Transformation in Staphylococcus aureus: role of bacteriophage and incidence of competence among strains.

When used in a helper phage capacity, phages 29, 52, 52A, 79, 80, 55, 71, 53, 83A, 85, 95, 96, phi11, and 80 alpha, all serological group B Staphylococcus phages, conferred competence for transformation to S. aureus 8325-4, a strain that does not normally become competent. Of the serological group A phages tested, only phage 3A showed significant competence-conferring activity. Phages 29, 55, 53, 83A, .85, 95, phi11, and 80 alpha showed an enhancement of competence-conferring activity if exposure to the cells occurred in the presence of nromal rabbit serum. All of the propagating strains for the Staphylococcus reference typing phages were rendered competent for transformation by exposure to at least one of these helper phages. The use of a helper phage to confer competence to S. aureus did not result in distortion of the genetic linkages observed in an inherently competent strain. Lysogenization by phages phi11 or 83A is shown not to be required for the expression of competence, and evidence is presented which indicates that competence in the inherently competent 8325 strain is due to a helper phage effect initiated by the adsorption to cells of phi11 virion parts [or phi11 particles in the case of the single lysogen 8325-4(phi11)] that have been liberated by prophage induction.     

Evidence for common binding sites for ferrichrome compounds and bacteriophage phi 80 in the cell envelope of Escherichia coli.

Mutants ton A and ton B of Escherichia coli K12, known to be resistant to bacteriophage phi80, were found to be insensitive as well to albomycin, an analogue of the specific siderochrome ferrichrome. Ferrichrome at micromolar concentrations strongly inhibited plaque production by phi80. Preincubation with ferrichrome did not inactivate the phage. At a concentration at which ferrichrome allowed 90% inhibition of plaque formation, the chromium analogue of ferrichrome showed no detectable activity. Similarly, ethylenediaminetetraacetic acid, ferrichrome A, and certain siderochromes structurally distinct from ferrichrome, such as ferrioxamine B, schizokinen, citrate, and enterobactin, did not show detectable inhibitory activity. However, rhodotorulic acid showed moderate activity. A host range mutant of phi80, phi80h, was also inhibited by ferrichrome, as was a hybrid of phage lambda possessing the host range of phi80. However, phage lambdacI- and a hybrid of phi80 possessing the host range of lambda were not affected by ferrichrome. Finally, ferrichrome and chromic deferriferrichrome were shown to inhibit adsorption of phi80 to sensitive cells, ferrichrome giving 50% inhibition of adsorption at a minimal concentration of 8 nM. It is suggested that a component of the ferrichrome uptake system may reside in the outer membrane of E. coli K12 and may also function as a component of the receptor site for bacteriophage phi80, and that ferrichrome inhibition of the phage represents a competition for this common site.     

Genetic transformation in Staphylococcus aureus: isolation and characterization of a competence-conferring factor from bacteriophage 80 alpha lysates.

The competence-conferring activity in crude lysates of the staphylococcal bacteriophage 80 alpha was concentrated and purified by (NH4)2SO4 precipitation, differential ultracentrifugation and rate-zonal centrifugation through Ficoll. This concentrated preparation exhibited lytic activity toward assay cells of Staphylococcus aureus 8325-4 that could not be attributed to the residual 80 alpha infectious particles present. Electron microscopic examination of the concentrated competence-conferring activity revealed an occasional intact but empty virion and large numbers of free phage tails. Sodium dodecyl sulfate-polyacrylamide gel analysis of this material confirmed that the competence-conferring activity contained only some, but not all, of the major virion proteins. The competence-conferring activity exhibited single-hit kinetics when assay cells and 80 alpha transfecting deoxyribonucleic acid were present in excess. The competence-conferring activity thus seems to be a unique morphogenic precursor of the 80 alpha virion that mediates transfection and transformation in the presence of 0.1 M CaCl2.     

鲍曼不动杆菌烈性噬菌体的分离与纯化

利用柱层析方法,纯化鲍曼不动杆菌（Acinetobacter baumannii）烈性噬菌体AB1。首先采用聚乙二醇6000沉淀方法,初步分离裂解液中的噬菌体,噬菌体纯度由6.1×1010 pfu/mg提高到37×1010 pfu/mg,噬菌体回收率为58.8%,蛋白质去除率为90.6%;噬菌体粗提样品经Sepharose 4B凝胶过滤层析柱进一步纯化,纯度提高到73×1010 pfu/mg,噬菌体回收率为95.7%,蛋白质去除率为48.1%;收集的噬菌体样品最后经DEAE-52阴离子交换层析柱处理,噬菌体纯度为40×1010 pfu/mg,回收率为50.8%,蛋白去除率15.6%。内毒素分析结果显示,Sepharose 4B凝胶过滤层析纯化的噬菌体样品中,内毒素含量为443.8 EU/mg,而DEAE-52阴离子交换层析纯化的噬菌体样品中,内毒素含量为544.4 EU/mg。实验结果显示,PEG沉淀方法与Sepharose 4B凝胶过滤方法能够有效地提高噬菌体纯度,而DEAE-52阴离子交换层析则不能提高噬菌体的纯度,也无法有效地去除样品中的内毒素。     

Molecular evidence for a new bacteriophage of Borrelia burgdorferi

We have recovered a DNase-protected, chloroform-resistant molecule of DNA from the cell-free supernatant of a Borrelia burgdorferi culture. The DNA is a 32-kb double-stranded linear molecule that is derived from the 32-kb circular plasmids (cp32s) of the B. burgdorferi genome. Electron microscopy of samples from which the 32-kb DNA molecule was purified revealed bacteriophage particles. The bacteriophage has a polyhedral head with a diameter of 55 nm and appears to have a simple 100-nm-long tail. The phage is produced constitutively at low levels from growing cultures of some B. burgdorferi strains and is inducible to higher levels with 10 microg of 1-methyl-3-nitroso-nitroguanidine (MNNG) ml(-1). In addition, the prophage can be induced with MNNG from some Borrelia isolates that do not naturally produce phage. We have isolated and partially characterized the phage associated with B. burgdorferi CA-11.2A. To our knowledge, this is the first molecular characterization of a bacteriophage of B. burgdorferi.     

Endo-N-acetylneuraminidase associated with bacteriophage particles.

A bacteriophage (phi 1.2) has been isolated for Escherichia coli K235 (O1:K1:H-). phi 1.2 is specific for the host capsular polysaccharide (colominic acid). The phage forms plaques with acapsular halos and thus carries a glycanase activity for colominic acid, a homopolymer of alpha (2 leads to 8)-linked N-acetylneuraminic acid (NeuNAc) residues. Upon incubation with purified phi 1.2 particles, a solution of K1 polysaccharide loses viscosity and consumes increasing amounts of periodate. Also, by gel filtration, the production of colominic oligosaccharides (down to a size of two to three NeuNAc residues) can be demonstrated. No NeuNAc monomers, however, are formed. The capsules of E. coli strains with the K92 antigen, which consists of NeuNAc residues linked by alternating alpha (2 leads to 8) and alpha (2 leads to 9) bonds, are also depolymerized by the phi 1.2 enzyme. Under the electron microscope, phage phi 1.2 is seen to belong to Bradley's morphology group C (D. E. Bradley, Bacteriol. Rev. 31:230-314, 1967); it has an isometric head, carrying a baseplate with six spikes. By analogy to other virus particles with host capsule depolymerase activity, it is probable that the phi 1.2 endo-N-acetylneuraminidase activity is associated with these spikes.     

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.     

Intracellular neutralization of bacteriophage T4 by antiphagic serum

Specific antiserum, introduced into the spheroplasts of Escherichia coli B infected with bacteriophage T4, has been shown to neutralize phage particles formed within the cells.     

Molecular relationships among serogroup B bacteriophages of Staphylococcus aureus.

The typing bacteriophages 55, 80, 83A, and 85 of Staphylococcus aureus, representative of the three major lytic groups of serological group B aureophages, have been examined for relatedness of their genomes and virion proteins. Phages 11 and 80 alpha were also examined to determine the relationship of phage 80 alpha to phages 11 and 80. Total genome hybridization measurements divided the phages into two groups. Phages 55 and 80, in the first group, had DNA homology of 50%. Phages 11, 80 alpha, 83A, and 85 formed a second group with 27 to 65% homology. Homology between the two groups was in the range of 14 to 22%. Phage 80 alpha is more closely related to phage 11 than to phage 80, though it is probably not a simple recombinant of phages 11 and 80. Restriction enzyme digestion and phage [32P]DNA hybridization analysis of the endonuclease-generated fragments from each phage DNA confirmed the findings of the DNA homology measurements. The endonuclease fragment patterns generated by EcoRI and HindIII were distinctive for each phage, confirming that none of the phages are closely related. Common sequences were present in most fragments from the phage DNAs when the labeled probe DNA was from a different phage in the same group. Cross-group probing of endonuclease fragments revealed both a diminished level of homology when similar sequences were present and the probable absence of some sequences. Virion proteins, examined by polyacrylamide gel electrophoresis, were similar in number and molecular weight for phages 11, 80 alpha, 83A, and 85, reflecting the DNA homology analyses. The virion proteins from phages 55 and 80, however, were more distinctive, and both differed from the phages in the other group.     

Assembly of bacteriophage phi X174: identification of a virion capsid precursor and proposal of a model for the functions of bacteriophage gene products during morphogenesis.

A capsomeric structure sedimenting with an S value of 108 in sucrose gradients was isolated from Escherichia coli infected with bacteriophage phi X174. The 108S material contained viral proteins F, G, H, and D, and the relative amounts of these proteins in the 108S material were similar to those in the infectious 132S particle, which has previously been described as a possible intermediate in the assembly of 114S phage particles. Electron micrographs indicated that the size and shape of the 108S material resemble those of the 132S particle. The 108S material contained no DNA, and its formation occurred independently of DNA synthesis. The 108S material accumulated in infected cells when viral DNA replication was prevented either by mutation in phage genes A or C or by removal of thymidine from a culture infected with wild-type phage or with a lysis gene E mutant. Upon restoration of thymidine to cells infected with the lysis gene E mutant and then starved of thymidine, the accumulated 108S material was converted to 132S particles and to 114S phage particles, implying that the 108S material is a precursor of phage particles. A model that proposes possible functions for the products of phi X174 genes A, B, C, D, F, and G during viral replication and phage maturation is described.     

Characterization of a bacteriophage that carries the genes for production of Shiga-like toxin 1 in Escherichia coli.

The Shiga-like toxin 1-converting bacteriophage H-19B was recently shown to carry the structural genes for the toxin and was shown to have DNA sequence homology with phage lambda. We present evidence that the linear genome of bacteriophage H-19B has cohesive termini which become covalently associated during prophage integration. Integration occurs through a site on a 4-kilobase-pair EcoRI fragment located near the center of the bacteriophage chromosome. The relationship between bacteriophages H-19B and lambda was examined by Southern hybridization. Homologous regions were mapped on the respective chromosomes which corresponded to the regions of the J gene, the int-xis area, and the O and P genes of phage lambda. The H-19B tox genes were mapped to the right of the O and P gene homology, which was far away from the phage attachment site. We concluded that H-19B is a lambdoid bacteriophage. Unlike other toxin-converting bacteriophages, the toxin genes were not located adjacent to the phage attachment site. It appeared that the Shiga-like toxin 1 genes were not picked up by a simple imprecise prophage excision. H-19B could, however, have acquired chromosomally located toxin genes by a series of events involving deletion and duplication followed by aberrant excision.     

Morphogenesis of Bacteriophage φ80: Identification of the Cistron 13 Product

An in vitro complementation reaction leading to the assembly of bacteriophage phi80 tails from component proteins is described. Tail assembly occurs when a lysate of any mutant in cistron 13 is mixed with a second lysate of a mutant in any of the other cistrons involved in tail formation. Lysates of mutants that are blocked in tail formation contain phage heads that can unite with free tails to form infective particles. The rate of the complementation reaction shows little dependence upon temperature, suggesting that the assembly depends largely upon the kinetic encounter of the interacting components. The tail component missing in cistron 13 mutant lysates was purified approximately 55-fold and shown to be, at least in part, a protein having a molecular weight of approximately 22,000. This protein was also released from highly purified infective phi80 particles after osmotic shock followed by heattreatment, suggesting that it most probably is an integral structural protein of the phage tail. Lysates of mutants of bacteriophage lambda that are defective in tail formation were shown to contain a tail component identical with or similar to the phi80 cistron 13 product.     

Bacteriophage and bacteriophage resistance in lactic acid bacteria

Abstract: The study of bacteriophage-host interactions has been instrumental in the development of genetic systems in many genera, and laid many of the foundations of modern molecular genetics. Research into bacteriophage and bacteriophage resistance in the lactic acid bacteria has moved into a new and exciting dimension in recent years. Mechanisms such as adsorption inhibition, restriction and modification, and abortive infection which have been detected and described phenotypically over the past decade are now being subjected to molecular analysis, and this has led to a better understanding of the nature and variety of resistance systems employed by lactic acid bacteria to combat phage attack. In addition, analysis of different bacteriophage has increased our knowledge of these ubiquitous particles to the point where it is possible to construct novel phage resistances based on the phage genome itself. This review outlines the recent progress in the molecular analysis of bacteriophage, bacteriophage resistance and counter resistance, and the construction of novel resistance mechanisms.     

SEROLOGY AND TRANSDUCTION IN STAPHYLOCOCCAL PHAGE.

Dowell, C. E. (The University of Texas, Dallas) and E. D. Rosenblum. Serology and transduction in staphylococcal phage. J. Bacteriol. 84:1071-1075. 1962.-A triply lysogenic strain of Staphylococcus aureus was shown to carry a serological group B phage capable of transduction. Three typing phages (53, 80, 42D), either belonging to serological group B or having a close association with it, were also shown to have transducing ability. A rapid screening method was used to isolate two new transducing phages, both of which belonged to serological group B. Propagating strain 42B/47C was found to carry a transducing phage that was neutralized by both group B and group F antisera. Nine other phages belonging to serological groups other than group B did not have generalized transducing ability, nor did three group B typing phages that were atypical in their calcium requirement. It was postulated that transducing ability is associated with staphylococcal phages of serological group B and with related phages of group F.     

Alkylation of T7 bacteriophage blocks superinfection exclusion

Alkylation of T7 bacteriophage by methyl methane sulfonate blocked superinfection exclusion. This blockage could be correlated with a delay in the synthesis of phage-specific proteins. Therefore we conclude that protein synthesis directed by the primary infecting phage is required for efficient exclusion of superinfecting phage particles.     

Isolation and characteristics of the bacteriophage from the vaccine strain Tohama phase I

Some properties of bacteriophage phi T isolated from the vaccine strain Bordetella pertussis Tohama phase I and propagated in Bordetella parapertussis 504 cells are presented. Phage phi T belongs to the IV group in accordance with Tikhonenko classification. The diameter of head and length of noncontractile tail sheath are 49.5 +/- 0.5 and 145 +/- 7 nm, respectively. Diameter of the tail sheath is 3.2 +/- 0.6 nm. Molecular mass of the phage DNA is 37 +/- 3 kb. Population of phi T phage is polymorphous and consists of particles the genomes or which vary from each other by the "insert" located 6.8 +/- 0.6 kb from the end of molecule. The blot hybridization has demonstrated that the bacteriophage genome is not inserted into the chromosome of the lysogenic strain. Autonomous location of the phage genome in the host cell is suggested. The temperature and hydrogen ions concentration effects on bacteriophage phi T stability were studied. The conditions for phage suspension storage are described.     

Knotting of DNA molecules isolated from deletion mutants of intact bacteriophage P4.

DNA molecules isolated from tailless phage particles (capsids) of bacteriophage P4 virl del10 are known to be knotted. We have found by electron microscopy that 80% of DNA molecules isolated from intact phage particles of P4 virl del10 also contained knots. This observation indicates that the predominant form of P4 virl del10 DNA within the intact phage particle is either knotted or in a configuration that permits knotting upon isolation. In comparison to P4 virl del10 (deleted 1000 basepairs), DNA molecules isolated from intact P4 virl del2 (deleted 650 basepairs) and P4 virl (non-deleted) contained 50% and 15% knots respectively, showing an association of decreased size of deletion of DNA with a decreased fraction of knotted genomes.     

Photoinactivation of bacteriophage PM2 by cyanine dyes]

Photoinactivation of the lipid-containing bacteriophage PM2 by visible light and cyanine dyes (carbo- and dicarbocyanines), aluminum phtalocyanine tetrasulfonate and methylene blue was studied. It was concluded that cyanine dye aggregates adsorbed on phage particles and oxygen are essential for phage photoinactivation.     

Alteration of bacteriophage attachment capacity by near-UV irradiation.

Near-UV (NUV) (300 to 400 nm) and far-UV (FUV) (254 nm) radiations damage bacteriophage by different mechanisms. Host cell reactivation, Weigle reactivation, and multiplicity reactivation were observed upon FUV, but not upon NUV irradiation. Also, the number of his+ recombinants increased with P22 bacteriophage transduction in Salmonella typhimurium after FUV, but not after NUV irradiation. This loss of reactivation and recombination after NUV irradiation was not necessarily due to host incapability to repair phage damage. Instead, the phage genome failed to enter the host cell after NUV irradiation. In the case of NUV-irradiated T7 phage, this was determined by genetic crosses with amber mutants, which demonstrated that either "all" or "none" of a T7 genome entered the Escherichia coli cell after NUV treatment. Further studies with radioactively labeled phage indicated that irradiated phage failed to adsorb to host cells. This damage by NUV was compared with the protein-DNA cross-link observed previously, when phage particles were irradiated with NUV in the presence of H2O2. H2O2 (in nonlethal concentration) acts synergistically with NUV so that equivalent phage inactivation is achieved by much lower irradiation doses.     

Superinfection exclusion by bacteriophage T7.

Only two of the early genes of bacteriophage T7 were found to play a significant role in exclusion of superinfecting bacteriophage T3 particles; genes 0.3 and 1. Protein synthesis by the preinfecting phage particle was not required for efficient exclusion. These findings are discussed with regard to the known functions of these genes during T7 development.