New method for evaluation of genotoxicity, based on the use of real-time PCR and lysogenic gram-positive and gram-negative bacteria

A method for the detection of the SOS response as measured by the liberation of resident prophages from the genomes of their hosts is described. It is based on the use of two converging oligonucleotides that flank the attP attachment site of the phage as primers for real-time PCR. Amplification was observed only after the phage DNA became excised. The system responds to both chemicals and physical conditions. Quantitative data on the concentration and/or potency of the genotoxic condition were obtained. Results can be achieved within 1 day and are less susceptible to possible toxic effects than phage generation or other methods that require DNA synthesis. The use of both gram-positive and gram-negative bacteria widens the range of compounds that can be tested because it eliminates impermeability problems derived from the particular composition of each cell wall type.     

Orientation of the DNA in the filamentous bacteriophage f1

The filamentous bacteriophage f1 consists of a molecule of circular single-stranded DNA coated along its length by about 2700 molecules of the B protein. Five molecules of the A protein and five molecules of the D protein are located near or at one end of the virion, while ten molecules of the C protein are located near or at the opposite end. The two ends of the phage can be separated by reacting phage fragments, which have been generated by passage of intact phage through a French press, with antibody directed against the A protein (Grant et al., 1981a). By hybridizing the DNA isolated from either end of ³²P-labeled phage to specific restriction fragments of fl replicative form I DNA, we have determined that the single-stranded DNA of the filamentous bacteriophage f1 is oriented within the virion. For wild-type phage, the DNA that codes for the gene III protein is located at the A and D protein end and that which corresponds to the intergenic region is located close to the C protein end of the particle. The intergenic region codes for no protein but contains the origins for both viral and complementary strand DNA synthesis. Analysis of the DNA orientation in phage in which the plasmid pBR322 has been inserted into different positions within the intergenic region of fl shows that the C protein end of all sizes of filamentous phage particles appears to contain a common sequence of phage DNA. This sequence is located near the junction of gene IV and the intergenic region, and probably is important for normal packaging of phage DNA into infectious particles. There appears to be no specific requirement for the origins of viral and complementary strand DNA synthesis to be at the end of a phage particle.     

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.     

Purification and characterization of the N gene product of bacteriophage lambda

The N protein (pN) specified by bacteriophage λ is an antitermination factor and is required for phage development. pN can be assayed by making use of the observation that the in vitro synthesis of trp mRNA in a reaction programmed with DNA template from λtrp transducing phage bearing N$The N protein (pN) specified by bacteriophage λ is an antitermination factor and is required for phage development. pN can be assayed by making use of the observation that the in vitro synthesis of trp mRNA in a reaction programmed with DNA template from λtrp transducing phage bearing N and fed mutations is pN dependent (Ishii et al., 1980). The assay has been used to purify pN. We have observed that pN forms a complex with E. coll protein(s) and is dissociated in the presence of urea. The complex is not formed in host bacteria bearing thenusA_nusB_ mutations. pN is a basic protein and heat-stable. Using these characteristics, we have purified pN to virtual homogeneity as judged by polyacrylamide gel electrophoresis in the presence of SDS. pN is a monomeric protein and its mol. wt. is approx. 14 000. The antiterminating activity of pNappears to be enhanced by complex formation with host-encoded protein(s) depending on the nusA and/or nusB gene function.      5. Phage-lambda-mediated transduction of non-conjugative plasmids is promoted by transposons   总被引：3，自引：0，他引：3   J C Piffaretti  O Fayet 《Gene》1981,13(3):319-325 Transduction experiments using phage λ as a vector have shown that non-conjugative plasmids can be transduced from one cell to the other, provided the phage or the plasmid DNA carries a copy of a Tn3-like transposon. The transduction is a result of replicon fusion between the phage and the plasmid DNA occurring during the transposition event.      6. Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda   总被引：6，自引：0，他引：6   M Blanco  R Devoret 《Mutation research》1973,17(3):293-305 Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA+ function, the red+ but not the int+ function can substitute for recA+ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.      7. Bacteriophage lambda derivatives carrying two copies of the cohesive end site   总被引：27，自引：0，他引：27   S W Emmons 《Journal of molecular biology》1974,83(4):511-525 A spontaneously arising tandem duplication derivative of bacteriophage lambda has been isolated, which carries two copies of the site where the cohesive ends are formed (designated cos). Its structure has been determined by electron microscopy of DNA heteroduplexes. These heteroduplexes reveal that the duplication is usually, but not always, carried on the left end of the chromosome. A second duplication phage having two copies of cos, constructed by Feiss &; Campbell (1974), has also been studied by electron microscopy and is found to have a similar property.Unlike most tandem duplication derivatives of phage λ, the mutant studied here is not stable during growth in the absence of generalized recombination, but segregates both the triplication and the parental phage. This verifies that both cos sites are functional. The triplication does not arise as a result of end-to-end aggregation of phage chromosomes or site-specific recombination catalyzed by the chromosome maturation system at cos. It must therefore result from the cutting of mature ι chromosomes from concatemeric replication intermediates. The pattern of cutting observed shows that the λ cohesive ends are not created by a free nuclease acting on unpackaged DNA. The cutting appears to be influenced by the amount of DNA previously packaged into a phage head. A model for λ packaging is presented which explains the results.The duplication phage of Feiss &; Campbell (1974) carries a novel addition containing self-complementary sequences.      8. Intracellular events during infection by Haemophilus influenzae phage and transfection by its DNA   总被引：8，自引：0，他引：8   N K Notani  J K Setlow  D P Allison 《Journal of molecular biology》1973,75(4):581-599 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.      9. Studies on the Pathway of Incorporation of 2-Aminopurine into the Deoxyribonucleic Acid of Escherichia coli   总被引：3，自引：1，他引：2       下载免费PDF全文 Eleanor G. Rogan  Maurice J. Bessman 《Journal of bacteriology》1970,103(3):622-633 A pathway for the incorporation of 2-aminopurine into deoxyribonucleic acid (DNA) was studied in cell-free extracts of Escherichia coli. It was demonstrated that the free base can be converted to the deoxynucleoside, and that the deoxynucleotide can be phosphorylated to the di- and triphosphates and then incorporated into the DNA. From a consideration of the individual reactions in crude extracts, it is likely that the rate-limiting step in this pathway is the formation of the deoxynucleotide. Of especial interest is the observation that 2-aminopurine may be viewed as an analogue of either guanine or adenine, depending on which enzymatic step is being considered. On the one hand, it resembles guanine in that it is specifically converted from the mono- to the diphosphate by guanylate kinase and not by adenylate kinase. On the other hand, it replaces adenine rather than guanine in the DNA synthesized with purified DNA polymerases. E. coli DNA polymerase utilizes aminopurine deoxynucleoside triphosphate as a substrate for DNA synthesis much better than does purified phage T5-induced DNA polymerase and is also much less inhibited by this analogue than the T5 enzyme. These experiments in vitro correlate with known differential effects of 2-aminopurine on E. coli and phage in vivo.      10. Isolation and Expression of the Lysis Genes of Actinomyces naeslundii Phage Av-1          下载免费PDF全文 Allan L. Delisle  Gerard J. Barcak    Ming Guo 《Applied microbiology》2006,72(2):1110-1117 Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these species-specific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective λ holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products.      11. A method for isolation of a large amount of a single-stranded DNA fragment   总被引：9，自引：0，他引：9   J R Patton  C B Chae 《Analytical biochemistry》1982,126(1):231-234 Single-stranded DNA inserts can be digested from recombinant phage DNA of M13mp7 with BamHI or EcoRI restriction endonucleases. The single-stranded DNA is satisfactory for DNA sequencing and nuclei acid hybridization.      12. Effects of DNA Heterologies on Bacteriophage λ Packaging      R. K. Pearson  M. S. Fox 《Genetics》1988,118(1):5-12 We have examined the impact of DNA heterologies on the packaging of λ DNA in vitro. Heterology-containing DNA molecules were constructed by denaturing and reannealing a mixture of DNA from cI(+) phage and DNA from phage carrying small insertion or deletion mutations in the cI gene. We found that molecules with heterologies of up to 19 base pairs (bp) can be packaged as viable heterozygous phage with approximately the same efficiency as molecules with a base pair mismatch. In contrast, with a heterology of 26-bp heterozygous plaque formers are rare. In principle, the absence of cI heterozygotes among packaged phage may be due either to a failure to encapsidate the DNA or a failure to inject the packaged DNA on infection. Southern blot analysis of DNA isolated from packaged phage indicates that DNA harboring a 26-bp heterology is almost completely absent in packaged phage. Thus, an upper limit has been established for the size of heterology that can be accommodated by the packaging apparatus. The size of the connector portal could be the basis for this limit.      13. Maturation of the head of bacteriophage T4. III. DNA packaging into preformed heads   总被引：13，自引：0，他引：13   U K Laemmli  N Teaff  J D'Ambrosia 《Journal of molecular biology》1974,88(4):749-765 An estimate was made of the amount of DNA packaged into gene 49-defective heads when P49 is activated by a temperature shift. The uptake of DNA into preformed heads following activation of P49 was studied using bromo-deoxyuridine as a label. The rate of inactivation by visible light of the phage matured in the presence of BrdU as well as their buoyant density in CsCl, indicate that over half of the particles package, on the average, at least 25% of the DNA complement following P49 activation. This is a minimum estimate, since the BrdU-labeled DNA has to compete with unlabeled DNA. Analysis on alkaline sucrose gradients of the size of the DNA extracted from phage matured in the presence of BrdU following irradiation reveals that extended irradiation at 313 nm breaks the DNA close to half of its original size. These experiments clearly show that up to half of the DNA can be packaged into the preformed heads made at high temperature following activation of the product of gene 49 (P49), strongly supporting the pathway for phage head maturation described by Laemmli &; Favre (1973).The so-called τ-particles, which accumulate in 24-defective cells, can serve as precursors of the mature phage (Bijlenga et al., 1973). We have measured the uptake of BrdU-labeled DNA into τ-particles during their maturation. We find that a very large proportion of DNA made after activation of P24 is apparently incorporated into preformed τ-particles as these particles are converted into mature heads. This indicates that the τ-particles contain very little or no DNA prior to P24 activation and supports the pathway described by Laemmli &; Favre (1973).      14. Some Properties of DNA from Phage-Infected Bacteria   总被引：15，自引：0，他引：15   M. G. Smith  A. Skalka 《The Journal of general physiology》1966,49(6):127-142 Replicating T5 or λ phage DNA has been labeled by adding tritiated thymidine for short periods to cultures of phage-infected Escherichia coli before isolation of intracellular DNA. Two procedures are described for separating T5 replicating DNA from DNA of intracellular phage particles. Both T5 and λ replicating DNA had the same bouyant density in cesium chloride as DNA from phage particles but sedimented faster when centrifuged in sucrose density gradients. The fast sedimentation did not appear to be caused by DNA protein or DNA-RNA complexes or by aggregation of DNA, but is probably due to DNA molecules of unusual structure. Experiments involving hydrodynamic shear and sucrose density gradient centrifugation at alkaline pH have suggested that with λ the replicating form of DNA is a linear molecule considerably longer than the DNA molecules of λ-phage particles. The constituent polynucleotide chains of λ but not T5 replicating DNA also appear to be longer than those of phage DNA.      15. Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion   总被引：21，自引：0，他引：21       下载免费PDF全文 Harald Brüssow  Carlos Canchaya    Wolf-Dietrich Hardt 《Microbiological reviews》2004,68(3):560-602 Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.      16. A fast and simple method for sequencing DNA cloned in the single-stranded bacteriophage M13.   总被引：74，自引：0，他引：74   P H Schreier  R Cortese 《Journal of molecular biology》1979,129(1):169-172 The chain termination DNA sequencing procedure of Sanger et al. (1977) requires single-stranded DNA as template. M13 phage DNA exists as a single strand and therefore every DNA sequence cloned in M13 can be easily obtained in this form. Here we show that M13 single-stranded DNA pure enough to be used as a template for sequence determination can be prepared by simple centrifugation of the phage particle and extraction with phenol.      17. Effect of enzymatic methylation on the chemical,physical, and biological properties of DNA      Jerrold Greenberg  Alvin I. Krasna 《Archives of biochemistry and biophysics》1976,177(2):468-479 DNA methylase was partially purified from Escherichia coli W and used to methylate DNA from Bacillus subtilis and bacteriophage φ105. The former DNA was methylated 1.17% and the latter 0.87%. The products were 6-methyladenine (85%) and 5-methylcytosine (15%) in both cases. The methylated DNA was stable toward depurination and viscosity loss at elevated temperatures. Methylation led to a 50% decrease in transforming activity in two strains of B. subtilis and no change in a third strain. The ability of phage φ105 DNA to rescue a defective phage strain was decreased 50% by methylation. No changes were observed in the ability of methylated DNA to serve as a template for DNA polymerase or RNA polymerase. The pattern of cleavage of DNA by a variety of restriction endonucleases was not affected by methylation. There were no changes in the physicochemical properties of DNA on methylation as measured by hyperchromicity on heating, formaldehyde denaturation, viscosity, and sedimentation.      18. Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway   总被引：29，自引：0，他引：29   J King  E V Lenk  D Botstein 《Journal of molecular biology》1973,80(4):697-731 We have identified and characterized structural intermediates in phage P22 assembly. Three classes of particles can be isolated from P22-infected cells: 500 S full heads or phage, 170 S empty heads, and 240 S “proheads”. One or more of these classes are missing from cells infected with mutants defective in the genes for phage head assembly. By determining the protein composition of all classes of particles from wild type and mutant-infected cells, and examining the time-course of particle assembly, we have been able to define many steps in the pathway of P22 morphogenesis.In pulse-chase experiments, the earliest structural intermediate we find is a 240 S prohead; it contains two major protein species, the products of genes 5 and 8. Gene 5 protein (p5) is the major phage coat protein. Gene 8 protein is not found in mature phage. The proheads contain, in addition, four minor protein species, PI, P16, P20 and PX. Similar prohead structures accumulate in lysates made with mutants of three genes, 1, 2 and 3, which accumulate uncut DNA. The second intermediate, which we identify indirectly, is a newly filled (with DNA) head that breaks down on isolation to 170 S empty heads. This form contains no P8, but does contain five of the six protein species of complete heads. Such structures accumulate in lysates made with mutants of two genes, 10 and 26.Experiments with a temperature-sensitive mutant in gene 3 show that proheads from such 3? infected cells are convertible to mature phage in vivo, with concomitant loss of P8. The molecules of P8 are not cleaved during this process and the data suggest that they may be re-used to form further proheads.Detailed examination of 8? lysates revealed aberrant aggregates of P5. Since P8 is required for phage morphogenesis, but is removed from proheads during DNA encapsulation, we have termed it a scaffolding protein, though it may have DNA encapsulation functions as well.All the experimental observations of this and the accompanying paper can be accounted for by an assembly pathway, in which the scaffolding protein P8 complexes with the major coat protein P5 to form a properly dimensioned prohead. With the function of the products of genes 1, 2 and 3, the prohead encapsulates and cuts a headful of DNA from the concatemer. Coupled with this process is the exit of the P8 molecules, which may then recycle to form further proheads. The newly filled heads are then stabilized by the action of P26 and gene 10 product to give complete phage heads.      19. Direct cloning of cDNA inserts from λgt11 phage DNA into a plasmid vector by a novel and simple method      《Gene Analysis Techniques》1990,7(1):18-23 Bacteriophage λgt11 has been used quite extensively for producing cDNA libraries. The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis. However, isolating the recombinant DNA of interest from the phage clones can be a tedious task. Since the E. coli strain Y1088 used for λgt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert. In this report, we describe a method in which cDNA inserts from λgt11 phage were cloned directly into the pBR322 plasmid vector, by-passing the time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method is likely to be extended to the cloning of DNA inserts derived from other phage λ vectors when bacteria containing endogenous pBR322 are used as host cells.      20. Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro      Dorothee Andres  Christin Hanke  Ulrich Baxa  Ana?t Seul  Stefanie Barbirz  Robert Seckler 《The Journal of biological chemistry》2010,285(47):36768-36775 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.      $

Phage-lambda-mediated transduction of non-conjugative plasmids is promoted by transposons

Transduction experiments using phage λ as a vector have shown that non-conjugative plasmids can be transduced from one cell to the other, provided the phage or the plasmid DNA carries a copy of a Tn3-like transposon. The transduction is a result of replicon fusion between the phage and the plasmid DNA occurring during the transposition event.     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

Bacteriophage lambda derivatives carrying two copies of the cohesive end site

A spontaneously arising tandem duplication derivative of bacteriophage lambda has been isolated, which carries two copies of the site where the cohesive ends are formed (designated cos). Its structure has been determined by electron microscopy of DNA heteroduplexes. These heteroduplexes reveal that the duplication is usually, but not always, carried on the left end of the chromosome. A second duplication phage having two copies of cos, constructed by Feiss &; Campbell (1974), has also been studied by electron microscopy and is found to have a similar property.Unlike most tandem duplication derivatives of phage λ, the mutant studied here is not stable during growth in the absence of generalized recombination, but segregates both the triplication and the parental phage. This verifies that both cos sites are functional. The triplication does not arise as a result of end-to-end aggregation of phage chromosomes or site-specific recombination catalyzed by the chromosome maturation system at cos. It must therefore result from the cutting of mature ι chromosomes from concatemeric replication intermediates. The pattern of cutting observed shows that the λ cohesive ends are not created by a free nuclease acting on unpackaged DNA. The cutting appears to be influenced by the amount of DNA previously packaged into a phage head. A model for λ packaging is presented which explains the results.The duplication phage of Feiss &; Campbell (1974) carries a novel addition containing self-complementary sequences.     

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.     

Studies on the Pathway of Incorporation of 2-Aminopurine into the Deoxyribonucleic Acid of Escherichia coli

A pathway for the incorporation of 2-aminopurine into deoxyribonucleic acid (DNA) was studied in cell-free extracts of Escherichia coli. It was demonstrated that the free base can be converted to the deoxynucleoside, and that the deoxynucleotide can be phosphorylated to the di- and triphosphates and then incorporated into the DNA. From a consideration of the individual reactions in crude extracts, it is likely that the rate-limiting step in this pathway is the formation of the deoxynucleotide. Of especial interest is the observation that 2-aminopurine may be viewed as an analogue of either guanine or adenine, depending on which enzymatic step is being considered. On the one hand, it resembles guanine in that it is specifically converted from the mono- to the diphosphate by guanylate kinase and not by adenylate kinase. On the other hand, it replaces adenine rather than guanine in the DNA synthesized with purified DNA polymerases. E. coli DNA polymerase utilizes aminopurine deoxynucleoside triphosphate as a substrate for DNA synthesis much better than does purified phage T5-induced DNA polymerase and is also much less inhibited by this analogue than the T5 enzyme. These experiments in vitro correlate with known differential effects of 2-aminopurine on E. coli and phage in vivo.     

Isolation and Expression of the Lysis Genes of Actinomyces naeslundii Phage Av-1

Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these species-specific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective λ holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products.     

A method for isolation of a large amount of a single-stranded DNA fragment

Single-stranded DNA inserts can be digested from recombinant phage DNA of M13mp7 with BamHI or EcoRI restriction endonucleases. The single-stranded DNA is satisfactory for DNA sequencing and nuclei acid hybridization.     

Effects of DNA Heterologies on Bacteriophage λ Packaging

We have examined the impact of DNA heterologies on the packaging of λ DNA in vitro. Heterology-containing DNA molecules were constructed by denaturing and reannealing a mixture of DNA from cI(+) phage and DNA from phage carrying small insertion or deletion mutations in the cI gene. We found that molecules with heterologies of up to 19 base pairs (bp) can be packaged as viable heterozygous phage with approximately the same efficiency as molecules with a base pair mismatch. In contrast, with a heterology of 26-bp heterozygous plaque formers are rare. In principle, the absence of cI heterozygotes among packaged phage may be due either to a failure to encapsidate the DNA or a failure to inject the packaged DNA on infection. Southern blot analysis of DNA isolated from packaged phage indicates that DNA harboring a 26-bp heterology is almost completely absent in packaged phage. Thus, an upper limit has been established for the size of heterology that can be accommodated by the packaging apparatus. The size of the connector portal could be the basis for this limit.     

Maturation of the head of bacteriophage T4. III. DNA packaging into preformed heads

An estimate was made of the amount of DNA packaged into gene 49-defective heads when P49 is activated by a temperature shift. The uptake of DNA into preformed heads following activation of P49 was studied using bromo-deoxyuridine as a label. The rate of inactivation by visible light of the phage matured in the presence of BrdU as well as their buoyant density in CsCl, indicate that over half of the particles package, on the average, at least 25% of the DNA complement following P49 activation. This is a minimum estimate, since the BrdU-labeled DNA has to compete with unlabeled DNA. Analysis on alkaline sucrose gradients of the size of the DNA extracted from phage matured in the presence of BrdU following irradiation reveals that extended irradiation at 313 nm breaks the DNA close to half of its original size. These experiments clearly show that up to half of the DNA can be packaged into the preformed heads made at high temperature following activation of the product of gene 49 (P49), strongly supporting the pathway for phage head maturation described by Laemmli &; Favre (1973).The so-called τ-particles, which accumulate in 24-defective cells, can serve as precursors of the mature phage (Bijlenga et al., 1973). We have measured the uptake of BrdU-labeled DNA into τ-particles during their maturation. We find that a very large proportion of DNA made after activation of P24 is apparently incorporated into preformed τ-particles as these particles are converted into mature heads. This indicates that the τ-particles contain very little or no DNA prior to P24 activation and supports the pathway described by Laemmli &; Favre (1973).     

Some Properties of DNA from Phage-Infected Bacteria

Replicating T5 or λ phage DNA has been labeled by adding tritiated thymidine for short periods to cultures of phage-infected Escherichia coli before isolation of intracellular DNA. Two procedures are described for separating T5 replicating DNA from DNA of intracellular phage particles. Both T5 and λ replicating DNA had the same bouyant density in cesium chloride as DNA from phage particles but sedimented faster when centrifuged in sucrose density gradients. The fast sedimentation did not appear to be caused by DNA protein or DNA-RNA complexes or by aggregation of DNA, but is probably due to DNA molecules of unusual structure. Experiments involving hydrodynamic shear and sucrose density gradient centrifugation at alkaline pH have suggested that with λ the replicating form of DNA is a linear molecule considerably longer than the DNA molecules of λ-phage particles. The constituent polynucleotide chains of λ but not T5 replicating DNA also appear to be longer than those of phage DNA.     

Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like “swarms” of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.     

A fast and simple method for sequencing DNA cloned in the single-stranded bacteriophage M13.

The chain termination DNA sequencing procedure of Sanger et al. (1977) requires single-stranded DNA as template. M13 phage DNA exists as a single strand and therefore every DNA sequence cloned in M13 can be easily obtained in this form. Here we show that M13 single-stranded DNA pure enough to be used as a template for sequence determination can be prepared by simple centrifugation of the phage particle and extraction with phenol.     

Effect of enzymatic methylation on the chemical,physical, and biological properties of DNA

DNA methylase was partially purified from Escherichia coli W and used to methylate DNA from Bacillus subtilis and bacteriophage φ105. The former DNA was methylated 1.17% and the latter 0.87%. The products were 6-methyladenine (85%) and 5-methylcytosine (15%) in both cases. The methylated DNA was stable toward depurination and viscosity loss at elevated temperatures. Methylation led to a 50% decrease in transforming activity in two strains of B. subtilis and no change in a third strain. The ability of phage φ105 DNA to rescue a defective phage strain was decreased 50% by methylation. No changes were observed in the ability of methylated DNA to serve as a template for DNA polymerase or RNA polymerase. The pattern of cleavage of DNA by a variety of restriction endonucleases was not affected by methylation. There were no changes in the physicochemical properties of DNA on methylation as measured by hyperchromicity on heating, formaldehyde denaturation, viscosity, and sedimentation.     

Mechanism of head assembly and DNA encapsulation in Salmonella phage P22. II. Morphogenetic pathway

We have identified and characterized structural intermediates in phage P22 assembly. Three classes of particles can be isolated from P22-infected cells: 500 S full heads or phage, 170 S empty heads, and 240 S “proheads”. One or more of these classes are missing from cells infected with mutants defective in the genes for phage head assembly. By determining the protein composition of all classes of particles from wild type and mutant-infected cells, and examining the time-course of particle assembly, we have been able to define many steps in the pathway of P22 morphogenesis.In pulse-chase experiments, the earliest structural intermediate we find is a 240 S prohead; it contains two major protein species, the products of genes 5 and 8. Gene 5 protein (p5) is the major phage coat protein. Gene 8 protein is not found in mature phage. The proheads contain, in addition, four minor protein species, PI, P16, P20 and PX. Similar prohead structures accumulate in lysates made with mutants of three genes, 1, 2 and 3, which accumulate uncut DNA. The second intermediate, which we identify indirectly, is a newly filled (with DNA) head that breaks down on isolation to 170 S empty heads. This form contains no P8, but does contain five of the six protein species of complete heads. Such structures accumulate in lysates made with mutants of two genes, 10 and 26.Experiments with a temperature-sensitive mutant in gene 3 show that proheads from such 3^? infected cells are convertible to mature phage in vivo, with concomitant loss of P8. The molecules of P8 are not cleaved during this process and the data suggest that they may be re-used to form further proheads.Detailed examination of 8^? lysates revealed aberrant aggregates of P5. Since P8 is required for phage morphogenesis, but is removed from proheads during DNA encapsulation, we have termed it a scaffolding protein, though it may have DNA encapsulation functions as well.All the experimental observations of this and the accompanying paper can be accounted for by an assembly pathway, in which the scaffolding protein P8 complexes with the major coat protein P5 to form a properly dimensioned prohead. With the function of the products of genes 1, 2 and 3, the prohead encapsulates and cuts a headful of DNA from the concatemer. Coupled with this process is the exit of the P8 molecules, which may then recycle to form further proheads. The newly filled heads are then stabilized by the action of P26 and gene 10 product to give complete phage heads.     

Direct cloning of cDNA inserts from λgt11 phage DNA into a plasmid vector by a novel and simple method

Bacteriophage λgt11 has been used quite extensively for producing cDNA libraries. The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis. However, isolating the recombinant DNA of interest from the phage clones can be a tedious task. Since the E. coli strain Y1088 used for λgt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert. In this report, we describe a method in which cDNA inserts from λgt11 phage were cloned directly into the pBR322 plasmid vector, by-passing the time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method is likely to be extended to the cloning of DNA inserts derived from other phage λ vectors when bacteria containing endogenous pBR322 are used as host cells.     

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.