Transducing Particles Research Articles

A quantitative model for nonrandom generalized transduction, applied to the phage P22-Salmonella typhimurium system.

A mathematical model for nonrandom generalized transduction is proposed and analyzed. The model takes into account the finite number of transducing particle classes for any given marker. The equations for estimation of the distance between markers from contransduction frequency data are derived and standard errors of the estimates are given. The obtained relationships depend significantly on the number of classes of transducing fragments. The model was applied to estimate the number of transducing fragment classes for a given marker in transduction with phage P22 of Salmonella typhimurium. It was found that the literature data on frequencies of contransduction in crosses with mutual substitution of selective and nonselective markers can be rationalized most accurately by assuming that the mean number of classes is equal to 2. An improved method for analysis of cotransduction data is proposed on the basis of our model and the results of calculation. The method relies on solving a set of algebraic equations for cotransduction frequencies of markers located within one phage length. The method allows a relatively precise determination of distances between markers, positions of transducing particle ends and deletion or insertion lengths. The approach is applied to the trp-cysB-pyrF and aroC-hisT-purF-dhuA regions of the Salmonella typhimurium chromosome.

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.

In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries.

Strains of Escherichia coli K-12 were constructed that permitted the amplification of in vitro-packaged recombinant cosmid-transducing particles by in vivo repackaging of recombinant cosmid molecules. Thermal induction of these thermoinducible, excision-defective lysogens containing recombinant cosmid molecules yielded high titers of packaged recombinant cosmids and low levels of PFU. These strains were used to amplify packaged recombinant cosmid libraries of Mycobacterium leprae, Mycobacterium vaccae, Salmonella typhimurium, and Streptococcus mutans DNA. Contiguous and noncontiguous libraries were compared for the successful identification of cloned genes. Construction of noncontiguous libraries allowed the dissociation of desired genes from genes that were deleterious to the survival of a cosmid recombinant and permitted selection for unlinked traits that resulted in a selected phenotype. In vivo repackaging of recombinant cosmids permitted amplification of the original in vitro-packaged collection of transducing particles, storage of cosmid libraries as phage lysates, facilitation of complementation screening, expression analysis of repackaged recombinant cosmids after UV-irradiated cells were infected, in situ enzyme or immunological screening, and facilitation of recovery of recombinant cosmid molecules containing transposon inserts.

Characterization of a defective phage system for the analysis of bacteriophage T4 DNA replication origins

We have developed a defective phage system for the isolation and analysis of phage T4 replication origins based on the T4-mediated transduction of plasmid pBR322. During the initial infection of a plasmid-containing cell, recombinant plasmids with T4 DNA inserts are converted into fully modified linear DNA concatamers that are packaged into T4 phage particles, to create defective phage (transducing particles). In order to select T4 replication origins from genomic libraries of T4 sequences cloned into the plasmid pBR322, we searched for recombinant plasmids that transduce with an unusually high efficiency, reasoning that this should select for T4 sequences that function as origins on plasmid DNA after phage infection. We also selected for defective phage that can propagate efficiently with the aid of a coinfecting helper phage during subsequent rounds of phage infection. which should select for T4 sequences that can function as origins on the linear DNA present in the defective phage. Several T4 inserts were isolated repeatedly in one or both of these selective procedures, and these were mapped to particular locations on the T4 genome. When plasmids were selected in this way from genomic libraries constructed using different restriction nucleases, they contained overlapping segments of the T4 genome, indicating that the same T4 sequences were selected. The inserts in two of the selected plasmids permit a very high frequency of transduction from circular plasmids: these have been shown to contain a special type of T4 replication origin.

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 λ docR 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 λ docL 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

Studies of cosmid transduction in Streptomyces lividans and Streptomyces parvulus.

Cosmid pR4Cl is a derivative of multicopy plasmid pIJ365 which has an insertion of the cos (cohesive end site) region of actinophage R4 [T. Morino, H. Takahashi and H. Saito, Mol. Gen. Genet., 198, 228 (1985)]. When the donor R4 phage was propagated in S. lividans carrying the plasmid, the phage lysate contained transducing particles which encapsulated head-to-tail concatemers of the plasmid DNA. These particles could mediate transfer of the plasmid at a high frequency. We examined conditions that gave a maximum transduction frequency of cosmid pR4Cl. Conditions which depress R4 phage propagation, such as incubation of recipient S. parvulus at a high temperature, improved the frequency. Obviously such conditions minimized the lethal effect of viable phage propogation. The highest transduction frequency obtained so far was around 3×10-3 transductants per infected phage when S. lividans was used as the recipient. This was about 30 per cent of the cosmid transducing particles estimated from the cosmid DNA content in the transducing lysate. The significance of cosmid transduction for gene manipulation in Streptomyces strains is also discussed.

Transduction of multi-copy plasmid pBR322 by bacteriophage Mu.

The temperate bacteriophage Mu transduces the 4363 bp multi-copy plasmid pBR322 at frequencies similar to those of chromosomal markers. Plasmid transducing particles contain DNA molecules of Mu DNA length. Plasmid DNA is transduced as a head-to-tail oligomer that becomes circularized in the recipient cell. The rec system of the donor strain participates in oligomer formation and the rec system of the recipient strain is required for oligomer circularization. Possible mechanisms that may explain the origin of plasmid transducing particles are discussed.

Pac sites are indispensable for in vivo packaging of DNA by phage P22.

F' pro+ plasmids were selected and used as donors to prepare P22 transducing phages. Two types of result were observed. pro+ from type I donors cannot be packaged by wild-type P22 to yield transducing particles unless a prophage pac site is introduced into the plasmid. Transposon Tn10 also allows initiation of packaging. pro+ from type II plasmids can be transduced with the same efficiency as pro+ DNA on the chromosome, indicating that a chromosomal pac site was included when the F' pro+ was excised from the Hfr strain. The usefulness of type I plasmids as a test substrate for pac signals is discussed.

Cosduction: Transduction of Bacillus subtilis with phage ϕ105 using a ϕ105 cosplasmid

Plasmid pC194 can be transduced with phage ϕ105 if a ϕ105 fragment is cloned on it. The use of a phage deletion mutant enhances the frequency with which transducing particles are formed; from <10-6 to about 10-3 per plaque forming unit. Evidence is presented showing the covalent integration of the plasmid into the phage genome and the requirement for recombination. A plasmid (pC0S1) with the cohesive ends from ϕ105 results in a much enhanced formation of transducing particles, about 4%. This plasmid was used as a vector for cloning of E. coli DNA which could then be transduced with ϕ105. It is proposed that this process is called cosduction.

A single copy, transducible system for complementation and dominance analysis in Bacillus subtilis

A complementation and dominance system for Bacillus subtilis has been designed by inserting a fragment of the SPβ prophage into the bifunctional replicon pHV14. This new plasmid, pFH7, integrates into the SPβ prophage genome upon transformation into B. subtilis. When integrated, pFH7 provides a region of homology with pBR322 related plasmids allowing integration of these plasmids within the SPβ prophage upon transformation into B. subtilis. Thus, plasmid pMB9 carrying a fragment of the B. subtilis chromosome was found to integrate either at its region of homology with its chromomal fragment or in the SPβ prophage when transformed into B. subtilis. Upon induction of SPβ from such transformants, specialized transducing particles were obtained that carried both plasmid pFH7 and plasmid pMB9 with B. subtilis insert. When induced, lysogens carrying only pFH7 give rise to viable, mature phage particles carrying the plasmid.

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.

Packaging signals for phage P22 on the chromosome of Salmonella typhimurium.

Numbers of transducing particles for a set of 28 different chromosomal markers were determined in a lysate of Salmonella phage P22. The results were plotted with regard to the map positions of the transduced loci. Maxima of the resulting curve are interpreted as positions of start signals ("pac-sites") for packaging series on the Salmonella chromosome. 5-6 pac-sites could be found as a minimum estimate.

Insertion of bacteriophage SP beta into the citF gene of Bacillus subtilis and specialized transduction of the ilvBC-leu genes.

We isolated a strain of Bacillus subtilis in which the SP beta c2 prophage is inserted into the citF (succinate dehydrogenase) gene. Defective specialized transducing particles for the ilvBC-leu genes were isolated from phage-induced lysates of this lysogen. We isolated a group of phages that differ in the amount of genetic material they carry from this region. Also, we incorporated mutant ilv and leu alleles into the genomes of several transducing phages. Our phage collection enables us to identify the cistron of new ilv and leu mutations by complementation analysis. In this process we discovered a fourth leu cistron, leuD. Characterization of the phages confirmed the published gene order: ilvB-ilvC-leuA-leuC-leuB; leuD lies to the right of leuB.

Mechanism of pBR322 transduction mediated by cytosine-substituting T4 bacteriophage.

A cytosine-substitution type mutant of bacteriophage T4 (T4dC phage) has been shown to mediate the transfer of plasmid pBR322. The transduction frequency was around 10(-2) per singly infected cell at low multiplicity of infection. The transductants contained either a monomer or multimers of pBR322. The transducing capacity of T4dC phage was resistant to methylmethanesulfonate treatment. The results of Southern blotting experiments have indicated that the pBR322 DNA exists as head-to-tail concatemers in the transducing particles. The mechanism of transfer of pBR322 mediated by T4dC phages is discussed.

Specialized transduction of the ilvD-thyB-ilvA region mediated by Bacillus subtilis bacteriophage SP beta.

Specialized transducing SP beta particles were found that carried the Bacillus subtilis genes lying to the left of the prophage attachment site. Three classes of transducing particles were differentiated, depending upon whether they carried ilvA only, thyB and ilvA, or ilvD, thyB, and ilvA. Lysates prepared by the induction of strains that carried both a transducing phage and a plaque-forming phage contained the two particles in a ratio of about 1:3,000. When the transducing particles were used to transduce a phage-sensitive auxotrophic strain to prototrophy, some of the transductants carried only the transducing phage genomes which, by themselves, were defective. One putative nondefective transducing phage (for ilvA only) is also described. SP beta can mediate specialized transduction even in the absence of the major (recE) bacterial recombination system.

Chromosomal location of att P7, the recA-independent p7 integration site used in the suppression of Escherichia coli dnaA mutations.

Conjugational and transductional analyses were used to determine the chromosomal location of attP7, the recA-independent integration site used by bacteriophage P7 to suppress host dnaA mutations. The site of integration was found to be between tolC and dnaG. An increase in transduction frequencies was observed for markers surrounding attP7 when P7 was integrated. Under these conditions, all pairs of markers in this region, including those separated by attP7, were cotransduced at frequencies higher than normal, indicating the possible production of P7 specialized transducing particles.

Bacteriophage P22 mutants that alter the specificity of DNA packaging

Bacteriophage P22 cleaves and packages the mature viral chromosome from a concatemeric precursor by sequential headfuls. Generalized transducing particles are formed when occasionally host rather than phage DNA is cut and packaged. Since infected cells contain about as much bacterial DNA as P22 DNA at late times, but most P22 particles contain only viral DNA, the packaging mechanism must be able to choose phage DNA in preference to host DNA as substrate for encapsulation. The HT mutants isolated by Schmieger (1971b, 1972) appear altered in this packaging specificity, since they form transducing particles at substantially increased frequencies. The HT mutants have now been shown to have reduced specificity for pac, the site on P22 DNA at which headful packaging normally initiates. One mutant, HT 12/4, no longer packages any phage DNA at pac, but recognizes a new site on P22 DNA. The HT 12/4 mutant is shown to make a gene 3 protein of altered specificity, suggesting that the gene 3 function may be required to recognize pac. These results indicate that recognition of pac is the mechanism by which phage DNA is packaged in preference to host DNA.

MP13, a generalized transducing bacteriophage for Bacillus megaterium.

The first generalized transducing bacteriophage reported for Bacillus megaterium has been characterized. Optimum conditions for lysate production and transduction procedures were established so that transducing frequencies of 8 x 10(-6) and higher are now possible. The phage, MP13, has a head diameter of 97 nm and a contractile tail (202 by 17 nm) and adsorbs to the periphery of the cell. MP13 was inactivated rapidly at 60 degrees C, but not at 55 degrees C, and was sensitive to toluene, ether, and chloroform. When centrifuged in a neutral CsCl gradient, two bands were observed, a major band of 1.490 g cm-3 and a minor band of 1.482 g cm-3 buoyant density. The major band contained only infective particles, whereas the minor band contained both infective and transducing particles. Phage DNA was resistant to several restriction endonucleases, but yielded 9 fragments with MboI, more than 34 with HindIII, and 7 with BstEII. The molecular weights for the fragments from MboI-BstEII double digests total 97 x 10(9).

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction.

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.

SP02 particles mediating transduction of a plasmid containing SP02 cohesive ends.

SP02 particles that mediate transduction of plasmid pPL1010, a 4.6-megadalton derivative of pUB110 containing an Eco RI endonuclease-generated fragment of SP02 deoxyribonucleic acid that spans the cohesive ends, exhibit three unusual features: the transducing particles have a lower buoyant density than infectious particles; the transduction of pPL1010 occurs at high efficiency; and the transducing activity of the particles is relatively resistant to ultraviolet irradiation when the recipient is recombination proficient. Evidence is presented which indicates that SP02(pPL1010) particles carry the plasmid predominantly as a linear multimer having a molecular mass comparable to that of infectious SP02 deoxyribonucleic acid (ca. 31 megadaltons). The plasmid monomers in the linear multimer appear oriented in the same polarity. The buoyant density difference between infectious and transducing particles appears to be due mainly to the buoyant density difference between pPL1010 (1.699 g/cm3) and SP02 deoxyribonucleic acid (1.702 gm/cm3).