Transducing Particles Research Articles

Fis is required for illegitimate recombination during formation of lambda bio transducing phage.

Specialized transducing particles of phage lambda are formed by illegitimate recombination during prophage induction. We examined the effects of an Esherichia coli int, xis, himA, himD, or fis mutation on illegitimate recombination during formation of lambda Spi- phage, a class of lambda bio transducing phage. This type of phage is distinguishable from the docL and docR particles, which contain one cohesive end and are formed by cutting of the cos site, by plaque formation of lambda bio on Escherichia coli P2 lysogens. The yields of lambda Spi- phage in the int, xis, int-xis deletion, and b2 deletion mutants were about 50- to 200-fold higher than that of the wild-type prophage when bacteria were irradiated with UV light. This result indicates that Int and Xis functions, and the att site, are not required for illegitimate recombination. The yield of lambda Spi- phage in the himA, himD, or fis mutant carrying lambda delta int-xis prophage was 2.6-, 3.3-, or 17-fold lower, respectively, than that in the wild-type bacteria under UV irradiation. Analysis of the nucleotide sequences of the junctions of the transducing phages indicates that recombination at the hotspots, as well as at non-hotspots, takes place between short homologous sequences. Because the growth of infecting phages was not suppressed by the himA, himD, or fis mutation, we conclude that Fis is required, but IHF is only partially required, for short-homology-dependent illegitimate recombination during the formation of lambda bio transducing phage.

Transduction of low-copy number plasmids by bacteriophage P22.

The generalized transducing bacteriophage of Salmonella typhimurium, P22, can transduce plasmids in addition to chromosomal markers. Previous studies have concentrated on transduction of pBR322 by P22 and P22HT, the high transducing mutant of P22. This study investigates the mechanism of P22HT transduction of low-copy number plasmids, namely pSC101 derivatives. We show that P22HT transduces pSC101 derivatives that share homology with the chromosome by two distinct mechanisms. In the first mechanism, the plasmid integrates into the chromosome of the donor by homologous recombination. This chromosomal fragment is then packaged in the transducing particle. The second mechanism is a size-dependent mechanism involving a putative plasmid multimer. We propose that this multimer is formed by interplasmidic recombination. In contrast, P22HT can efficiently transduce pBR322 by a third mechanism, which is independent of plasmid homology with the chromosome. It has been proposed that the phage packages a linear concatemer created during rolling circle replication of pBR322, similar in fashion to phage genome packaging. This study investigates the role of RecA, RecD, and RecF recombination proteins in plasmid/plasmid and plasmid/chromosome interactions that form packageable substrates in the donor. We also examine the resolution of various transduced plasmid species in the recipient and the roles of RecA and RecD in these processes.

A Salmonella phage-P22 mutant defective in abortive transduction.

In the course of a lytic infection the Salmonella phage P22 occasionally encapsulates bacterial DNA instead of phage DNA. Thus, phage lysates include two classes of viral particles. Phage particles carrying bacterial DNA are referred to as transducing particles and deliver this DNA to a host as efficiently as particles carrying phase DNA. Once injected, the transduced DNA can either recombine with the recipient chromosome to form a "complete" transductant, or it can establish itself as an expressible, nonreplicating genetic element and form an "abortive" transductant. In this work, we describe a P22-phage mutant with reduced ability to form abortive transductants. The mutation responsible for this phenotype, called tdx-1, was found as one of two mutations contributing to the high-transducing pheno-type of the P22-mutant HT12/4. In addition, the tdx-1 mutation is lethal when combined with an erf am mutation. The tdx-1 mutation has been mapped to a region of the P22 genome that encodes several injected proteins and may involve more than one mutant locus. The phenotypes of the tdx-l mutation suggest that the Tdx protein(s) normally assist in the circularization of the P22 genome and also contribute to the formation of DNA circles thought to be required for abortive transduction.

A novel adenovirus-adeno-associated virus hybrid vector that displays efficient rescue and delivery of the AAV genome.

Adenovirus and adeno-associated virus (AAV) are eukaryotic DNA viruses being developed as vectors for human gene therapy. The strengths of each system have been exploited in a novel vector that is based on an adenovirus-AAV hybrid virus incorporated into a plasmid-based molecular conjugate. Efficient rescue and replication of the recombinant AAV genome in this hybrid required transient expression of rep. This feature was incorporated into the transducing particle by conjugating a rep expression plasmid to the hybrid virus through a polylysine bridge. The resulting particle is an attractive vehicle for gene therapy because it is easily manufactured and capable of efficiently transducing cells with the end result being rescue and replication of the recombinant AAV genome. This particle is also useful in the production of recombinant AAV resulting in yields 10-fold greater than that achieved with transfection-based protocols.

A Discontinuous Headful Packaging Model for Packaging Less Than Headful Length DNA Molecules by Bacteriophage T4

Bacteriophage T4 and other double-stranded DNA-containing bacteriophages package DNA by the classical headful packaging mechanism. In this mechanism, the packaging machinery cuts a DNA concatemer and packages a single unit length genome within the viral capsid. The length of the packaged DNA molecule is determined by the size of the viral capsid. Surprisingly, during large DNA cloning experiments, we observed that the in vitrophage T4 packaging system can package and transduce DNA molecules that are much smaller than the T4 headful size. We analyzed this phenomenon by using defined plasmid DNAs as substrates for in vitropackaging. The data showed that phage T4 can successfully package and transduce 4 to 29 kb plasmid DNA molecules. When two plasmid DNAs with different antibiotic markers were added to the packaging reaction mixture, transductants that are resistant to both the antibiotics were obtained, suggesting that both the plasmid DNAs are packaged within the same head. Analysis of the transducing particles by equilibrium CsCl density-gradient centrifugation showed that the particles have the same density as the wild-type phage. That the less than headful length molecules were not converted to T4 headful length prior to packaging was established by a number of independent approaches. Finally, unit length plasmid DNA molecules of appropriate size were isolated from the in vitropackaged particles. Based on these data, we propose a discontinuous headful packaging model for packaging less than headful length molecules. In this model, the packaging machinery packages the first available less than headful length DNA molecule and generates a partially full head. The partially full head then reinitiates packaging on a second DNA molecule. This process continues until the head is filled with DNA.

Effects of Suspended Particulates on the Frequency of Transduction among Pseudomonas aeruginosa in a Freshwater Environment

Transduction has been shown to play a significant role in the transfer of plasmid and chromosomal DNA in aquatic ecosystems. Such ecosystems contain a multitude of environmental factors, any one of which may influence the transduction process. It was the purpose of this study to show how one of these factors, particulate matter, affects the frequency of transduction. In situ transduction rates were measured in lake water microcosms containing either high or low concentrations of particulate matter. The microcosms were incubated in a freshwater lake in central Oklahoma. Transduction frequencies were found to be enhanced as much as 100-fold in the presence of particulates. Our results suggest that aggregations of bacteriophages and bacterial cells are stimulated by the presence of these suspended particulates. This aggregation increases the probability of progeny phages and transducing particles finding and infecting new host cells. Consequently, both phage production and transduction frequencies increase in the presence of particulate matter.

Densovirus of Aedes aegypti as an Expression Vector in Mosquito Cells

We have constructed an infectious DNA clone containing the genome of Aedes aegypti densovirus (AeDNV) in a bacterial plasmid. When this clone was transfected into Aedes albopictus C6/36 mosquito cells, the AeDNV genome rescued from the plasmid and replicated as the wild-type virus. To investigate the cloned virus as an expression vector, the reporter gene encoding β-galactosidase (β-gal) was inserted into four large open reading frames (ORF) observed in the AeDNV genome. When these recombinant constructs were transfected into Aedes albopictus C6/36 cells, the β-gal was expressed efficiently from the right ORF (encoding capsid proteins, Vps) and the mid ORF (encoding putative nonstructural protein 2). A low level of expression was found from the left ORF (encoding nonstructural protein 1, NS1), and no expression was detected from the ORF observed on the minus strand of the AeDNV genome. The expression from the right, mid, and left ORFs can be trans-activated with NS1. A putative nuclear targeting sequence observed in the N-terminus of the AeDNV Vps is presumed to be responsible for transport of the chimeric β-gal into nucleus. The recombinant AeDNV genomes (carrying the β-gal gene) supplied with the AeDNV capsid proteins can be packaged into infectious transducing particles. Our results indicate that the genome of AeDNV can serve as a vector for delivery and expression of foreign genes in mosquito cells with subsequent targeting of the product to the desired cell compartment.

5187061 Transducing particles and methods for their production: Neal I Gutterson, William T Tucker, Paul K Wolber assigned to DNA Plant Technology Corporation

5187061 Transducing particles and methods for their production: Neal I Gutterson, William T Tucker, Paul K Wolber assigned to DNA Plant Technology Corporation

Bacteriophage P22 transduction of integrated plasmids: single-step cloning of Salmonella typhimurium gene fusions

Transcriptional fusions to Salmonella typhimurium chromosomal genes were constructed by integration of a suicide fusion vector into the chromosome by homologous recombination with random cloned chromosomal fragments. We describe here a transductional method using the generalized transducing phage of S. typhimurium, P22, to clone these fusions directly from the bacterial chromosome, in a single step, without the use of restriction enzymes. In this transduction, the phage packages the chromosomal fragment containing the integrated plasmid. Once introduced into the recipient, the plasmid circularizes by homologous recombination between the duplicated region determined by the cloned fragment. Although RecA mediates the majority of these events, the plasmid can circularize in a recA recipient. However, in this case, the event occurs at a much lower frequency and only when the transduction is done at a high multiplicity of infection. In addition to integrated fusion constructs, we also show that autonomously replicating low-copy-number plasmids can be transduced. In this case, transduction is dependent on homologous recombination between the plasmid and the donor chromosome via cloned sequences, in which the transducing particle effectively traps the integrated plasmid.

DNA sequences necessary for packaging bacteriophage T3 DNA

A recombinant plasmid, pUCE1-TR, carrying a target for processing of the concatemer joint (TR) and sequences to the left of the target (E1), is efficiently packaged into transducing particles during T3 phage infection. Using this plasmid packaging/transduction system, the minimal sequences necessary for packaging of T3 DNA were determined. The TR sequence contains the targets for initiation cleavage and termination cleavage of concatemer processing ( pacCR and pacCL, respectively). A plasmid lacking pacCL was packaged as efficiently as pUCE1-TR but one deleted for pacCR was packaged at a very low efficiency, showing that pacCR is essential for production of transducers but that pacCL is dispensable. DNA from transducing particles carrying a recombinant plasmid lacking pacCL or pacCR had the same right or left end as T3 DNA, respectively, but its other end was not unique. In the absence of pacCL, packaging is initiated from the DNA end created by cleavage at the pacCR and terminated at any sequence after packaging a headful of DNA. In the absence of pacCR, packaging is initiated from the DNA end created by nonspecific, inefficient cleavage and terminated by cleavage at the pacCL after packaging a headful of DNA. A 23-bp segment flanking the site where the mature right end is formed was found to support efficient formation of transducing particles. A 53-bp sequence, including a consensus sequence for the promoter for T3 RNA polymerase, was a responsible element in the El sequence for packaging of plasmid DNA. Deletions of the 5′-upstream sequence of the promoter sequence from the left decreased the promoter and packaging activities in parallel, but with those of the 3′-downstream sequence from the right, the packaging activity was impaired before the promoter activity, indicating that transcription from the promoter is necessary but not sufficient for T3 DNA packaging.

Rapid mapping in Salmonella typhimurium with Mud-P22 prophages.

A new method for mapping mutations in the Salmonella typhimurium chromosome is described and applied to the localization of novel regulatory mutations affecting expression of the nirB (nitrite reductase) gene. The mapping technique is also illustrated by the mapping of mutations in genes affecting carbohydrate catabolism and biosynthetic pathways. The new mapping method involves use of the hybrid phage MudP and MudQ (together referred to as Mud-P22), originally constructed by Youderian et al. (Genetics 118:581-592, 1988). This report describes a set of Mud-P22 lysogens, each member of the set containing a different Mud-P22 insertion. The insertions are scattered along the entire Salmonella genome. These lysogens, when induced by mitomycin C, generate transducing lysates that are enriched (45- to 1,400-fold over the background, generalized transducing particle population) for transducing particles containing bacterial DNA that flanks one side of the insertion. We demonstrate that within the set of lysogens there can be found at least one Mud-P22 insertion that enriches for any particular region of the Salmonella chromosome and that, therefore, all regions of the chromosome are discretely enriched and represented by the collection as a whole. We describe a technique that allows the rapid and facile determination of which lysate contains enriched sequences for the repair of a mutant locus, thereby allowing the determination of the map position of the locus. This technique is applicable to those mutations for which the wild-type allele is selectable. We also describe a procedure whereby any Tn10 insertion can be mapped by selecting for the loss of Tetr.

Selection and characterization of randomly produced mutants of gene V protein of bacteriophage M13.

Gene V protein of bacteriophage Ff (M13, f1, fd) is a master regulator of phage DNA replication and phage mRNA translation. It exerts these two functions by binding to single-stranded viral DNA or to specific sequences in the 5' ends of its target mRNAs, respectively. To study the structure/function relationship of gene V protein, M13 gene V was inserted in a phagemid expression vector and a library of missense and nonsense mutants was constructed by random chemical mutagenesis. Phagemids encoding gene V proteins with decreased biological activities were selected and the nucleotide sequences of their gene V fragments were determined. Furthermore, the mutant proteins were characterized both with respect to their ability to inhibit the production of phagemid DNA transducing particles and their ability to repress the translation of a chimeric lacZ reporter gene whose expression is controlled by the promoter and translational initiation signals of M13 gene II. From the data obtained, it can be deduced that the mechanism by which gene V protein binds to single-stranded DNA differs from the mechanism by which it binds to its target sequence in the gene II mRNA.

Bacteriophage T7 DNA Packaging: I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection

Bacteriophage T7 DNA is a linear duplex molecule with a 160 base-pair direct repeat (terminal redundancy) at its ends. During replication, large DNA concatemers are formed, which are multimers of the T7 genome linked head to tail through recombination at the terminal redundancy. We define the sequence that results from this recombination, a mature right end joined to the left end of T7 DNA, as the concatemer junction. To study the processing and packaging of T7 concatemers into phage particles, we have cloned the T7 concatemer junction into a plasmid vector. This plasmid is efficiently (at least 15 particles/infected cell) packaged into transducing particles during a T7 infection. These transducing particles can be separated from T7 phage by sedimentation to equilibrium in CsCl. The packaged plasmid DNA is a linear concatemer of about 40 x 10(3) base-pairs with ends at the expected T7 DNA sequences. Thus, the T7 concatemer junction sequence on the plasmid is recognized for processing and packaging by the phage system. We have identified a T7 DNA replication origin near the right end of the T7 genome that is necessary for efficient plasmid packaging. The origin, which is associated with a T7 RNA polymerase promoter, causes amplification of the plasmid DNA during T7 infection. The amplified plasmid DNA sediments very rapidly and contains large concatemers, which are expected to be good substrates for the packaging reaction. When cloned in pBR322, a sequence containing only the mature right end of T7 DNA is sufficient for efficient packaging. Since this sequence does not contain DNA to the right of the site where a mature T7 right end is formed, it was expected that right ends would not form on this DNA. In fact, with this plasmid the right end does not form at the normal T7 sequence but is instead formed within the vector. Apparently, the T7 packaging system can also recognize a site in pBR322 DNA to produce an end for packaging. This site is not recognized solely by a "headful" mechanism, since there can be considerable variation in the amount of DNA packaged (32 x 10(3) to 42 x 10(3) base-pairs). Furthermore, deletion of this region from the vector DNA prevents packaging of the plasmid. The end that is formed in vector DNA is somewhat heterogeneous. About one-third of the ends are at a unique site (nucleotide 1712 of pBR322), which is followed by the sequence 5'-ATCTGT-3'. This sequence is also found adjacent to the cut made in a T7 DNA concatemer to produce a normal T7 right end.

Bacteriophage T7 DNA Packaging: II. Analysis of the DNA sequences required for packaging using a plasmid transduction assay

Recombinant plasmids carrying a bacteriophage T7 origin of DNA replication and sequences from the T7 concatemer junction are efficiently packaged into transducing particles during phage infection. With some constructs, as many as 50 transducing particles are produced per infected cell. We have used this plasmid packaging system to determine which T7 DNA sequences are required for the processing and packaging of the plasmid concatemers and to investigate the effects of altering the spacing and orientation of the required sequences. An origin of T7 DNA replication is essential for high-efficiency transduction, presumably to form the plasmid concatemers that are the substrates of the packaging reaction. In addition, two short sequences from the concatemer junction are required, one flanking the site where the right end of T7 DNA is formed (pacR) and the other flanking the site for formation of the left end (pacL). The spacing between pacR and pacL is not important, but the sequences must be positioned in the same orientation on the plasmid. With certain deletions of pacL, the specificity of end formation is reduced but the efficiency of packaging is near normal. Plasmids that contain only one of the two pac sites are packaged at about 10% of the efficiency of those with both sites. The residual packaging of these plasmids results from regeneration of the other packaging site by recombination with T7 phage DNA. To function in plasmid packaging, the sequences from the concatemer junction must be positioned on the plasmid in the same orientation relative to the T7 replication origin as is found in T7 DNA. This apparently results from a requirement for transcription through these sequences in the rightward direction from the T7 promoter that is associated with the replication origin. Such transcription from another T7 promoter (phi 10), that is not itself a replication origin, allows packaging when the origin is in the opposite orientation.

Transduction of a plasmid containing the bacteriophage D3 cos site in Pseudomonas aeruginosa

Plasmids harboring the cos sequences of bacteriophage D3 can be transferred, by bacteriophage D3, into Pseudomonas aeruginosa by a mechanism which is insensitive to DNase. Transducing activity was separated from the plaque-forming particles by CsCl equilibrium gradient centrifugation. Restriction endonuclease digestion patterns suggest that the transducing particles contain plasmid concatemers.

Regulation of icosahedral virion capsid size by the in vivo activity of a cloned gene product.

Determination of icosahedral virion capsid size can be directly studied during helper-dependent lytic development of satellite P4 because the assembly pathway specified by the P2 helper virus is altered to yield smaller-sized capsids. Size determination (sid) mutations identify a P4-encoded function regulating this process. To determine whether the sid gene product is necessary and sufficient to redirect the assembly pathway, we (i) cloned the sid structural gene in a plasmid vector (pMA30) under the control of an inducible promoter and (ii) constructed a packaging substrate (pMA1), a P4 genome-sized plasmid containing only that region of P4, the cos site, necessary for encapsidation. Superinfection by P2 of a host carrying pMA30 under induced conditions resulted in a shift from large to small capsid production. P2 superinfection of a host carrying the cos plasmid pMA1 plus pMA30 under induced conditions yielded pMA1-transducing particles of P4 capsid size. These cloning-based analyses directly demonstrate that sid protein is the only P4 gene product required for small-capsid size determination. In the absence of the P2 O gene product no capsids of any size are assembled during solo infection by P2. Nevertheless, P2 Oam mutant superinfection of a host carrying pMA1 and pMA30 under induced conditions yielded small P4-sized transducing particles. We therefore propose that (i) the sid gene product competes with the O gene product to determine the assembly of small vs. large capsid sizes and (ii) both gene products probably function as temporary scaffolding proteins.

Packaging and transduction of non-T3 DNA by bacteriophage T3

A defined in vitro system for packaging T3 DNA also packaged other linear DNAs, including T4, λ, and plasmid DNAs. The packaging capacity was determined to be 40 kb (kilobase pairs) by measuring the packaged length of T4 DNA. Packaged λ and plasmid DNAs were injected into host cells to form plaques and transductants, respectively. The yield of transducers increased by using artificially ligated plasmid oligomers. The T3 mutant in gene 3 endonuclease (T3 3 −) packaged plasmid DNA during abortive infection and transduced it into the recipient. Transduction of recombinant plasmids was not affected by the presence of the terminally redundant sequence (TR sequence) but increased by 4 orders of magnitudes when the genetic right-end 2.7-kb sequences, containing gene 19 (El) but lacking TR, were present and by 7 orders when both El and TR sequences were present. However, these sequences did not increase transduction of these plasmids by T7 3 −. Analysis of the structure of transduced plasmid DNAs indicates that transducing particles carry head-to-tail oligomers of plasmid DNA with the same termini as those of T3 genomic DNA. The mechanism of formation of transducing particles is discussed.

Construction and genetic characterization of lambda specialized transducing particles carrying therglB gene ofEscherichia coli K-12

TherglB gene ofEscherichia coli codes for a restriction activity that cleaves the hydroxymethylated DNA of T2 and T4 phages. Earlier mapping data placed the gene at 98.39 min counterclockwise to thehsd operon. Genetic analysis of the in vivo gene fusions with λ fusion-transducing phages established the location of therglB gene next to thehsdS gene of thehsdRMS cluster. The methodology used in this study could be extended to similar in vivo physical mapping of closely linked genes.

Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.

A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.

Small Staphylococcus aureus plasmids are transduced as linear multimers that are formed and resolved by replicative processes

The molecular processes involved in the transduction of small staphylococcal plasmids by a generalized transducing phage, φ11, have been analysed. The plasmids are transduced in the form of linear concatemers containing only plasmid DNA; plasmid-initiated replication is required for their generation but additive interplasmid recombination is not. Concatemers are probably generated by the interaction of one or more phage functions with replicating plasmid DNA. Insertion of any restriction fragment of the phage into the plasmid causes an approximately 10 5-fold increase in transduction frequency, regardless of the size or genetic content of the fragment. The resulting transducing particles (Hft particles) contain mostly pure linear concatemers composed of tandem repeats of the plasmid: phage chimera, and their production requires active plasmid-initiated replication. The high frequency of transduction is a consequence of homologous recombination between the linear chimeric and phage concatemers, which has the effect of introducing an efficient pac site into the former. Following introduction into lysogenic recipient bacteria, the transducing DNA is first converted to the supercoiled form, then processed to monomers by a mechanism that requires the active participation of the plasmid replication system.