Generalized Transducing Phage Research Articles

The genetic organization of arginine biosynthesis in Pseudomonas aeruginosa

Six loci coding for arginine biosynthetic enzymes in Pseudomonas aeruginosa strain PAO were identified by enzyme assay: argA (N-acetylglutamate synthase), argB (N-acetylglutamate 5-phosphotransferase), argC (N-acetylglutamate 5-semialdehyde dehydrogenase), argF (anabolic ornithine carbamoyl-transferase), argG (argininosuccinate synthetase), and argH (argininosuccinase). One-step mutants which had a requirement for arginine and uracil were defective in carbamoylphosphate synthase, specified by a locus designated car. To map these mutations we used the sex factor FP2 in an improved interrupted mating technique as well as the generalized transducing phages F116L and G101. We confirmed earlier studies, and found no clustering of arg and car loci. However, argA, argH, and argB were mapped on a short chromosome segment (approx. 3 min long), and argF and argG were cotransducible, but not contiguous. N-Acetylglutamate synthase, the enzyme which replenishes the cycle of acetylated intermediates in ornithine synthesis of Pseudomonas, appears to be essential for arginine synthesis since argA mutants showed no growth on unsupplemented minimal medium.

The frequency of P1 transduction of the genes of Escherichia coli as a function of chromosomal position: Preferential transduction of the origin of replication

The frequencies with which the generalized transducing phage P1 transduced 26 selected markers on the E. coli. chromosome were measured. The frequencies were found to vary relative to argH+ = 1 from a maximum of 6.8 near the origin of replication to a minimum of 0.23 for a marker not far from the terminus. The low frequencies obtained for some markers were shown not to result from poor expression under the selective conditions employed. When plotted as a function of marker position on the chromosome the frequencies were found to exhibit a series of peaks and troughs which correspond to those in gene density noted by Bachmann et al. (1976). The possible relationship of these results to the structure of the E. coli chromosome and to the mechanism of generalized transduction are discussed.

Transduction of a Proteus vulgaris strain by a Proteus mirabilis bacteriophage.

Only Proteus vulgaris strain PV127 out of many P. vulgaris, P. morganii and Providence strains was transduced to kanamycin resistance by high-frequency transducing variants, 5006MHFTk and 5006MHFTak, of phage 5006M, a general transducing phage for P. mirabilis strain PM5006. The phages adsorbed poorly to strain PV127 and did not form plaques. The transduction frequency of PV127 by these phages was 5 x 10(-8)/p.f.u. adsorbed. Phage 5006M increased the transduction frequencies. Abortive transductants were not detected. Transductants segregated kanamycin-sensitive clones at high frequency and this, together with data from the inactivation of transducing activity of lysates by ultraviolet irradiation, indicated that transduction was by lysogenization. The general transducing property of the phages was not expressed in transductions to auxotrophs of PV127. Transductants (type I) resulting from low multiplicities of phage input adsorbed phage to the same extent as PV127. This suggested a defect in the transducing particles (or host) because single phage 5006M infection converted strain PM5006 to non-adsorption of homologous phage. Type I transductants did not liberate phage, suggesting a defective phage maturation function. Transductants (type II) which arose from higher multiplicities of phage input did not adsorb phage, indicating possible heterogeneity among transducing particles. Phage derived from type II transductants adsorbed poorly to PV127 and transduced it to kanamycin resistance at frequencies similar to those of phages 5006MHFTk and 5006MHFTak, ruling out host-controlled modification as a cause of the low transduction frequencies. This phage transduced PM5006 to antibiotic resistance at high frequencies but generalized transduction was again not detected. It was suggested that general transduction could be performed by particles which, due to a different composition and/or mode of chromosomal integration, made material they carried susceptible to host-cell modification.

Revision of the linkage map of Bacillus subtilis 168: indications for circularity of the chromosome.

A revision of the linkage map of the Bacillus subtilis 168 chromosome has been undertaken with the use of the generalized transducing phage PBS1. The mapping of four new markers (narB1, mtlB1, aroI906, and tre-12) has allowed a determination of the relative orientation of the purB-dal segment and its linkage with the lin markers. The chromosomal segment comprised between the sacQ36 and gtaA12 markers has been linked with the narA1, ctrA1, and sacA321 markers. The recA1 marker has been mapped relative to the thyA and citB17 markers. Indications of linkage have been found between the tre-12 and catA markers and the aroG932 and sacQ36 markers. According to these results, a circular genetic map of the chromosome of B. subtilis 168 is presented. Taken together, the transduction data and the order of marker replication determined by Harford in the accompanying paper support strongly the hypothesis of a symmetrical and fully bidirectional mode of replication for the B. subtilis 168 chromosome.

Genetic analysis of amidase mutants of Pseudomonas aeruginosa.

SUMMARYMutants ofPseudomonas aeruginosa, which differed in amide growth phenotype from the wild-type strain, were subjected to genetic analysis using the generalized transducing phage F116. The map order of some mutational sites was determined by 3-factor crosses in which a mutation in the linked regulator geneamiRwas used as the outside marker to determine the relative order of mutations in the amidase structural geneamiE. Acetamide-positive transductants were recovered in crosses between amidase-negative strains and strains PhB3(PAC377), V2(PAC353) and V5(PAC356) producing mutant amidases which hydrolyse phenylacetamide and valeramide but not acetamide. Some recombinants carried the mutationamiE16 determining the properties of the mutant B amidase produced by strain B6(PAC351) from which both PhB and V class mutants were derived, while other recombinants produced A amidase determined by the wild-typeamiEgene.

The order of replication of chromosomal markers in Pseudomonas aeruginosa strain 1. I. Marker frequency analysis by transduction.

SUMMARYThe generalized transducing phage F116 has been used to prepare lysates from fast- and slow-growing cultures ofPseudomonas aeruginosastrain 1. These lysates have been used to transduce a number of auxotrophic markers to prototrophy and the ratios of the numbers of transductants obtained with each lysate have been determined. Since the markers are those which have been mapped by conjugation in previous studies it has been possible to compare the ratios obtained for each marker with the relative position of the marker on the chromosome map. If the assumption is made that there is only one circular chromosome inP. aeruginosastrain 1 it is possible to suggest a way in which two apparently unlinked segments might be joined together. It is also possible to suggest that the chromosome replicates sequentially in two directions from a fixed origin.

Transduction with integration-defective mutants of Salmonella typhimurium bacteriophage KB1.

Phage KB1 has gained use as a generalized transducing phage for Salmonella typhimurium, including strains resistant to phage P22. Integration-defective mutants of KB1 have now been isolated; one of these, int-1, is recommended for transduction when nonlysogenic recombinants are desired.

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.

Structure of a new pyrimidine from Bacillus subtilis phage SP-15 nucleic acid.

THE unusual modified base1 of the nucleic acid of the large, generalized transducing phage of Bacillus subtilis SP-15 was predicted from the low denaturation temperature of the native nucleic acid. The base was detected as a fifth ultraviolet-absorbing component once the nucleic acid had been hydrolysed with formic acid; it partially replaces the thymine and represents 12 mol per cent of all the bases1. It is responsible for the low Tm of the nucleic acid, since once all other unusual components have been removed, the remaining material melts at the same temperature as the native nucleic acid. We report here the isolation and characterization of this new pyrimidine.

Unique properties of nucleic acid from Bacillus subtilis phage SP-15.

BACTERIOPHAGE SP-15 is a large, generalized transducing phage of Bacillus subtilis and B. licheniformis. The DNA extracted from the purified phage has unusual physical properties: its melting temperature (Tm) in 0.15 M NaCl, 0.015 M sodium citrate (SSC) is very low, 61.5° C and its buoyant density in neutral CsCl is very high, 1.761 g/ml.1. We describe here additional unique features of SP-15 DNA: the presence of (1) a new modified pyrimidine which partially replaces the thymine; (2) a compound which reacts with orcinol as a pentose; (3) alkali-sensitive phosphodiester bonds; and (4) glucose.

A novel transducing phage. Its role in recognition of a possible new host-controlled modification system in Pseudomonas aeruginosa.

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.

A New Transducing Phage Related to P22 of Salmonella typhimurium

SUMMARY Properties of a new general transducing phage, PSA68, isolated from a commercial digestive are described in comparison with other transducing phages, L, MG40, and P22. The morphology, the latent period, and the average burst size of PSA68 were indistinguishable from those of P22. This phage most resembled L serologically and MG40 in immunity properties; P22 was dominant over the other three phages, and PSA68 and MG40 over L. The buoyant density of PSA68 was 1.510 g./cm.3, identical to L and MG40, that of P22 being 1.520 g./cm3. The relative number of abortive transductants produced by the nonmotile double mutants was not significantly different among the four phages, though the transduction frequencies per p.f.u. were different from each other, implying the presence of genetically identical lengths of chromosome fragments.

Appearance of transducing activity in P1-infected Escherichia coli

CP-T1 is the only described generalized transducing bacteriophage for the intestinal pathogen Vibrio cholerae, yet many of its basic biological parameters remain unknown. Due to low frequencies of transduction and pseudolysogen formation, CP-T1 has not been widely used as a genetic tool. To overcome these limitations, we have isolated a conditional mutant of CP-T1 that exhibits temperature-sensitive plaque formation. Several biological properties of CP-T1ts were determined, including its restrictive temperature, adsorbance profile to host cells, burst time, and burst size. Based on these properties, an optimized transduction protocol was designed which resulted in several fold higher transduction frequencies for a variety of genetic markers from a number of chromosomal loci. Generalized transduction was also demonstrated between classical and E1 Tor biotype strains of V. cholerae.

Growth and cultivation of the unusual generalized transducing Bacillus bacteriophage SP-15.

Additional properties of SP-15, a generalized transducing bacteriophage notable for the ability to transfer an unusually large fragment of deoxyribonucleic acid (DNA) to Bacillus subtilis and B. licheniformis, are presented together with improved methods that enhance its utility. Simple means have been found to provide the rigid control over moisture that is necessary for the assay of plaque-forming units (PFU). Reproducible procedures for propagating transducing phage, which depend upon an appropriate mixing of PFU with uninfected bacteria, have replaced less reliable methods that utilized infected spores. Transduction of B. subtilis W-23 increased linearly when MgSO(4) in recipient cell-SP-15 mixtures was increased from 0.005 to 0.03 m. Methods have been developed that protect SP-15 from the damaging effects of CsCl and of osmotic shock subsequent to dilution. Evidence that the PFU and transducing particles of lysates decay at the same slow rate during extended storage suggests that the decay is a result of damage to protein rather than to DNA. One-step growth experiments, in which SP-15 was propagated on B. subtilis W-23-S(r)/1 mg, indicated a latent period of 100 min, a rise period of 60 min, and a burst size of 25 to 34 PFU per infected cell. These findings suggest explanations for some of the technical difficulties SP-15 has presented.

Transduction of the nitrogen-fixation genes in Klebsiella pneumoniae.

The bacteriophage P1 infects and functions as a generalized transducing phage for nitrogen-fixing strains of the coliform bacterium Klebsiella pneumoniae. Bacterial mutants (nif(-)) unable to grow on molecular nitrogen as a nitrogen source were found to be deficient in nitrogenase activity as assayed by the conversion of acetylene to ethylene. These mutants regained normal nitrogenase activity and the ability to grow on N(2) after transduction with lysates of P1 phage prepared from wild-type bacteria. Transductional analysis with P1 revealed that several nif genes are located on the genetic linkage map of Klebsiella near the histidine operon.

Isolation and characterization of a new generalized transducing bacteriophage different from P1 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.

Growth and Cultivation of the Unusual Generalized Transducing Bacillus Bacteriophage SP-15

Additional properties of SP-15, a generalized transducing bacteriophage notable for the ability to transfer an unusually large fragment of deoxyribonucleic acid (DNA) to Bacillus subtilis and B. licheniformis, are presented together with improved methods that enhance its utility. Simple means have been found to provide the rigid control over moisture that is necessary for the assay of plaque-forming units (PFU). Reproducible procedures for propagating transducing phage, which depend upon an appropriate mixing of PFU with uninfected bacteria, have replaced less reliable methods that utilized infected spores. Transduction of B. subtilis W-23 increased linearly when MgSO(4) in recipient cell-SP-15 mixtures was increased from 0.005 to 0.03 m. Methods have been developed that protect SP-15 from the damaging effects of CsCl and of osmotic shock subsequent to dilution. Evidence that the PFU and transducing particles of lysates decay at the same slow rate during extended storage suggests that the decay is a result of damage to protein rather than to DNA. One-step growth experiments, in which SP-15 was propagated on B. subtilis W-23-S(r)/1 mg, indicated a latent period of 100 min, a rise period of 60 min, and a burst size of 25 to 34 PFU per infected cell. These findings suggest explanations for some of the technical difficulties SP-15 has presented.

ES18, a general transducing phage for smooth and nonsmooth Salmonela typhimurium

Bacteriophage ES18, derived from typing phage 18 of the Salmonella typhimurium phage-typing scheme of Callow, is hear-labile and serologically and morphologically unlike phage P22 (a typical phage of Boyd's group A). Unlike phage P22 it attacks all classes of nonsmooth, as well as smooth, derivatives of LT2 lines “cured” of prophage Fels 2. A host-range variant, ES18. h1, overcomes the (partial) resistance conferred by lysogeny for Fels 2. Phage ES18 does not lysogenize pro deletion mutants lacking the preferred P22 prophage integration site. Lysogeny for P22 confers immunity to ES18, but ES18 lysogens are still sensitive to P22. Phage ES18 is a general transducing phage, effecting both complete and abortive transduction. Experiments to demonstrate transduction must take account of the very slow adsorption rate of this phage. The observed frequency of (complete) transduction of various loci by ES18 was lower, by a factor of 10–100, than the corresponding rates of transduction by phage P22. Lysogeny of the recipient for P22 increased the yield of transductants evoked by ES18 (though it lowered the yield of P22 transductants). For several pairs of linked loci the rates of cotransduction by ES18 and by P22 were about the same; this suggests that the chromosome fragments present in transducing particles of the two phages are of similar lengths. The activity of ES18 and ES18. h1 on nonsmooth mutants makes them useful for the transductional mapping of rfa genes in S. typhimurium.

New generalized transducing bacteriopahge in Echerichia coli.

A new generalized transducing bacteriophage similar to P1 was isolated from Salmonella oranienburg.

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.