Phage-typing ofSalmonella panama according to the method of Craigie and Yen

A phage-typing scheme forSalmonella panama has been established. This was done according to the method of Craigie and Yen by means of phage preparations which are all derived from a single phage. So far, eight phage types and a restgroup Z could be demonstrated by means of phage 47 and seven adaptations derived from it.     

Isolation of nonlysogenic bacteria from lysogenic ones using a phage carrying resistance to antibiotics

The method for obtaining nonlysogenic bacteria from lysogenic ones by means of a phage carrying resistance to antibiotics is proposed. Solitary nonlysogenic cells in a lysogenic culture are lysogenized after the infection of the culture with a labeled phage and then harvested on a selective medium: under special conditions the phage is eliminated from the cells.     

The interaction of phage and biofilms

Biofilms present complex assemblies of micro-organisms attached to surfaces. they are dynamic structures in which various metabolic activities and interactions between the component cells occur. When phage come in contact with biofilms, further interactions occur dependent on the susceptibility of the biofilm bacteria to phage and to the availability of receptor sites. If the phage also possess polysaccharide-degrading enzymes, or if considerable cell lysis is effected by the phage, the integrity of the biofilm may rapidly be destroyed. Alternatively, coexistence between phage and host bacteria within the biofilm may develop. Although phage have been proposed as a means of destroying or controlling biofilms, the technology for this has not yet been successfully developed.     

细菌群体感应系统与噬菌体的相互作用机制研究进展

细菌在与噬菌体的长期共进化过程中形成多种抵抗噬菌体侵染的机制,其中群体感应参与的细菌抵御噬菌体侵染机制成为近年来的研究热点。群体感应与噬菌体之间的相互作用是复杂和多样的,本文将重点综述群体感应在噬菌体侵染中的作用、调控在噬菌体裂解-溶源转变的作用,以及群体感应与噬菌体的其他相互影响等内容,为噬菌体在细菌性疾病的治疗提供理论依据。     

Reparation of UV-damaged bacteriophage ed]

A repair of UV-damaged phage DNA in the "phage-host" system in accordance with the excision reparative mechanism is demonstrated by means of centrifugation in alkaline sucrose gradient of virulent 3H-thymidine labelled phage sd. The increase of the transfectants quantity of UV-irradiated DNA on uvr+ bacteria compatibly to uvr- bacteria evidences that the bacterial host participates in phage reparation. Caffeine inhibition of UV-irradiated phage sd survival confirms the participation of cell-host in reparation of UV-damaged phage.     

In utero detection of T7 phage after systemic administration to pregnant mice

The phage is used as a scaffold to display recombinant libraries of peptides, which provides the means to rescue and amplify peptides that bind target macromolecules. Many reports showed that the T7 phage display method can be used to obtain a ligand-binding peptidefor tissue-targeted therapies in adult animals. In utero tissue targeting of fetal tissues may help in the correction of many genetic and metabolic diseases. Here we demonstrate the distribution and detection of T7 phage displaying the C-X7-C peptide library in mouse fetal tissues after systemic injection of T7 phage into pregnant mouse tail vein. T7 phage was recovered from fetal tissues 15 min after injection of T7 phage. Our results suggest that T7 phage may be a useful tool in selecting the tissue-specific ligand-binding peptide for fetal tissues. This approach may be helpful in designing in utero tissue-targeted therapies.     

Application of phage-displayed single chain antibodies in western blot

A phage display single chain fragment variable library constructed on pⅢ protein of M13 filamentous phage was screened using B-lymphocyte stimulator and FP248 as selective molecules. After four rounds of panning, there was a remarkable enrichment in the titer of bound phages. Twenty phage clones were selected from the last round and screened by means of phage-ELISA. With the antibody phages as primary antibodies in Western blot, we developed a method for detecting the specific antigen. The dilutions of antibody phages depend on the affinity between antibody-displayed phage particles and antigens.     

Interchangeability of the adsorption proteins of bacteriophages Ff and IKe.

The wild-type adsorption protein (g3p) of filamentous phage IKe cannot be exchanged with its analogous protein in the related Ff (M13, fd, and f1) phage particles. Deletion mutants of the protein, however, are assembled into Ff phage particles. These hybrid Ff phage particles bearing deleted IKe g3p attach to N pili, thus conserving the host attachment property of the protein but not its infection-initiating function. This means that the attachment specificity is determined by IKe g3p independently of other phage components in contact with it. Infection initiation function, the process in which phage DNA is released into the host, in contrast seems to require either more complex structural features of the protein (for example, a certain oligomeric structure) provided only in the original particle, or a concerted action of g3p with another particle component, not replaceable by its homologous counterpart in the related phage.     

A specialized transducing phage, lambda psrlA, for the sorbitol phosphotransferase of Escherichia coli K12

A specialized transducing phage for the srlA gene, specifying the sorbitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system, was constructed and its DNA was analysed by restriction endonuclease digestion. Phage construction involved four steps: (1) integration of lambda into the srlA gene; (2) selection of phage carrying (a) the left and (b) the right end of the srlA gene by means independent of the function of the new DNA acquired; (3) reconstitution of the srlA gene in a dilysogen of these two phage; and (4) the excision, using the heteroimmune lambdoid phage 21, of a plaque-forming srlA+ phage from the dilysogenic chromosome. Comparison of the DNA restriction digests of the transducing phage with those of its parents and of wild-type lambda revealed fragments consisting partly of lambda and partly of Escherichia coli DNA. The junction points in the intermediate phage define a site that must lie within the reconstituted gene of the final phage. This technique should be of general application in relating genes, cloned by our method, to DNA sequences.     

Optimizing bacteriophage plaque fecundity

Bacteriophages (phages), the viruses of bacteria, form visible lesions within bacterial lawns (called plaques), which are employed ubiquitously in phage isolation and characterization. Plaques also can serve as models for phage population growth within environments that display significant spatial structure, e.g. soils, sediments, animal mucosal tissue, etc. Furthermore, phages growing within plaques, in experimental evolution studies, may become adapted to novel conditions, may be selected for faster expansion, or may evolve toward producing more virions per plaque. Here, we examine the evolution of the latter, greater plaque fecundity, considering especially tradeoffs between phage latent period and phage burst size. This evolution is interesting because genetically lengthening latent periods, as seen with phage lysis-timing mutants, should increase phage burst sizes, as more time is available for phage-progeny maturation during infection. Genetically shortening latent periods, however, is a means toward producing larger phage plaques since phage virions then can spend more time diffusing rather than infecting. With these larger plaques more bacteria become phage infected, resulting in more phage bursts. Given this conflict between latent period's impact on per-plaque burst number versus per-infection burst size, and based on analysis of existing models of plaque expansion, we provide two assertions. First, latent periods that optimize plaque fecundity are longer (e.g. at least two-fold longer) than latent periods that optimize plaque size (or that optimize phage population growth within broth). Second, if increases in burst size can contribute to plaque size (i.e. larger plaques with larger bursts), then latent-period optima that maximize plaque fecundity should be longer still. As a part of our analysis, we provide a means for predicting latent-period optima-for maximizing either plaque size or plaque fecundity-which is based on knowledge of only phage eclipse period and the relative contribution of phage burst size versus latent period toward plaque size.     

Orientation of the tox gene in the prophage of corynebacteriophage beta.

The orientation of the gene for diphtheria toxin, tox, in the prophage of converting corynebacteriophage beta has been determined. The orientation of tox in prophage and that reported simultaneously by Holmes (1976) for vegetative phage are compatible with the hypothesis that beta phage is inserted into the chromosome of its bacterial host by means of a mechanism similar to that described for lambda phage, and that the phage attachment site lies between the tox and imm genes. The position of three tox mutations that are phenotypically CRM- has also been determined. Relative to the tox-45 mutation, they are located more proximally to the end of the tox structural gene that corresponds to the amino terminal of diphtheria toxin.     

60Co irradiation of Shiga toxin (Stx)-producing Escherichia coli induces Stx phage

Shiga toxin (Stx)-producing Escherichia coli (STEC), an important cause of hemolytic uremic syndrome, was completely killed by (60)Co irradiation at 1 x l0(3) gray (1 kGy) or higher. However, a low dose of irradiation (0.1-0.3 kGy) markedly induced Stx phage from STEC. Stx production was observed in parallel to the phage induction. Inactivation of Stx phage required a higher irradiation dose than that for bacterial killing. Regarding Stx, cytotoxicity was susceptible to irradiation, but cytokine induction activity was more resistant than Stx phage. The findings suggest that (1). although (60)Co irradiation is an effective means to kill the bacteria, it does induce Stx phage at a lower irradiation dose, with a risk of Stx phage transfer and emergence of new Stx-producing strains, and (2). irradiation differentially inactivates some activities of Stx.     

Encapsulation of Bacteriophage in Liposome Accentuates Its Entry in to Macrophage and Shields It from Neutralizing Antibodies

Phage therapy has been a centre of attraction for biomedical scientists to treat infections caused by drug resistant strains. However, ability of phage to act only on extracellular bacteria and probability of interference by anti-phage antibodies in vivo is considered as a important limitation of bacteriophage therapy. To overcome these hurdles, liposome were used as delivery vehicle for phage in this study. Anti-phage antibodies were raised in mice and pooled serum was evaluated for its ability to neutralize free and liposome entrapped phage. Further, ability of phage and liposome-entrapped phage to enter mouse peritoneal macrophages and kill intracellular Klebsiella pneumoniae was compared. Also, an attempt to compare the efficacy of free phage and liposome entrapped phage, alone or in conjunction with amikacin in eradicating mature biofilm was made. The entrapment of phage in liposome provided 100% protection to phage from neutralizing antibody. On the contrary un-entrapped phage got neutralized within 3 h of its interaction with antibody. Compared to the inability of free phage to enter macrophages, the liposome were able to deliver entrapped phage inside macrophages and cause 94.6% killing of intracellular K. pneumoniae. Liposome entrapped phage showed synergistic activity along with amikacin to eradicate mature biofilm of K. pneumoniae. Our study reinforces the growing interest in using phage therapy as a means of targeting multidrug resistant bacterial infections as liposome entrapment of phage makes them highly effective in vitro as well as in vivo by overcoming the majority of the hurdles related to clinical use of phage.     

Genetic and Immunological Studies of Bacteriophage T4 Thymidylate Synthetase

Thymidylate synthetase, which appears after infection of Escherichia coli with bacteriophage T4, has been partially purified. The phage enzyme is immunologically distinct from the host enzyme and has a molecular weight of 50,000 in comparison to 68,000 for the host enzyme. A system has been developed to characterize T4 td mutants previously known to have impaired expression of phage thymidylate synthetase. For this system, an E. coli host lacking thymidylate synthetase was isolated. Known genetic suppressors were transduced into this host. The resulting isogenic hosts were infected with phage T4 td mutants. The specific activities and amounts of cross-reacting material induced by several different types of phage mutants under conditions of suppression or non-suppression have been examined. The results show that the phage carries the structural gene specifying the thymidylate synthetase which appears after phage infection, and that the combination of plaque morphology, enzyme activity assays, and an assay for immunologically cross-reacting material provides a means for identifying true amber mutants of the phage gene.     

Molecular genetics of SaPI1--a mobile pathogenicity island in Staphylococcus aureus

The Staphylococcus aureus gene for toxic shock toxin (tst) is carried by a 15 kb mobile pathogenicity island, SaPI1, that has an intimate relationship with temperate staphylococcal phage 80alpha. During phage growth, SaPI1 is excised from its unique chromosomal site, attC, replicates autonomously, interferes with phage growth, and is efficiently encapsidated into special small phage heads commensurate with its size. Upon transfer to a recipient organism, SaPI1 integrates at attC by means of a self-coded integrase. One or more phage functions are required for excision, autonomous replication and encapsidation of the element and, thus, the overall relationship between SaPI1 and 80alpha is similar to that between coliphages P4 and P2. Among other staphylococcal phages tested, only phi13 interacts with SaPI1, inducing excision but not replication or transfer of the element.     

A helper phage to improve single-chain antibody presentation in phage display

We show here that the number of single-chain antibody fragments (scFv) presented on filamentous phage particles generated with antibody display phagemids can be increased by more than two orders of magnitude by using a newly developed helper phage (hyperphage). Hyperphage have a wild-type pIII phenotype and are therefore able to infect F(+) Escherichia coli cells with high efficiency; however, their lack of a functional pIII gene means that the phagemid-encoded pIII-antibody fusion is the sole source of pIII in phage assembly. This results in an considerable increase in the fraction of phage particles carrying an antibody fragment on their surface. Antigen-binding activity was increased about 400-fold by enforced oligovalent antibody display on every phage particle. When used for packaging a universal human scFv library, hyperphage improved the specific enrichment factor obtained when panning on tetanus toxin. After two panning rounds, more than 50% of the phage were found to bind to the antigen, compared to 3% when conventional M13KO7 helper phage was used. Thus, hyperphage is particularly useful in stoichiometric situations, when there is little chance that a single phage will locate the desired antigen.     

Antibody VH and VL recombination using phage and ribosome display technologies reveals distinct structural routes to affinity improvements with VH-VL interface residues providing important structural diversity

In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.     

Filamentous bacteriophage display of a bifunctional protein A::scFv fusion

Filamentous bacteriophage display is a powerful and widely used technology for the selection of affinity ligands. However, the commonly used phagemid systems result in the production of a population of phage of which those displaying the ligand of interest represent only a small proportion. Through simple dilution and nonspecific binding effects, the presence of large numbers of ligand-free phage reduces the likelihood that weak binders will be successfully selected from a ligand library. To provide a means of avoiding such problems, we have introduced an affinity handle into the phage that permits the purification of ligand-displaying phage. The IgG binding domains ofStaphylococcus ciureus protein A (SpA) were fused to a ligand (single chain Fv[scFv]) which is displayed as a fusion with the phage surface protein ApIII. Phage-displaying SpA were separated by affinity chromatography using immobilized human IgG from non-displaying phage and the purified phage were shown to possess functional scFv. Comparisons of fusion proteins in which either the scFv or the affinity handle occupied the amino terminus of the fusion protein showed that, whereas SpA function was unaffected by position, scFv function was compromised when the scFv did not occupy the amino terminus.     

Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains.

The display of proteins on the surface of phage offers a powerful means of selecting for rare genes encoding proteins with binding activities. Recently we found that antibody heavy and light chain variable (V) domains fused as a single polypeptide chain to a minor coat protein of filamentous phage fd, could be enriched by successive rounds of phage growth and panning with antigen. This allows the selection of antigen-binding domains directly from diverse libraries of V-genes. Now we show that heterodimeric Fab fragments can be assembled on the surface of the phage by linking one chain to the phage coat protein, and secreting the other into the bacterial periplasm. Furthermore by introducing an amber mutation between the antibody chain and the coat protein, we can either display the antibody on phage using supE strains of bacteria, or produce soluble Fab fragment using non-suppressor strains. The use of Fab fragments may offer advantages over single chain Fv fragments for construction of combinatorial libraries.     

Behaviour of temperate phage Mu in Salmonella typhi

We have developed a convenient system for genetic analysis of Salmonella typhi exploiting the properties of the mutator phage Mu. In spite of the fact that wild-type Salmonella typhi strains do not allow Mu to form plaques on them, we have shown that these strains are actually sensitive to the phage. It proved possible to use Mu to induce mutations and to promote intra- and interspecific genetic transfer, without having to introduce the phage into the bacteria by means other than infection. Furthermore, we isolated Salmonella typhi derivatives on which Mu formed plaques, and studied the behaviour of Mu in these and wild-type strains.