High-frequency Transducing Lysates Research Articles

Specialized transduction in Bacillus subtilis by the phages IG1, IG3, and IG4

The temperate phages IG1, IG3, and IG4 of Bacillus subtilis were found to mediate specialized transduction of a lambda-like type. High frequency transducing lysates were also obtained. In many instances the transductants were shown to be heterogenotes, segregating the mutant alleles. They are presumably the result of transduction by addition. These findings are discussed together with data on other members of the group III of temperate bacteriophages aiming to a deeper understanding of that group, and of its evolutionary history.

Specialized transduction of Pseudomonas aeruginosa PAO by bacteriophage D3.

D3, a temperate bacteriophage of Pseudomonas aeruginosa PAO, was found to specifically transduce the alleles met-49 and met-117. Induction of established lysogens with UV light was necessary for the production of transducing lysates. Transduced cells were immune to superinfection by phage D3 and could give rise to high-frequency transducing lysates. Cotransduction of these two alleles could not be demonstrated. met-117 was mapped to 26 min on the PAO genetic map. Complementation studies using the generalized transducing phage F116L indicated that met-49 is an allele of met-9011 which maps at 55 min. The integrated D3 prophage was shown to be coinherited with met-117 and with met-49.

Intra-species tranduction with Proteus mirabilis high frequency transducing phages.

The properties of three additional Proteus mirabilis hosts for the high frequency transducing (HET) phages 5006MHFTkappa and 5006MHFTak are described. The phages transduce resistance to kanamycin and to ampicillin plus kanamycin, respectively, and were produced by ultraviolet induction of derivatives of P. mirabilis strain PM5006. Strain PM804 could not be shown to adsorb the phages although it yielded a few transductants. All transductants, even those produced at low multiplicities of phage input were lysogenic and segregated markers at high frequency. Ultraviolet induced phage lysates of these transductants transduced PM804 at higher frequencies and PM5006 at lower frequencies than the original phages. Strain PM804 or its derivatives did not adsorb phage from these lysates. Tranmission experiments through PM5006 of phage in the transductant lysates confirmed that PM804 had a host-controlled modification system which modified HFT phage from PM5006. That PM804 also possessed a restriction system was inferred from the greater numbers of transductants obtained with page which bore a compatible modification pattern. Strain N and a restrictionless mutant of it named NHI, adsorbed the HET phages avidly and failed to modify their host range. Transduction frequencies of the phage markers were about 10(-4)/p.f.u. adsorbed to strain NHI and only about tenfold lower to strain N which did not plate the phages. Tranductants also had the features of heterogenotes and those obtained at low multiplicites of infection were non-lysogenic. The latter transductants adsorbed homolgous phage while NHI transductants also plated the HFT lysates. Strain NHI, lysogenized by the parent phage 5006M, did not adsorb the HFT phages. These findings suggest that the HFT phages were most likely defective for lysogenic conversion to homologous phage non-adsorption. The postulated restriction enzymes of PM804 and strain N could not be shown to be thermolabile.

Specialized transduction of kanamycin resistance in a Providence strain.

Properties of a transducing system with a phage able to transduce a kanamycin-resistance marker of the T compatibility group plasmid R394 at a frequency of 2 times 10(-2)/plaque-forming unit adsorbed are described. The phage was detected in Providence strain P29 transduced to kanamycin resistance by Providence phage PL25 grown on this strain harbouring the R factor. Four P29 transductants, specially selected at the lowest multiplicities of infection of the high frequency transducing (HFT) phage, were defective lysogens. They plated PL25 with an efficiency of I and only one liberated low-titre phage spontaneously or on u.v. induction. The defect in maturation function could be corrected by introduction of a wild PL25 prophage. The transducing phage was serologically frequency was increased by the simultaneous presence of homologous non-transducing phage. Transductants did not transfer the kanamycin-resistance marker by conjugation, and produced kanamycin-sensitive segregants at a moderate rate. These segregants could be transduced to kanamycin resistance by the HFT phage. Irradiation of HFT lysates by u.v. produced an exponential fall in transduction frequency. It was concluded that the defective phage transduced by lysogenization. Kanamycin-resistant transductants could themselves be transduced by streptomycin resistance by PL25 reared on a streptomycin-resistant mutant. Lysogenic transductants produced by the HFT phage did not always liberate HFT phage on u.v. induction. Possible explantations are considered.

Genetic and physical studies of lambda transducing bacteriophage carrying the beta subunit gene of the Escherichia coli ribonucleic acid polymerase.

The prophage lambdac1857 was inserted into the bfe gene located near rif (the structural gene for the beta subunit of deoxyribonucleic acid [DNA]-dependent ribonucleic acid polymerase) on the Escherichia coli chromosome. Induced lysates (low-frequency transducing lysates) of such a lysogen contained defective lambda phage particles (lambdadrif+) that can specifically transduce the wild-type rif+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harboring both the wild-type and the mutant rif genes were isolated. Rec+ derivatives of these heterogenotes produce high-frequency transducing lysates that contain lambdadrif+ and normal active phages at a ratio of 1 to 2. The results of marker rescue experiments and of density determination with several transducing phages indicate that most of the late genes are deleted and replaced by a segment of the chromosomal DNA carrying the bfe-rif region. The length of the chromosomal segment seems to vary between approximately 0.5 and 0.6% of the total bacterial DNA among the three independently isolated lambdadrif+ phages. Electron microscopy of heteroduplex DNA consisting of one strand from lambdadrif+-6 and the other from lambdaimm-21 phages directly confirmed that most of the phage DNA of the "left arm" was replaced by the bacterial DNA. The heteroduplex study also demonstrated that the integration of prophage lambda into the bfe region occurred at the normal cross-over point within the phage attachment site.

Isolation and characterization of lambda transducing bacteriophages for the su1+ (supD minus) amber suppressor of Escherichia coli.

Specialized lambda transducing phages for the sul+ (supD-) amber suppressor in Escherichia coli K-12 have been isolated, using a secondary site lambda-cI857 lysogen in which we have shown the prophage to be closely linked to sul+.sul+ transducing particles were detected frequently, at 10-5 per plaque-forming unit, in lysates prepared from the secondary-site lysogens. High-frequency transducing lysates were obtained from several independently isolated sul+ transductants and were analyzed by CsCl equilibrium density gradient centrifugation. The transducing phages are defective; marker rescue analysis indicates that the lambda-N gene is not present. In lambda-cI857DELTANdSul+, a bio-type transducing phage, the genes specifying recombination and excision functions have been replaced by bacterial deoxyribonucleic acid.

Isolation of specialized transducing bacteriophages for gluconate 6-phosphate dehydrogenase (gnd) of Escherichia coli.

Specialized transducing phages for gluconate 6-phosphate dehydrogenase (gnd), a constitutive enzyme in Escherichia coli, have been isolated using a method previously described for other genes. The gnd-his region, carried on an F' episome, was first transposed to tonB. Rare phages carrying gnd were selected, by transduction, from phi80 lysogens of these strains; one phage also carried his (phi80gndhis). From the transductants, high-frequency transducing lysates were obtained; low multiplicity of infection then yielded defective lysogens. tonB deletion analysis of the phi80dgndhis lysogen shows the order of genes in the prophage to be imm80...hisOGD...gnd; according to a marker rescue experiment most phage late genes have been replaced by bacterial deoxyribonucleic acid. A heat-inducible, lysis-defective lambda-phi80 hybrid derivative of phi80dgndhis has been prepared.

Specialized transduction of tetracycline resistance by phage P22 in Salmonella typhimurium: II. Properties of a high-frequency-transducing lysate

A high-frequency-transducing (HFT) lysate for tetracycline resistance ( tet R) was obtained by inducing an unusual tet R P22 lysogen which had been made by transducing Salmonclla typhimurium LT-2 to tet R with P22 phage grown on an LT-2 strain carrying the R factor 222. The HFT lystate contains defective P22 particles, called P22 Tc-10, which cannot grow or lysogenize (i.e., transduce tet R) upon single infection. However, two defective P22 Tc-10 particles can cooperate with each other for growth or for lysogeny (i.e., transduction of tet R), “Wild-type” phages which can grow on single infection and which no longer transduce tet R can be found at a frequency of 5 × 10 −5 PFU/particle in the HFT lysate. Five percent of these “wild-type” phages are also integration deficient. At high multiplicities of infection the tet R transductants made with an HFT lysate appear to be similar to a P22 Tc-10 lysogen (i.e., they are immune to P22 and yield HFT lysates upon induction); at low multiplicities of infection, many of the tet R transductants are tet R P22 prophage deletions. To account for these observations, a model is proposed in which the genes for tet R are inserted into the intact P22 genome in the P22 Tc-10 prophage. The consequences of such an insertion are discussed with regard to the structure of phage P22 DNA and the topological requirements for successful DNA replication, phage growth, and lysogenization.

Transduction mechanisms of bacteriophage epsilon 15. I. General properties of the system.

SUMMARYBacteriophageεγis capable of transduction both by replacement of a genetic segment of the recipient by the homologous genetic material from the donor strain and by the formation of defective transducing particles capable of lysogenizing the recipient strain ofS. anatum.The isolation of strains carrying such prophages, which have incorporated the lactose or arabinose operons, is reported. Lysogenic strains, carrying both normal and defective transducing prophage, form high-frequency transducing lysates. Other strains, carrying only defective prophage, show evidence that the association of prophage genes and transduced materials is stable since the loss of one frequently entails loss of the other.

Autonomous replication of a defective transducing phage in Pseudomonas putida

The mandelate gene cluster for four enzymes has been transduced from Pseudomonas putida strain PRS1 (ATCC 12633) to the camphor-growing, mandelate-deleted strain PUG2 (ATCC 17452). Whereas crosses within strain PRS1 using phage pf16h2 yield principally phage-sensitive transductants, the interstrain crosses of PRS1 and PUG2 yield mostly immune ones, unstable for the mandelate and immune characters. The mandelate cluster is segregated at an approximate frequency of 10 −3 per bacterium per division during the cellular life cycle of the transductants. The immune heterogenotic transductants do not produce active vegetative phages on induction, but upon UV irradiation and superinfection with viable nontransducing phages they produce lysates which transduce at frequencies of 10 −5 to 10 −6 instead of the usual 3 × 10 −8. The release of transducing phages is dependent on the multiplicity of the superinfecting phages and on the dose of UV irradiation. Phage-sensitive transductants of PUG2 also occur, and these also produce high-frequency transducing lysates without producing viable phages. We conclude that the transducing particles consist of different amounts of the phage genome, with or without an immunity-controlling region, and lack some essential phage function. These defective phages have been termed pf dm in analogy to λ dg or Pl dl. In CsCl gradient the infective particles in high-frequency transducing lysates have a characteristic density of 1.519 g/cm 3, whereas three of the pf dm elements tested vary in density from 1.526 to 1.534 g/cm 3, suggesting compositional heterogeneity of the transducing particles. Phage-sensitive transductants of PUG strains can be cured of the defective pf dm particles by ultraviolet irradiation. They contain a satellite DNA band in addition to the bacterial DNA in a CsCl density gradient. These cells overproduce mandelate enzymes, suggesting that pf dm particles replicate autonomously in the phage-sensitive PUG cells.

CHARACTERISTICS OF THE TRANSDUCING ELEMENTS OF BACTERIOPHAGE PHI-80.

Abstract The density of bacteriophage φ80 was measured by density gradient equilibrium centrifugation in CsCI solutions. This density was the same in phages derived from a lysogenic or a lytic growth cycle. The distribution of low-frequency transducing (LFT) particles of φ80 in a CsCl density gradient was shown to be much broader than that of plaque-forming particles. The transducing phages in the LFT lysates appear to transmit their characteristic densities, as well as definite try markers, to the φ80 dt particles in the high-frequency transducing lysates derived from them. The φ80 dt particles can be divided into two classes according to their requirement for helper phages in transduction. One class transduces more efficiently in the presence of active helper phage, while with the other class the active phage does not enhance or even reduces the transduction frequency. A new transducing phage type, which is not defective since it multiplies without help from the active phage, has been isolated. This phage, called φ80 pt (plaque-forming tryptophan), forms a small turbid plaque. A tryptophan auxotroph lysogenized by φ80 pt becomes try+, although it always segregates out some try− nonlysogenic cells. The buoyant density of φ80 pt in CsCI solution is greater than that of φ80, suggesting that a larger genetic fragment is carried in this phage.

The specific gravity of transducing particles of bacteriophage P1

The density of the particles of coliphage P1 and of the transducing particles derived from this phage was studied by density-gradient centrifugation in CsCl solutions. The normal and the transducing phage particles formed single-peak bands, the transducing particles being slightly heavier. Differences among the transducing particles in various types of high-frequency transducing lysates for the lac + marker were observed. The particles of the antigen-converting Salmonella phage ϵ 15 vir and of a nonconverting mutant ϵ y were indistinguishable in their banding properties in CsCl gradient.

Transduction by coliphage λ of the galactose marker

Phages obtained by induction of strain C600 Gal + made lysogenic for λ ++ when infecting strain 112 Gal − sensitive transduce Gal + with low frequency. The strain C600 Gal + is stable; it does not segregate Gal −. The transformed strains are “heterogenotes.” These heterogenotes have the following properties: 1. 1. They are Gal + and lysogenic. 2. 2. They segregate Gal − lysogenic. 3. 3. They do not segregate defective lysogenic or sensitives. 4. 4. They produce primary HFT (high frequency transducing) lysates after induction. These primary HFT lysates have the following properties: 1. 1. The burst size is about 100 phages per bacterium. 2. 2. They produce lytic or lysogenic responses in sensitive cells in the same proportion as normal lysates. 3. 3. Among the lysogenized cells there are a large number which have been transformed to Gal +. 4. 4. All the Gal + are lysogenic. 5. 5. The “Gal + titer” of the lysate is much higher than the titer of the induced bacteria which produced the lysate. 6. 6. When the primary HFT lysate is passed through one cycle of growth on C600 Gal + sensitive, on 112 Gal + sensitive, or on Gal − sensitives, the phage titer and the Gal + titer go up nearly in parallel; the efficiency of multiplication of HFT in the Gal − cells seems smaller than in the Gal + cells. 7. 7. The HFT lysate transduces Gal + into 112 Gal − lẏsogenic cells with a much lower efficiency than they transduce it into sensitive cells. 8. 8. All these transformed lysogenic cells are heterogenotes and the vast majority of them, upon induction, liberate HFT lysates whose phages do not have the genetic markers of the phages used to transform them, but have the genetic markers of the prophage that was carried by these cells.