Selective transduction of recombinant plasmids with cloned pac sites by Salmonella phage P22

Summary Recombinant plasmids having PstI fragments of P22 DNA inserted in the vector pBR322 can be transduced efficiently by Salmonella phage P22, irrespective of the cloned phage sequences. When the rec function of the donor cells and the corresponding recombination system erf of the infecting phage are simultaneously inactivated, only plasmids containing the P22 pac site can be transduced. By this selective, generalized transduction an EcoRV DNA fragment of the P22 related phage L has been identified that carries a base sequence recognized by phage P22 as a packaging signal. Experiments in which only one of the two recombination systems was inactivated, showed that the bacterial rec system obviously promotes cointegrate formation between plasmid and phage DNA much more efficiently than the phage-coded erf system, allowing the specialized plasmid transduction observed by Orbach and Jackson (1982).     

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.     

Absence of a protein constituent and occurrence of an oversized DNA genome in a high density mutant particle of phage P22

Summary Mutants of P22 phage with abnormal density in CsCl solution (P22ndc phage) were analyzed in detail for this report. Two dimensional polyacrylamide gel electrophoresis revealed that wild-type P22ndc ⁺ phage virions contained a new protein (gpU) in addition to nine already identified proteins, while P22ndc lacked gpU. The molecular weight of gpU was essentially the same as that of gp5 (45 500), and one mature virion of phage P22ndc ¹ contained as many as 30–50 molecules of gpU. As P22ndc is a plaque-forming phage, gpU cannot be essential for the growth and assembly of P22 phage. Both genetical and biochemical analysis of the phage DNA in the virion revealed that P22ndc phage contained 2%–4% longer DNA than wild type P22ndc ⁺. A model is presented to account for the formation of P22ndc phage.     

Über die Mehrfachreaktivierung des Infektions- und Lysogenisierungsvermögens des Phagen P22 von Salmonella typhimurium nach UV-Bestrahlung

Summary It has been shown, that uv-lesions in the DNA of the temperate Salmonella bacteriophage P22 can undergo multiplicity reactivation. The multiplicity reactivation is very efficient and does not seem to be hindered by recombination events between phage and host genetic material as it is the case with the Coli phage T1. The ability of P22 to lysogenize its host cell is very sensitive to low doses of UV; its inactivation by UV can also undergo multiplicity reactivation.     

The origin of the DNA in a special class of generalized transducing particles of Salmonella-phage P22

Summary A special class of transducing particles in lysates of P22 is described. When DNA synthesized after infection is density labelled with bromodeoxyuridine, this class of particles exhibit a higher density in CsCl-gradients than normal transducing particles in the same lysates. It is shown that the DNA of these transducing particles carries the normal amount of bacterial information and is synthesized in the semiconservative mode after phage-infection. From the efficiency of BU-incorporation we conclude that the DNA molecules of these particles are not products of normal bacterial replication, but might be replicated under the control of the phage.     

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full‐length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.     

Replication and maturation of phage P22 in a mutant of Salmonella typhimurium temperature sensitive in initiation of DNA replication.

Summary TB37 is a dna A-mutant of Salmonella typhimurium in which the initiation of DNA replication at the origin is stopped at 42°C. DNA synthesis in uninfected cells of this strain and in cells infected by phage P22 was followed by the pulse labelling technique. DNA replication ceases completely after about 50 minutes at the high temperature. After lytic infection with P22 (c2) at this time, DNA synthesis starts immediately and increases at a rate well comparable to the permissive control. Obviously the temperature sensitive function of the dnaA-product is dispensable for P22 DNA replication, especially for its initiation. This result is confirmed by the normal yield of phage particles under these conditions, provided that a late step in P22 maturation which naturally is temperature sensitive can proceed at low temperature. If TB37 is infected at 42°C with P22 wild type, an unexpected high rate of phage controlled DNA synthesis is observed. Preliminary results seem to indicate that the process of integration is a prerequisite for part of this synthesis.     

Specialized Transduction by Bacteriophage P22 in SALMONELLA TYPHIMURIUM: Genetic and Physical Structure of the Transducing Genomes and the Prophage Attachment Site

P22pro-1 and P22pro-3 are specialized transducing derivatives of phage P22 that carry the proA and proB genes of Salmonella typhimurium. These genes lie immediately adjacent to the prophage attachment site on the bacterial chromosome. By examining DNA heteroduplexes in the electron microscope, we found that DNA molecules from P22pro-1 and P22pro-3 each contain a substitution which adds length to the composite genome making the intracellular replicated genome too long to fit into a single phage particle. In this respect, and in many of their biological properties, the proline-transducing phages resemble P22Tc-10, another specialized transducing phage with an oversize, intracellular replicated genome which carries a tetracycline-resistance determinant from an R-factor.—Unlike P22Tc-10, however, P22pro-1 and P22pro-3 fail to integrate normally during lysogenizing infections, even when provided with all known integration functions. These results suggest that the proline substitutions have created a defect in the phage attachment site and suggest that the Campbell model for the formation of specialized transducing phages is applicable to phage P22 with the additional feature that oversize genomes can be produced and propagated.—A physical and genetic map of the P22 genome near the prophage attachment site was constructed which shows that the insertion from the R-factor in P22Tc-10 is not at the attachment site: it is therefore unlikely that P22Tc-10 was formed in an abnormal prophage excision event as envisioned in the Campbell model, but was instead the result of a direct translocation from the R-plasmid to P22.     

Phage P22-mutants with increased or decreased transduction abilities

Summary The properties of mutants of the Salmonella phage P22 are described which show decreased or increased frequencies for generalized transduction. It is shown that not only the markers used for detection of the mutants are affected by the mutation but also other markers tested. Evidence is presented that the altered T/P-ratios reflect changes in the actual numbers of transducing particles and that other factors like integration in the recipient cells cannot account for these alterations. Quantitative estimations of the amount of bacterial DNA converted to transducing fragments are shown for several phage mutants. Difficulties in the isolation procedure are discussed.     

Autoplaque Formation in a Pseudomonas fluorescensStrain: Phagelike Particles and Transactivation of the Defective Phage

Natural bacteriophages ofPseudomonas fluorescensare rare and its temperate phages have not been described so far. In search for these phages, we have found that one of the P. fluorescensstrains forms numerous small transparent autoplaques of different size and shape, which contained material reproducible on the same strains. When centrifuged in a cesium chloride gradient, this material yielded a band in the density zone of about 1.3 g/cm³, where protein components or bacteriophages with a relatively low content of nucleic acid are usually located. In the band material, electron microscopy revealed phagelike particles with empty and mostly undamaged heads and tails carrying in their distal region a formation resembling contracted sheath. DNA isolated from the preparation consisted of two components: a distinct 54-kb fragment, and a diffuse fragment ranging in size from 20 to 9.5 kb. Treatment of the large DNA fragment with various endonucleases yielded 42.2- and 29.5-kb fragments (on average for different endonucleases); whereas the same treatment of the diffuse fragment yielded two- to three distinct fragments with the overall molecular sizes of 8.9 and 6.2 kb (for different nucleases). We have suggested that cells harbor two different genetic elements whose interaction results in the autoplaque appearance and in the formation of negative colonies after infection with the autoplaque material. One of the two elements displays properties of a defective prophage with disturbed DNA synthesis and assembly, whereas the other exhibits the properties of a transposable phage. After complementation or some other interaction between these elements (transactivation, prophage induction caused by repressor inactivation), a bulk of defective phage particles devoid of DNA and a few DNA-containing particles were produced. It remains unclear whether both DNA types are contained in the same or different particles. The phage (or a system of elements) referred to as PT3 is noninducible. The phage mutants forming larger negative colonies (NCs) were also revealed. Some of bacterial mutants resistant to PT3 infection produce the mutant phage with small and turbid NCs. PT3 produces no NCs on the lawns of other strains of the same or other pseudomonad species. This is the first case of describing a natural temperate bacteriophage in P. fluorescens.The two different elements of this phage may represent the same genome of the defective prophage divided into two portions within a bacterial chromosome, each of which is capable of packaging into the phage head.     

Infection of transformable cells ofHaemophilus influenzae by bacteriophage and bacteriophage DNA

Summary A phage HP1, infecting transformable cells ofHaemophilus influenzae Rd, has been isolated. The general properties of the wild type and of a clear plaquemutantc1 employed for most of the experiments are described. Phage DNA is infective for transformableHaemophilus cells with an efficiency (plaqueforming units of the original phage recovered as DNA-infected cells) of up to 6×10^–3. The competence ofHaemophilus cells for infection with phage DNA parallels the competence for transformation with bacterial DNA.Both HP1 and thec1 mutant are able to lysogenize their host, and the lysogenic cells are readily induced by UV. Competent non-lysogenicHaemophilus cells can be infected by DNA of lysogenic cells, thereby giving rise to phage progeny. Thus, the phage genetic material can be introduced into competentHaemophilus cells in three different ways: injection from intact phage, and infection with either phage DNA or with bacterial DNA carrying the prophage.The UV inactivation curves for infectious phage DNA and for complete phages are similar, both indicating the occurrance of host-cell reactivation. Photoreactivationin vitro of infectious phage DNA takes place to about the same high extent as observed with bacterial transforming DNA.The usefulness of this system for investigating bacterial transformation and biological effects ofin vitro treatment of DNA is discussed.with the technical assistance ofSandra J. Antoine With 4 Figures in the TextPreliminary report presented at the 7th Annual Bacterial Transformation Meeting, Aspen, Colorado, June 17–19, 1963.Supported by a travel grant from the Deutsche Forschungsgemeinschaft.Supported by Research Carreer Development Award GM-K3-7500 and Research Grant RH 00221 from the U.S. Public Health Service.     

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.     

Salmonella typhimurium mutants affecting establishment of lysogeny

Summary Salmonella typhimurium mutants have been isolated in which phage P22 fails to establish lysogeny. These appear to be defective in cAMP metabolism. A phage mutation overcoming the bacterial defect has been mapped between gene c ₁ and gene 12.     

Phage P22 helps phage MB78 to overcome blockage of DNA maturation in rifampicin-resistant host

Bacteriophage MB78, a virulent phage ofSalmonella typhimurium cannot grow in rifampicin-resistant mutant (rif-39) of the host having altered RNA polymerase. The temperate phage P22 which cannot multiply in presence of the virulent phage MB78 can, however, help MB78 to overcome replication inhibition in rif-39. The processing of concatemeric phage DNA to monomer is blocked in this nonpermissive host. Superinfection with P22 induces synthesis of at least five P22 specific polypeptides which help phage MB78 in the processing of the concatemeric DNA and maturation of phage particles.     

Characterization of Deoxyribonucleic Acid of Virulent Bacteriophage and its Infectivity to Host Bacteria, Pseudomonas solanacearum

Virulent bacteriophage PK-101 was isolated from soil infested with strain K-101 of Pseudomonas solanacearum and nucleic acid was prepared from the phages. Some chemical properties of phage nucleic acid and its infectivities to various strains of P. solanacearum were examined in the present study. By digestion with restriction endonucleases, phage nucleic acid was shown to be linear duplex DNA approximately 35 kb long. Restriction fragment length polymorphism was observed when electrophoresis patterns of enzyme-digested PK-101 DNA were compared with those of DNA prepared from different phage isolates. Transfection of host strains by PK-101 DNA was carried out, and it was infectious not only to host strain K-101, but also to other strains which were resistant to phage particles. Transfection efficiency was considerably enhanced by directly introducing phage DNA into bacterial cells by means of an electroporation. The electroporation technique was also effective to transform P. solanacearum with large-size plasmid DNA.     

Evidence for an early regulatory function in phage P22

Summary A temperature sensitive mutant of P22 phage (ts X) was isolated and studied.This mutant seems to have a basic regulatory function: it is defective in an early function like the typical DNA^- mutant ts 12.1; it is unable to direct the phage DNA synthesis and does not lyse infected or induced cells.Unlike ts 12.1, the mutation ts X seems to involve a gene product necessary for the expression of any vegetative function, since no phage protein synthesis, no alteration of host DNA synthesis, and no cell killing can be observed under non-permissive conditions.The possible functional similarity between the N-cistron of the phage and the present X-cistron in P22 is discussed.     

Electrogenic cyclic redox chain in bacterial photosynthesis: Two regimes of operation in intact cells of Rhodospirillum rubrum

Bacteriophage K7 is specific for Escherichia coli strains harbouring R factors of incompatability group W, including hybrid coliphage P1-Myxococcus virescens plasmids. The phage has an unusual morphology with an isometric head and long tail of variable length. The tail lengths appear to fall into classes corresponsing to simple multimers of a unit length. Partially purified lysates of the phage include material that may represent phage particles in the process of biogenesis and other material demonstrating attachment of phage to cell envelope. Newly released phage DNA contains single standed ends. In the course of work, E. coli strains that harbour R factor Sa were found to be apparently restrictive.     

Participation of the host protein(s) in the morphogenesis of bacteriophage P22

Summary Spontaneous mutants of S. typhimurium resistant to thiolutin are conditionally non-permissive for phage P22 development (Joshi and Chakravorty 1979). At 40° C non-infective phage particles are produced. Phage development in two nonpermissive hosts (18/MC4 and 153/MC4) has been studied in detail. The steps at which the phage morphogenesis is interfered with differ in the two mutants. The electron micrograph of the particles produced in the mutant 18/MC4 reveals the presence of normal-looking particles; these particles contain phage DNA, adsorb to the permissive host but fail to inject their DNA. The particles produced in the mutant 153/MC4 which fail to adsorb to the host are found to be tail fibre-less. These observations indicate the involvement of host protein(s) in phage P22 morphogenesis.     

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 phage 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 phenotype 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-1 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.     

Atomic structure of bacteriophage Sf6 tail needle knob

Podoviridae are double-stranded DNA bacteriophages that use short, non-contractile tails to adsorb to the host cell surface. Within the tail apparatus of P22-like phages, a dedicated fiber known as the “tail needle” likely functions as a cell envelope-penetrating device to promote ejection of viral DNA inside the host. In Sf6, a P22-like phage that infects Shigella flexneri, the tail needle presents a C-terminal globular knob. This knob, absent in phage P22 but shared in other members of the P22-like genus, represents the outermost exposed tip of the virion that contacts the host cell surface. Here, we report a crystal structure of the Sf6 tail needle knob determined at 1.0 Å resolution. The structure reveals a trimeric globular domain of the TNF fold structurally superimposable with that of the tail-less phage PRD1 spike protein P5 and the adenovirus knob, domains that in both viruses function in receptor binding. However, P22-like phages are not known to utilize a protein receptor and are thought to directly penetrate the host surface. At 1.0 Å resolution, we identified three equivalents of l-glutamic acid (l-Glu) bound to each subunit interface. Although intimately bound to the protein, l-Glu does not increase the structural stability of the trimer nor it affects its ability to self-trimerize in vitro. In analogy to P22 gp26, we suggest the tail needle of phage Sf6 is ejected through the bacterial cell envelope during infection and its C-terminal knob is threaded through peptidoglycan pores formed by glycan strands.