Physical characterization of bacteriophage MX4, a generalized transducing phage for Myxococcus xanthus.

A generalized transducing bacteriophage of Myxococcus xanthus has been examined. The phage particle consists of an isometric head and a contractile tail. The genome of the phage is a linear DNA molecule of molecular weight 39 ± 2.1 × 10⁶, which contains the normal DNA bases 70% of which are guanosine + cytosine. No overall heterogeneity of base composition is present. The DNA does not carry easily detectable cohesive ends nor is it cyclically permuted. It does contain a large and somewhat variable terminal redundancy. Heating phage particles in the presence of EDTA causes tail sheath contraction and ejection of DNA, some of which remains attached to the tail. Digestion of tail-bound DNA with restriction enzymes shows that the phage tail can be attached to either end of the DNA. Thus the DNA probably contains recognition sites for the packaging of its DNA at both ends. These results suggest possible mechanisms for the genesis of transducing particles by phage MX4.     

The molecular structure of the transducing particles of Salmonella phage P22. II. Density gradient analysis of DNA

Summary CsCl density gradient analysis showed that the DNA of plaque forming particles ofSalmonella phageP22 is lighter than the host DNA. The DNA of transducing phages exhibits an intermediate density, but close to host DNA. BU labelling of DNA synthesized in the cells after phage infection resulted in a density increase of transducing DNA of about 0.004 gxcm^-3, whereas infectious DNA increased by about 0.045 gxcm^-3. Shearing of isolated DNA molecules from unlabelledP22 lysates demonstrated that transducing DNA consists of two pieces of DNA of different density: 90% stem from the bacterial host whereas 10% are phage DNA and therefore responsible for the BU lable in transducing phages.     

New transducing phage with RNA polymerase beta- and beta'-subunit genes derived from a hybrid phage lambda att80: isolation, genetic analysis and physical mapping]

A hybrid lambda att 80 phage with the genetic structure lambda (A-J) phi 80 (att-int-xis) imm lambda..cI857s7 is shown to be a convenient vector for creating transducing phages. On the one hand, the restriction analysis indicates that it has 3 restriction sites for EcoRI in comparison with 5 and 9 sites for parental phages lambda and phi 80 respectively. On the other hand, its buoyant density is less than that of phage lambda and under centrifugation it is easier separated from the phage transducing particles. When lambda att 80 prophage was excluded from the bfe locus of Escherichia coli, transducing phages with genes of two RNA polymerase beta-subunits (rpoB and rpoC) were isolated. To identify the latter, a convenient genetic test was worked out. A physical map of lambda att 80 drifd 35 transducing phage, carrying rpoB and rpoC genes has been constructed using endonucleases EcoRI and HindIII. A comparison of this map and the corresponding maps of transducing phages lambda drifd 18 and lambda drifd 47, studied earlier, led to the discovery of two integration sites of phage lambda within the locus bfe spaced apart by about 1800 nucleotide pairs. At all the sites both phages (lambda and lambda att 80) have integrated in the locus bfe in the counter clockwise order.     

Molecular characterization of ilvC specialized transducing phages of Escherichia coli K-12

A series of lambda defective ilvC specialized transducing phage has been isolated which carry regions of isoleucine and valine structural and regulatory genes derived from the ilv cluster at minute 83 on the linkage map of the chromosome of Escherichia coli K-12. The ilv genes carried by these phages and their order have been determined by transduction of auxotrophs. The ilvC+ lysogen of an ilvC- strain gave rise, after heat induction of the lysogen, to transducing particles which carried the wild-type allele of the cya-marker. Further experiments have shown that the lambda defective ilvC phages were able to cotransduce a rho-15ts mutation as well as a rep-5 mutation. Hence, the order of the clockwise excision of the ilv cluster was found to be ilvC-rho-rep-cya. Enzyme levels in strains carrying the lambda defective ilvC phages indicated the the ilvC gene was not altered by the insertion of lambda into the ilv cluster. The isolation and digestion of lambda defective ilvC DNA by EcoRI and HindIII restriction endonucleases demonstrated that the specialized transducing phages carried part of the genome from the E. coli K-12 chromosome.     

Isolation of P22 Specialized Transducing Phage following F''-Episome Fusion

A P22 specialized transducing phage has been constructed which carries the structural gene for aspartate transcarbamylase (ATCase). This gene (pyrB) was first brought close to the P22 attachment site by fusing an F' pyrB⁺ episome to an F' prolac episome which carries a P22 prophage attachment site. A prophage was added to these fused F' episomes and the lysogen was UV-induced. The specialized transducing phage was isolated from the resulting lysate. The phage also carries argI, the structure gene for ornithine transcarbamylase.     

The isolation and characterization of plaque-forming arabinose transducing bacteriophage lambda.

Non-defective arabinose transducing phage, λpara, were isolated in two steps: first, Escherichia coli strains containing rare insertions of λ DNA into the arabinose C or B genes were selected; and second, these lysogens were induced and transducing phage were selected from the resulting lysates. The approximate location of the bacterial substitution on the phage and the ara gene content of the substitution were determined genetically. The precise location of the substitution was determined by electron microscopy of DNA heteroduplexes.Transducing phage, derived from the strain possessing λ inserted into the araC gene, carried part of the araC gene, the ara regulatory region, and all of the araB gene. Transducing phage, derived from eight independent strains possessing λ inserted in the same orientation and in the same position in the araB gene, carried a portion of the araB gene, the ara regulatory region and all of the araC gene. In these nine cases the ara DNA on the phage was immediately adjacent to the normal phage attachment site, indicating that the transducing phage were formed by the same type of abnormal excision which produces gal or bio transducing λ phage. The relative orientations of ara and phage genes were deduced from the topology of such excisions. One anomalous transducing phage was also characterized.     

Fine structure analysis of the threonine operon in Escherichia coli K-12.

Summary A fine structure analysis of the threonine operon in Escherichia coli K-12 was performed by deletion mapping. Lambda transducing bacteriophages carrying various parts of the threonine operon were isolated from strains in which the lacZ gene was fused to a thr gene. We tested for recombination between deletions of the threonine promotor extending into the threonine operon, carried by the phage, and bacterial thr auxotrophs. The relative order of thrO (operator) mutations was established. We propose that an operator region is located between a promoter region and the structural genes. Mutations leading to the desensitization of the aspartokinase I-homoserine dehydrogenase I towards threonine were localized in two different regions of the thrA gene.     

Glutamine synthetase subunit mixing and regulation in Bacillus subtilis partial diploids

A specialized transducing phage, SP beta c2 dglnA2, of Bacillus subtilis was used to construct partial diploids with various glutamine auxotrophs. The overproduction of manganese-stimulated glutamine synthetase no longer occurred in the diploids. The kinetics of heat inactivation of the enzyme extracted from two diploids suggests that there was subunit mixing.     

Isolation and Characterization of a Generalized Transducing Bacteriophage for Acinetobacter

A series of bacteriophages which grow in various strains of Acinetobacter have been isolated. One of these, phage P78, which forms turbid plaques on Acinetobacter strain 78 is specific for this particular host and fails to attack 389 other independently isolated strains of Acinetobacter. Phage P78 appears to be a temperate phage which lysogenizes its host. Various agents such as N-methyl-N'-nitro-N-nitrosoguanidine, diethyl sulfate, mitomycin C, and ultraviolet light are effective inducers of the lysogen. Phage lysates of wild-type cells are capable of transducing auxotrophs of strain 78 to prototrophy at frequencies ranging from 0.3 x 10(-7) to 34 x 10(-7) per plaque-forming unit adsorbed. To date, no linkage has been detected between any of the markers studied in two-factor crosses. Donor phage grown in one particular mutant, strain 78 (arg-1), has been shown to give rise to significantly higher transduction frequencies than when phage is grown on wild-type or other auxotrophic strains. Phage P78 is rapidly adsorbed to its bacterial host and has a latent period of 25 min, and infection results in a burst size of approximately 50. Some of the physical properties of phage P78 and its DNA are described.     

Cotransduction and Cotransformation of Genetic Markers in Bacillus subtilis and Bacillus licheniformis

Bacteriophage SP-15, a large generalized transducing phage of Bacillus, was compared with phages PBS-1 and SP-10 for the ability to cotransduce pairs of genetic markers exhibiting different degrees of linkage. When auxotrophs of B. subtilis W-23 were used as recipients, SP-15 and PBS-1 effected a much higher frequency of cotransduction than did SP-10 with markers that were not closely linked. With more closely linked loci, the differences were not as great. SP-15 cotransduced linked markers at a higher mean frequency than PBS-1, suggesting that SP-15 is able to transfer a larger fragment of the Bacillus genome than any phage heretofore described. The frequency of the joint transfer of genetic markers in B. licheniformis was lower via transforming deoxyribonucleic acid than by transduction with phage SP-10. The availability of three procedures for genetic exchange-transduction by SP-15 and SP-10 as well as transformation-each of which reveals a different degree of linkage, makes B. licheniformis 9945A especially amenable to genetic analysis.     

Interactions of Tn7 and temperate phage F116L of Pseudomonas aeruginosa

Summary Tn7 insertions into the genome of F116L, a Pseudomonas aeruginosa generalized transducing phage, were isolated by repeated cycles of transducing phage, were of strains lysogenic for F116cts mutants with selection for trimethoprim resistance (Tp¹). Two non-defective F116Lcts:Tn7 phage were characterized. They have reduced plaquing ability, produced non-lysogenic Tp^r transductants, and have yielded a deletion mutant of the phage genome upon selection for plaque formation in single infection. F116L DNA is circularly permuted and terminally redundant. A circular restriction map of 61.7 kb has been defined, and a cleavage site common to many enzymes has been identified at coordinate 23.3 kb on the map. It is presumed that this site represents the sequence for the initiation of DNA encapsidation by a headful packaging mode. The Tn7 insertion targets and a 13.4 kb deletion define regions of the F116L genome non-essential for either vegetative growth or lysogenization. The restriction map of Tn7 has been determined for five enzymes. Non-lysogenic Tp^r transuctants reveal a Tn7 insertion hot-spot in the P. aeruginosa genome.     

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: identification of different types of specialized transducing particles.

The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanism. Phage lysates produced by induction of lysogenic strains contain very high frequencies of supQ newD- and proA,B-specialized transducing particles (10(-2)/PFU and 10(-3)/PFU, respectively), most of which are produced by independent aberrant excision events of various types. In a model, 12 different modes of transduction mechanisms were characterized by: (i) the structure of the specialized transducing genomes after injection into a new host cell, i.e., linear or circular, and (ii) the requirements for the transduction process, i.e., host recombination functions, phage integration functions, or presence of a prophage. By using different recipient strains and phage helper strains, it was possible to show that most specialized transducing particles (ca. 99%) contain linear genomes that cannot circularize upon injection into a new host cell and that require the presence of an integrated prophage as a site for a recombinational event to give rise to a transductant. Only 0.1% of all specialized transducing particles were shown to transduce by integration, suggesting that transducing genomes containing terminally redundant ends represent only a minor fraction of all transducing particles that are produced. However, it should be pointed out that the frequency (approximately 10(-5)/PFU) of these specialized transducing genomes that can circularize upon injection into a new host cell is as high as or even higher than the frequency of specialized transducing particles of phage lambda. The remaining approximately 1% of all specialized transducing particles can transduce by any one of the other mechanisms described.     

A hotspot of spontaneous and UV-induced illegitimate recombination during formation ofλbio transducing phage

To study the mechanism of spontaneous and UV-induced illegitimate recombination, we examined the formation of theλbio specialized transducing phage inEscherichia coli. Because mostλbio transducing phages have double defects in thered andgam genes and have the capacity to form a plaque on anE. coli P2 lysogen (Spi^? phenotype), we selectedλbio transducing phage by their Spi^? phenotype, rather than using thebio marker. We determined sequences of recombination junctions ofλbio transducing phages isolated with or without UV irradiation and deduced sequences of parental recombination sites. The recombination sites were widely distributed onE. coli bio andλ DNAs, except for a hotspot which accounts for 57% of UV-inducedλbio transducing phages and 77% of spontaneously inducedλbio transducing phages. The hotspot sites onE. coli andλ DNAs shared a short homology of 9 bp. In addition, we detected direct repeat sequences of 8 by within and near both thebio andλ hotspots. ArecA mutation did not affect the frequency of the recombination at the hotspot, indicating that this recombination is not a variant ofrecA-dependent homologous recombination. We discuss a model in which the short homology as well as the direct repeats play essential roles in illegitimate recombination at the hotspot.     

Isolation and characterization of a lambda transducing bacteriophage carrying the cysE gene of Escherichia coli K-12

A specialized lambda transducing phage carrying the cysE and gpsA genes of E. coli K-12 has been isolated. The transducing phage has been separated from the helper phage on equilibrium gradients and has been shown to be defective. Evidence is presented that the phage kil gene is not expressed.     

Gal transduction by phage lambda: on the origin and nature of LFT transducing genomes

Summary There are at least two classes of transducing particles made on the induction of normal lysogens: the first is capable of transducing by the insertion of the whole transducing genome into the host chromosome, so its genome must be capable of circularizing; the second transduces less well by insertion—perhaps not at all; if it does not transduce by insertion then its genome need not be linear.The formation of a transducing genome can be accomplished in three steps: (a) breaking the lysogenic bacterial chromosomes in two places, (b) joining the fragment ends together to form a circular structure, (c) opening the circle (by ter) to form a linear genome. If the resultant structure meets the requirements for packaging, it may be formed into a transducing phage, like a bougus .Any meaningful rearrangement of these steps in which step (b) is omitted or delayed leads to the formation of genomes, which are (1) unable to transduce by insertion (because both of its mature ends are unexposed) and (2) are on the average smaller than genomes which are capable of transducing by insertion (so the resultant transducing phage is less dense). Consequence (2) has been confirmed.We assume that the red function of catalyzes the joining of broken DNA molecules to each other. So red is responsible for rehealing the product of (a) back into a lysogenic chromosome and for catalyzing step (b), the healing of fragment ends into a circular structure. The much elevated level of stable transductants on induction of red ^– lysogens hereby is explained.Supported by grant E-2862 of the U.S.P.H.S. to Dr. Allan Campell.     

Isolation and Characterization of a New Generalized Transducing Bacteriophage Different from Pl in Escherichia coli

A new generalized transducing bacteriophage in the Escherichia coli system was isolated and characterized. This phage, designated D108, makes clear plaques on E. coli K-10, K-12, K-12(P1kc), K-12(D6), B/r, C, and 15 T(-), and Shigella dysenteriae. The plaque of phage D108 is larger in size than that of phage P1kc. Electron-microscopic observation revealed that phages D108 and P1kc are morphologically different from each other, suggesting that phage D108 belongs to a phage group different from phage P1. The fact that all of the 10 markers tested were transduced by phage D108 indicates that this phage is a generalized transducing phage in the E. coli system. The transduction frequency by phage D108 of chromosomal markers and of a drug resistance factor (R factor) ranged from 2 x 10(-6) to 3 x 10(-8) and 3 x 10(-9) to 6 x 10(-10) per phage, respectively. The cotransduction frequency of the thr and leu markers was 2.8% for phage P1kc and 1.5% for phage D108. The CM and TC markers (chloramphenicol-resistant and tetracycline-resistant markers, respectively) of the R factor were not cotransduced by phage D108, but the markers were generally cotransduced by phage P1kc. The results suggest that the transducing particle of phage D108 contains a smaller amount of host deoxyribonucleic acid than does phage P1kc.     

A novel assay for illegitimate recombination in Escherichia coli: Stimulation of λbio transducing phage formation by ultra-violet light and its independence from RecA function

We developed a novel assay system for illegitimate recombination, in which the frequency of the formation of λ Spi⁻ phages formed during prophage induction was measured with an E. coli P2 lysogen as the indicator bacteria. Since almost all of the λ Spi⁻ phages thus detected contain attR, they have essentially the same structures as λbio transducing phages, indicating that this assay system enables us to detect specialized transducing phages that produce heterogenote transductants, thus ignoring the occurrences of docL and docR particles which carry only one cohesive end. The following results on the formation of specialized transducing phages have been obtained by this assay system to date. (1) Irradiation with UV light greatly enhanced the formation of λ Spi⁻ phages. (2) Treatments with other DNA-damaging agents also enhanced the formation of λ Spi⁻ phages. (3) Illegitimate recombination during prophage induction does not require the RecA function, indicating that enhancement of λ Spi⁻ phage formation is not controlled by the SOS regulatory system. (4) Preliminary results suggested that DNA gyrase is involved in the formation of λ Spi⁻ phage during prophage induction. Since the above results were consistent with most of the previous observations on the illegitimate recombination in other systems, the Spi⁻ assay system can provide important clues to the mechanism of illegitimate recombination.     

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.     

Characterization of SE1, a new general transducing phage of Salmonella typhimurium

A transducing phage, SE1, which is able to infect Salmonella typhimurium was isolated from a Salmonella enteritidis strain. SE1 is a temperate phage which is heteroimmune with respect to phages P22, L, KB1 and ES18. It is similar in morphology and size to phages P22, L and KB1 and is serologically related to phages P22 and L but not to KB1. Efficiencies of generalized transduction effected by phage SE1 are similar to those for P22HT (int7), a mutant which mediates a high frequency of chromosomal gene transduction. The lengths of chromosomal DNA transduced by SE1 and P22HT (int7) are similar. Furthermore, the SE1 prophage does not exclude the transducing particles from cells it has lysogenized; consequently it is possible to use both SE1 lysogens and non-lysogenic strains as recipients in SE1-mediated transduction experiments, and obtain similar transduction efficiencies. However, the SE1 prophage gives rise to a lysogenic conversion that decreases the rate of adsorption of SE1 and L phages by about 50%, but does not affect adsorption of P22. Altogether these results suggest that phage SE1 may be a useful tool in the genetic manipulation of S. typhimurium.