Intracellular events during infection by Haemophilus influenzae phage and transfection by its DNA

Intracellular events following infection of competent Haemophilus influenzae by HPlcl phage, or transfection by DNA from the phage, were examined. Physical separation of a large fraction of the intracellular phage DNA from the bulk of the host DNA was achieved by lysis of infected or transfected cells with digitonin, followed by low-speed centrifugation. The small amount of bacterial DNA remaining with the phage DNA in the supernatants could be distinguished from phage DNA by its ability to yield transformants. After infection by whole phage, three forms of intracellular phage DNA were observable by sedimentation velocity analysis: form III, the slowest-sedimenting one; form II, which sedimented 1.1 times faster than III, and form I, which sedimented 1.6 times faster than III. It was shown by electron microscopy, velocity sedimentation in alkali, and equilibrium sedimentation with ethidium bromide, that forms I, II and III are twisted circles, open circles, and linear duplexes, respectively.After the entry of phage DNA into wild-type cells in transfection, the DNA is degraded at early times, but later some of the fragments are reassembled, resulting in molecules that sediment faster than the monomer length of phage DNA. Some of the fast-sedimenting molecules are presumably concatemers and are generated by recombination. In strain rec₁^? the fast-sedimenting molecules do not appear and degradation of phage DNA is even more pronounced than in wild-type cells. In strain rec₂^? there is little degradation of phage DNA, and the proportion of fast-sedimenting molecules is much smaller than in wild-type cells. Since rec₁^? and rec₂^? are transfected with much lower efficiency than wild type, our hypothesis is that both fragmentation and generation of fast-sedimenting phage DNA by recombination are required for more efficient transfection.     

Underwound loops in self-renatured DNA can be diagnostic of inverted duplications and translocated sequences

DNA that contains inverted duplications separated by non-inverted sequences often can form characteristic “underwound loops” when it is denatured and reannealed. An underwound loop is a partially double-stranded, partially denatured segment between the inverted duplications and is produced as follows. During the early stages of the reannealing, intrastrand stem-loop structures form with first-order kinetics when the inverted duplications pair. In a slower second-order reaction, complementary strands (each with a stem-loop) reanneal. The stem-loop structures produce a cruciform in the hybrid. Because of the unpaired sequences in the loop, the cruciform is unstable. It can isomerize to a linear duplex by double-strand exchange of complementary sequences in the stems. This process requires co-ordinated axial rotation of the stems and the flanking duplexes as well as rotation of the loops. If, however, complementary sequences in the loops start to pair, axial rotation is prevented and the stem-loop structures are trapped in a metastable state. The strands of separate, closed rings cannot interwind when they pair. Consequently, the loops observed by electron microscopy have variable patterns of single-stranded denaturation bubbles and duplex segments with both right-handed and left-handed winding.We have used underwound loops to identify a short inverted duplication flanking the γδ recombination sequence of Escherichia coli F factor (isolated on φ80 d₃ilv⁺ transducing phage) and to study DNA from phages Mu and P1 in which the G segments are flanked by inverted duplications. When deproteinized adenovirus-2 DNA was denatured and reannealed, some underwound circles the length of the entire chromosome were observed by electron microscopy. These resulted from the restricted interaction of complementary single-stranded rings generated when pairing of the short inverted terminal duplications closed the ends of single strands. Another type of underwound loop was seen in heteroduplexes containing complementary insertion loops located at different positions in the hybridized strands, such as occurs with P1 cam DNAs. All these underwound structures are similar in appearance to the hybrids formed when topologically separate, complementary single-stranded circles of Colicin E₁ DNA were allowed to anneal.     

Electron microscope study of the structures of the Bacillus subtilis prophages, SPO2 and phi105

Circular duplex structures of the correct length are observed in the electron microscope in hybridization mixtures of lysogen DNA and mature phage DNA for the case of the temperate Bacillus subtilis bacteriophage SPO2. This result shows that the sequence order of the prophage is a circular permutation of that of the mature phage. By making heteroduplexes of prophage DNA with that of the SPO2 deletion mutants, R90 and S25, the att site of the phage has been mapped at 61.2 ± 0.6% from one end of the mature phage DNA, which has a length of 38,600 base pairs. In the same co-ordinate system, the R90 deletion extends from 58.9 ± 0.7 to 66.8 ± 0.8% on the SPO2 chromosome, whereas the S25 deletion extends from 63.2 ± 0.6 to 66.9 ± 0.7%. In similar experiments with lysogen and mature phage DNA's of the temperate B. subtilis phage, φ105, no circular structures were seen. This result shows that the sequence order in the prophage and the phage are colinear, without circular permutation.     

Broad-host-range Yersinia phage PY100: genome sequence, proteome analysis of virions, and DNA packaging strategy

PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage AaΦ23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.     

Tests of spool models for DNA packaging in phage lambda

Experiments are reported which bear on two spool models proposed for packaging the DNA of phage lambda. Both spool models fill an assumed spherical cavity with DNA wrapped in cylindrical or quasi-cylindrical layers composed of adjacent circular turns. In the curved-spool model, a single continuous segment of DNA, about 20% of the DNA length and probably located near the left end of the DNA, is in contact with the coat protein of the phage capsid. In the straight spool model, there are several DNA segments in contact with the capsid; they are concentrated in one half (probably the left half) of lambda DNA. We have identified the loci on the DNA which are in contact with the capsid by chemical crosslinking, induced by ultraviolet-irradiation of phage containing 5-bromodeoxyuridine in place of thymine.In an electron microscope experiment, phage are first lysed with EDTA, and then spread in a cytochrome c film by the formamide method. The disrupted capsid, which has the appearance of a phage ghost, serves as a marker showing where the DNA is crosslinked to the coat. The left end of the DNA is not distinguished from the right end, and so the map of DNA-capsid contacts is folded over on itself. Contacts are found nearly randomly over the entire map.In a second experiment, DNA from lysed, crosslinked phage is cut either with EcoRI or HindIII restriction endonucleases and the cut restriction fragments are labeled at their ends with ³²P. Density centrifugation in a CsCl gradient separates free DNA from restriction fragments crosslinked to protein. After digestion with proteinase k, the DNA fragments previously crosslinked to protein are identified by size after agarose gel electrophoresis. DNA fragments from all parts of the genome are found.These two experiments show that, if the DNA of each phage is packaged identically, then the curved-spool model is ruled out and the straight spool model is unlikely. Alternatively, the manner of packaging the DNA may vary from one phage to the next. These results agree with other recent experiments on λ DNA packaging by Hall & Schellman (1982a,b), and by Haas et al. (1982).A different experiment is also reported. The psoralen derivative aminomethyltrioxalen (AMT) is allowed to intercalate into λ phage and then the DNA strands are crosslinked by ultraviolet-irradiation after the rapid phase of AMT intercalation is complete. The DNA is subsequently denatured by glyoxal modification and spread for electron microscopy in a cytochrome c film by the formamide method. Sites of AMT crosslinking appear duplex; uncrosslinked regions appear as single-stranded loops. AMT is found to intercalate throughout the λ DNA. Patterns of reacted sites appear different from one DNA molecule to the next, and no consistent pattern can be found. More extensive intercalation occurs with the deletion mutant λb221 than with phage of wild-type DNA length, and free DNA shows much more reaction than the DNA inside either phage type. In order for intercalation to occur, the DNA helix must unwind and become further extended. This experiment shows that regions throughout the entire DNA molecule can unwind and be extended by intercalation, which is not confined to a single DNA segment or to segments in one half of the DNA molecule, as would be expected for the two spool models if only the DNA in contact with the capsid were accessible to the dye.     

Identification of functional DNA variants in the constitutive promoter region of MDM2

Although mutations in the oncoprotein murine double minute 2 (MDM2) are rare, MDM2 gene overexpression has been observed in several human tumors. Given that even modest changes in MDM2 levels might influence the p53 tumor suppressor signaling pathway, we postulated that sequence variation in the promoter region of MDM2 could lead to disregulated expression and variation in gene dosage. Two promoters have been reported for MDM2; an internal promoter (P2), which is located near the end of intron 1 and is p53-responsive, and an upstream constitutive promoter (P1), which is p53-independent. Both promoter regions contain DNA variants that could influence the expression levels of MDM2, including the well-studied single nucleotide polymorphism (SNP) SNP309, which is located in the promoter P2; i.e., upstream of exon 2. In this report, we screened the promoter P1 for DNA variants and assessed the functional impact of the corresponding SNPs. Using the dbSNP database and genotyping validation in individuals of European descent, we identified three common SNPs (?1494?G?>?A; indel 40?bp; and ?182?C?>?G). Three major promoter haplotypes were inferred by using these three promoter SNPs together with rs2279744 (SNP309). Following subcloning into a gene reporter system, we found that two of the haplotypes significantly influenced MDM2 promoter activity in a haplotype-specific manner. Site-directed mutagenesis experiments indicated that the 40?bp insertion/deletion variation is causing the observed allelic promoter activity. This study suggests that part of the variability in the MDM2 expression levels could be explained by allelic p53-independent P1 promoter activity.     

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.     

Detailed Characterization and Comparison of Four Lactic Streptococcal Bacteriophages Based on Morphology, Restriction Mapping, DNA Homology, and Structural Protein Analysis

Bacteriophages uc1001 and uc1002, which are lytic for Streptococcus cremoris UC501 and UC502, respectively, were characterized in detail. Comparisons were made with a previously characterized phage, P008, which is lytic for Streptococcus lactis subsp. diacetylactis F7/2, and uc3001, which is a lytic phage for S. cremoris UC503. Phages uc1001 and uc1002 had small isometric heads (diameters, 52 and 50 nm, respectively) and noncontractile tails (lengths, 152 and 136 nm, respectively), and uc1002 also had a collar. Both had 30.1 ± 0.6 kilobase pairs (kbp) of DNA with cross-complementary cohesive ends. Restriction endonuclease maps made with seven endonucleases showed no common fragments. Despite this there was a very high level of homology between uc1001 and uc1002, and results of cross-hybridization experiments showed that the organization of both phage genomes was similar. Heteroduplex analysis confirmed this and quantified the level of homology at 83%. The regions of nonhomology comprised 2.1−, 1.1−, and 1.0-kbp deletion loops and 13 smaller loops and bubbles. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic structural protein profiles were related, with a major band of about 40,000 molecular weight and minor bands of 35,000 and 34,000 molecular weight in common. There were also differences, however, in that uc1001 had a second major band of 68,000 molecular weight and two extra minor bands. Except for the restriction maps, which were strain specific, phages uc1001, uc1002, and P008 were closely related by all the criteria listed above. Their DNAs also showed a very significant bias against the cleavage sites of 9 of 11 restriction endonucleases. Phage uc3001 was unrelated to uc1001, uc1002, or P008 in that it had a prolate head (53 by 39 nm) and a shorter tail (105 nm), contained approximately 22 kbp of DNA, had unrelated cohesive ends, showed no DNA homology with the isometric-headed phages, and displayed a very different structural protein profile.     

T1 Genes Which Affect Transduction

Amber mutants of T1 were grown on each of three donor strains which were identical except that they carried different suppressors: respectively, supD, supE, and supB. The efficiency with which the mutants were able to transduce was tested after growth on each donor. In general, it was found that functions which control the synthesis of phage DNA usually caused significant increases in the efficiency of transduction (EOT). A few mutants located in genes essential for head production caused significant decreases in EOT. The presence of a particular suppressor in a donor can cause noteworthy changes in the EOT by certain of the mutant phages. Amber mutations in gene 3 of T1 were extremely sensitive to the particular suppressor present in the donor, showing a 17-fold decrease in EOT compared with other mutants after growth in donors with the supD suppressor and a 75-fold increase after growth in supE donors. Increases in EOT by early genes of T1 do not seem to be caused by a lack of competition of bacterial DNA with phage DNA during packaging since, in most instances, infective phage were produced in relatively normal amounts compared with wild-type T1. Phage DNA synthesis and degradations of the host chromosome are closely coupled in T1 infections; we believe that increases in EOT by mutants of early functions are due to inefficient degradation of the host chromosome.     

Bacteriophage Resistance Plasmid pTR2030 Inhibits Lytic Infection of r1t Temperate Bacteriophage but Not Induction of r1t Prophage in Streptococcus cremoris R1

The effects of pTR2030 on the replication of four small isometric bacteriophages were examined in Streptococcus cremoris R1. Three lytic phages (652, 720, and 751), which were isolated independently over a 29-year period, were unable to form plaques on a pTR2030 transconjugant of S. cremoris R1. The fourth phage evaluated, phage r₁t, was a temperate phage induced from S. cremoris R1 by treatment with mitomycin C. A prophage-cured derivative of S. cremoris R1, designated R1Cs, was isolated and served as a lytic indicator for phage r₁t. Strain R1Cs and a derivative of this strain that was relysogenized with r₁t, designated R1Cs(r₁t), were used as conjugal recipients for transfer of the phage resistance plasmid pTR2030. pTR2030 transconjugants of strains R1Cs and R1Cs(r₁t) were evaluated for sensitivity to r₁t phage and induction of r₁t prophage, respectively. The temperate phage r₁t adsorbed eficiently but did not form plaques on the prophage-cured, pTR2030 transconjugant strain T-R1Cs. However, in the r₁t lysogen [T-R1Cs(r₁t)], pTR2030 did not inhibit prophage induction with mitomycin C, cell lysis, or production of infective r₁t phage particles. The data demonstrated that pTR2030-induced resistance inhibited lytic infection by r₁t phage from without but did not retard lytic development after prophage induction within the cell. It was suggested that pTR2030-encoded phage resistance to small isometric phages may, therefore, act at the cell surface or membrane to prevent phage DNA passage into the host cell or inhibit early events required for lytic replication of externally infecting phage.     

Mechanisms involved in the formation of plaque-forming derivatives from over-sized hybrid phages between bacteriophage P1 and the R plasmid NR1

Three case histories document how subsequent events of genomic rearrangements and selection interplay in the evolution of infectious bacteriophage genomes carrying acquired genes. Two of the phages studied were plaque-forming P1CmTc recombinants derived from P1Cm1 and P1Tc1, both of which are hybrids between phage P1 and the R plasmid NR1. In the formation of the P1CmTc4 genome a postulated intermediate underwent IS1-mediated deletion formation. From the same intermediate P1CmTc1 must have evolved by IS1-mediated inversion followed by homologous recombination with a parental phage DNA. The third case documents formation of the P1Cm2 genome by “illegitimate” intramolecular recombination in the genome of P1-r-det, a hybrid between P1 and NR1.     

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.