A novel transducing phage

Summary Two newly isolated generalized transducing phages, F126 and F130, are in many respects similar to the previously described phage F116 of P. aeruginosa strain PAO. They also share with F116 the property of not responding to host-specific restriction in strain PAO. However, while the transduction ability of phages F116 and F130 is also inert to PAO-specific restriction, transduction mediated by phage F126 is 8–120 fold reduced in restrictive conditions. In experiments designed to explore the conditions necessary to obtain restriction in F126 transduction it was found that it differed from those previously known to specify host-controlled restriction in P. aeruginosa PAO (Rolfe and Holloway, 1968). These observations suggest that more than one independent restriction system operates in strain PAO.     

Characterization of a glpK transducing phage

Summary A specialized glpK transducing phage, dglpK100, has been isolated and characterized with respect to DNA structure. The glpK component of the glpKF operon has been localized within a 2.0 kilobase pair (kbp) region of the approximately 8.24 kbp bacterial DNA insert, and the positions of BamHI, EcoRI and HindIII restriction sites within this DNA have been identified.     

In vivo titration of araC protein.

The requirement for araC protein in the induction of the araBAD operon was investigated. Strains of Escherichia coli carrying an araC(Am) mutation and temperature-sensitive amber suppressors were used to vary the intracellular level of araC protein. The levels of araC protein studied ranged from 0.007 to 1.8 times the normal amount. The results indicate that the normal level of araC protein is just sufficient to provide maximal expression of the araBAD operon.     

A generalized transducing phage of Pseudomonas cepacia

A generalized transducing phage, named CP75, was derived from a lysogenic strain of Pseudomonas cepacia. The frequency of transduction per phage particle ranged from 1.0 X 10(-6) to 2.0 X 10(-6) for a given marker. About half of the 105 P. cepacia strains tested were sensitive to the phage. The molecular size of the CP75 genome was approximately 52 kb.     

A method for detection of phage mutants with altered transducing ability

Summary A method is described which allows the detection of phage mutants with increased or decreased transduction frequencies. Some properties of such mutants are presented.     

The production of generalized transducing phage by bacteriophage lambda

Generalized tranduction has for about 30 years been a major tool in the genetic manipulation of bacterial chromosomes. However, throughout that time little progress has been made in understanding how generalized transducing particles are produced. The experiments presented in this paper use phage λ to assess some of the factors that affect that process. The results of those experiments indicate: (1) the production of generalized transducing particles by bacteriophage λ is inhibited by the phage λ exonuclease (Exo). Also inhibited by λ Exo is the production of λdocR particles, a class of particles whose packaging is initiated in bacterial DNA and terminated at the normal phage packaging site, cos. In contrast, the production of λdocL particles, a class of particles whose packaging is initiated at cos and terminated in bacterial DNA, is unaffected by λ Exo; (2) λ-generalized transducing particles are not detected in induced lysis-defective (S⁻) λ lysogens until about 60–90 min after prophage induction. Since wild-type λ would normally lyse cells by 60 min, the production of λ-generalized transducing particles depends on the phage being lysis-defective; (3) if transducing lysates are prepared by phage infection then the frequency of generalized transduction for different bacterial markers varies over a 10–20-fold range. In contrast, if transducing lysates are prepared by the induction of a λ lysogen containing an excision-defective prophage, then the variation in transduction frequency is much greater, and markers adjacent to, and on both sides of, the prophage are transduced with much higher frequencies than are other markers ; (4) if the prophage is replication-defective then the increased transduction of prophage-proximal markers is eliminated; (5) measurements of total DNA in induced lysogens indicate that part of the increase in transduction frequency following prophage induction can be accounted for by an increase in the amount of prophage-proximal bacterial DNA in the cell. Measurements of DNA in transducing particles indicate that the rest of the increase is probably due to the preferential packaging of the prophage-proximal bacterial DNA.

These results are most easily interpreted in terms of a model for the initiation of bacterial DNA packaging by λ, in which the proteins involved (Ter) do not recognize any particular sequence in bacterial DNA but rather     

Characterization of dnaA gene carried by lambda transducing phage

Summary Specialized transducing phages dnaA were obtained by inducing lysogens in which tna was integrated at the tnaA region of the Escherichia coli chromosome; the tnA region is located in the vicinity of the dnaA gene. The dnaA ^- deletion derivatives of dnaA were isolated from the lysate of dnaA grown on bacteria carrying a transposon Tn3.The structures of various transducing phages thus obtained were determined by heteroduplex DNA mapping. From these results, the transducing fragment of 13.8-kb-long was divided into nine domains. Upon infection of UV-irradiated cells with the phage, production of polypeptides of 49 kD and 42 kD was specifically associated with infections by the dnaA and recF transducing phages. Polypeptides of 49 kD and 42 kD appeared to be coded for by dnaA and recF genes, respectively. The dnaA gene was assigned to the region of 2.8-kb-long which extends by 2.4 kb in the counterclockwise direction on the E. coli genetic map and 0.4 kb in the opposite direction, as measured from the nearest HindIII site close to the tnaA gene. The recF gene was also discovered to lie very close to dnaA in the order of tnaA-dnaA-recF.Merogenotes heterozygous for the dnaA gene were constructed by introducing F100-12 carrying dnaA into the recipients with different mutations at or near dnaA. For combinations, F(dnaA ⁺)/dnaA46 and F(dna ⁺)/dna-83, dnaA ⁺ was trans-dominant, whereas the dnaA ⁺ was recessive for F(dnaA ⁺)/dna-5. For F(dnaA ⁺)/dna-167, the result of the transdominance test was affected by the growth media employed; dnaA ⁺ was dominant on a -broth plate, and dna-167 was dominant on an M9-minimal plate. Thus, transdominance of dnaA ⁺ in heterozygotes is affected by difference in mutations and growth media.     

Plaque assay for lambda transducing phage carrying the E. coli metB gene

A halo plaque assay has been developed for the detection of nondefective lambda transducing phage carrying functional alleles of the metB gene of Escherichia coli K12. The assay is based upon the production of phage plaques on lawns of metB- bacterial cells which are supplemented with limiting amounts of methionine and upon the subsequent transduction of methionine-starved cells in the lawn surrounding the plaques. The resulting prototrophic transductants give rise to a halo of bacterial growth surrounding the plaque. A precise genotype can be ascribed to the characteristic morphologies of selected haloes. This technique has general application for all biosynthetic markers.     

The construction in vitro of transducing derivatives of phage lambda

Summary Methods are described for the construction of plaque-forming, transducing derivatives of phage lambda, using appropriate receptor genomes and fragments of DNA generated by the restriction enzymes endo R.EcoRI and endo R.HindIII. The general properties of the transducing derivatives are described and discussed. Plaque-forming phages carrying the E. coli trp, his, cysB, thyA, supD, supE, supF, hsd, tna and lig genes have been isolated.     

Bxz1, a new generalized transducing phage for mycobacteria

We have isolated and characterized a new generalized transducing phage, Bxz1, from soil sampling at a neighboring Wildlife Preservation Park. The hosts of the phage, measured by the formation of plaques, include fast growing Mycobacterium smegmatis and Mycobacterium vaccae. Bxz1 is capable of transducing chromosomal markers, point mutations, and plasmids at frequencies ranging from 10(-8) to 10(-6) per plaque forming unit between strains of M. smegmatis. We also demonstrated cotransduction of a transposon insertion linked to a point mutation of the ndh gene.