and to staphylophages of serological group A: 3A, 3B, 3C, 6, 7, 42E, 47, 54 of the basic set of international lysotypy.

A special case of lysogeny in Mycobacterium avium.

Phage-producing colonies of cells resistant to homologous phage F4II have been isolated after infection ofMycobacterium avium. About one per cent phage-free, sensitive clones can usually be obtained after treatment of the cell suspension with phage antiserum. Because of the relatively high free phage titer in the culture, the presence of very dense particles, probably phage equivalents, in the cytoplasm of many cells and the varied sensitivity of intracellular phage to UV irradiation at different stages of growth, one can assume that a great part of the cells in the population burst and liberate phages. The relationship between phage and the host cell is discussed as a particular case of abortive lysogeny.

Effect of Antigenic Stimulation on the Specificity of Antibody Produced by Rabbits Immunized with Bacteriophage MS-2

Abstract Repeated immunization of rabbits with the RNA coliphage MS-2 resulted in the appearance of an increasing amount of neutralizing antibodies that showed an increased rate of association (i.e., affinity) with the homologous phage antigen. There was a progressive narrowing of the specificity of the antibodies produced after each antigenic stimulation as evidenced by a decrease in the extent of cross-reactions observed with related heterologous RNA phages. These results are discussed in relation to the nature of the antibody response and the antigenic structure of phage MS-2.

Effect of chloramphenicol on host-bacteriophage relationships in the lactic streptococci.

Chloramphenicol (CM)-resistant mutants of Streptococcus lactis strain ML3 were obtained either as a consequence of continuous transfer of the bacteria in broth containing increasing amounts of CM or by selecting for high-level resistant derivatives after mutagenic treatment of the bacteria. Some CM-resistant cells obtained by the first method were also resistant to the homologous bacteriophage. Cells trained to grow in the presence of CM developed resistance to some heterologous attacking phages but not to phage ml(3). Mutants selected for phage resistance were not resistant to CM. There appear to be two different loci for CM resistance on the bacterial chromosome: the one for high-level resistance is associated with the phage-resistance locus and the other is independent of it. A concentration of CM (280 mug/ml) that was bacteriostatic for ML3 inhibited the intracellular growth of ml(3) phage in strain ML3-CM(r)I, which had been trained to grow in the presence of that CM concentration, despite the fact that cells of this strain were not phage-resistant per se. The drug had no direct virucidal action and did not prevent adsorption to or penetration of phage into the bacterium. Lysogenization did not occur. It is concluded that the block in phage development probably involves inhibition of synthesis of phage components, either involving deoxyribonucleic acid at an early stage or the phage coat protein at a later one.

Effect of spermine on host-cell lysis and reproduction by a lactic streptococcal bacteriophage.

A method was tested for protecting a Streptococcus lactis strain, ML3, used as a starter in the manufacture of Cheddar cheese, against the lytic activity of its homologous phage, ml(3). At a concentration of 10(-2)m, a naturally occurring polyamine, spermine, in the form of its hydrochloride, protected ML3 against lysis-from-without and lysozyme activity and against lysis by the phage when added at the time of infection or up to 21 min after infection. It was found that the latter protective effect could be accounted for in terms of the spermine preventing the formation of mature particles rather than preventing the escape of viable phage. Single colonies selected from a culture of ML3 cells that had been previously infected with phage ml(3), in the presence of spermine, were all found to have acquired resistance to phage ml(3). They retained this resistance during a 3-month period of daily subculture in broth and, in the absence of spermine, could not be induced to liberate phage or phage components either by the techniques normally used for inducing lysogens or by artificial disruption of the cells. It is concluded that when spermine is added to ML3 cells before a certain critical stage of the phage infection cycle, the process of phage synthesis is irreversibly halted and the cells retain the infecting phage as a defective prophage that confers on the cells immunity to infection by the homologous phage. Phage-resistant cultures did not inherit reduced starter activity in association with their acquired resistance characteristic.

Absence of immunity to superinfection in spheroplasts of a lysogen.

Penicillin-induced spheroplasts of Salmonella typhimurium strain LT2 lysogenic for bacteriophage P22, appear to lose immunity to superinfection by homologous phage.

Effect of Spermine on Host-Cell Lysis and Reproduction by a Lactic Streptococcal Bacteriophage

A method was tested for protecting a Streptococcus lactis strain, ML3, used as a starter in the manufacture of Cheddar cheese, against the lytic activity of its homologous phage, ml(3). At a concentration of 10(-2)m, a naturally occurring polyamine, spermine, in the form of its hydrochloride, protected ML3 against lysis-from-without and lysozyme activity and against lysis by the phage when added at the time of infection or up to 21 min after infection. It was found that the latter protective effect could be accounted for in terms of the spermine preventing the formation of mature particles rather than preventing the escape of viable phage. Single colonies selected from a culture of ML3 cells that had been previously infected with phage ml(3), in the presence of spermine, were all found to have acquired resistance to phage ml(3). They retained this resistance during a 3-month period of daily subculture in broth and, in the absence of spermine, could not be induced to liberate phage or phage components either by the techniques normally used for inducing lysogens or by artificial disruption of the cells. It is concluded that when spermine is added to ML3 cells before a certain critical stage of the phage infection cycle, the process of phage synthesis is irreversibly halted and the cells retain the infecting phage as a defective prophage that confers on the cells immunity to infection by the homologous phage. Phage-resistant cultures did not inherit reduced starter activity in association with their acquired resistance characteristic.

Effect of Chloramphenicol on Host-Bacteriophage Relationships in the Lactic Streptococci

Chloramphenicol (CM)-resistant mutants of Streptococcus lactis strain ML3 were obtained either as a consequence of continuous transfer of the bacteria in broth containing increasing amounts of CM or by selecting for high-level resistant derivatives after mutagenic treatment of the bacteria. Some CM-resistant cells obtained by the first method were also resistant to the homologous bacteriophage. Cells trained to grow in the presence of CM developed resistance to some heterologous attacking phages but not to phage ml(3). Mutants selected for phage resistance were not resistant to CM. There appear to be two different loci for CM resistance on the bacterial chromosome: the one for high-level resistance is associated with the phage-resistance locus and the other is independent of it. A concentration of CM (280 mug/ml) that was bacteriostatic for ML3 inhibited the intracellular growth of ml(3) phage in strain ML3-CM(r)I, which had been trained to grow in the presence of that CM concentration, despite the fact that cells of this strain were not phage-resistant per se. The drug had no direct virucidal action and did not prevent adsorption to or penetration of phage into the bacterium. Lysogenization did not occur. It is concluded that the block in phage development probably involves inhibition of synthesis of phage components, either involving deoxyribonucleic acid at an early stage or the phage coat protein at a later one.

Bacteriophages of psychrophilic pseudomonads. II. Host range of phage active against Pseudomonas putrefaciens.

Forty phages active against 4 terrestrial and against 10 of 24 marine strains ofP. putrefaciens were isolated from raw municipal sewage, fish-pier water, and refrigerated haddoch fillets. With one exception, phages active against marine strains were obtained only from pier water and fish fillets. Only two strains were attacked solely by their homologous phage while five of the marine strains were not attacked by any phage. Lytic reactions revealed no obvious differences between marine and terrestrial strains.

Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus

A high-frequency transducing element for erythromycin resistance in Staphylococcus aureus has been found. This element, P11 de, is apparently the result of a recombinational event between a temperate phage, P11, and a penicillinase plasmid, γ; it lacks substantial sections both of the plasmid and of the bacteriophage. The phage moiety is cryptic, conferring neither lysogeny nor superinfection immunity upon host cells carrying it; helper phage is required for the production of transducing particles but not for the transduction process. Demonstrable phage-related properties include reactivation of ultraviolet-inactivated homologous phage and rescue of temperature-sensitive phage mutants. The plasmic moiety includes an erythromycin resistance marker, and the mc region, which is responsible for plasmid compatibility, is involved in a specific host-plasmid interaction and is required for plasmid replication; all the other known plasmid markers are missing. The autonomous replication of P11 de as well as its intracellular behavior vis-à-vis prophages and other plasmids appear to be governed by its mc determinant. In these respects P11 de is similar to the parental γ plasmid. This plasmid control, however, is superseded by active P11, which induces P11 de to multiply extensively. P11 de can recombine with wild-type plasmids; the resulting recombinants are not transduced at high frequency, nor do they reactivate UV inactivated phage particles. Based on these findings, a possible genetic structure for P11 de is presented and compared with maps of naturally occurring plasmids.

Adsorption of Staphylococcal Bacteriophage by Milk Proteins

Propagation of homologous bacteriophage in a culture of Staphylococcus aureus (1:1 ratio of phage to bacteria) in Trypticase Soy Broth (TSB) and in skim milk indicated more activity of phage in TSB. Early lysis of bacteria in skim milk followed by a pronounced rise in bacterial population suggested that staphylococcal phages were being inactivated by milk. Titration of phages from skim milk, whey, and TSB indicated about 90% adsorption of phages by acid- and heat-precipitable proteins of skim milk, whereas numbers recovered from whey were quite comparable to those recovered from TSB. Reducing the p H from 6.5 to 4.0 increased the percentage of phages recoverable from skim milk from 10 to 56%. Apparently, the changes in electrical charges on the casein micelles at this low p H were responsible for release of many phages from their complex with casein.