Cutaneous hypersensitivity test to evaluate phage display anti-tick borne vaccine antigen candidates

Early experiments performed by our group with the phage display technique revealed the potential for using epitope-displaying phages (mimotopes) as a tool for tick antigen discovery. Thus, as a preliminary study, inflammatory reactions induced by phage display tick-borne candidates were investigated by using the cutaneous hypersensitivity test. The profile of selected Rhipicephalus microplus mimotopes was assessed on tick field-exposed cattle and our data indicated a pattern similar to immediate hypersensitivity reaction and not a delayed immune response as expected. However, the wild-type phage inoculation surprisingly induced a strong immediate response on its own. Such reactions indicate that the wild-type phage may have hidden many of the potential reactions raised by the mimotopes. The study of the inflammatory reactions to these phage mimotopes in tick-infested hosts may provide basic information about the immune reaction. Finally, this work is of relevance for when considering research alternatives for finding and characterization of antigens by the phage display technique.     

The formation of bacteriophages in mixed cultures by recombination of genetic elements; characterisation of phage types ofSalmonella paratyphi B by phage reactions and lysogenic properties

Summary It was shown that bacteriophages, generated in mixed cultures of strains ofS. paratyphi B of different types are formed by recombination of elements from both strains. The characteristics of the bacteriophages found depend upon the “phage type” of the strains inoculated in the medium. Types ofS. paratyphi B can be characterised by a combination of phage reactions and lysogenic properties.     

In vitro and in vivo recombination-related reactions of Escherichia coli recA protein and glucosyl-hydroxymethyl-deoxycytidine DNA

Summary Recombination of T4 phage is not controlled by the host recA gene but by an analogous phage gene, uvsX. We have tested the hypothesis that recA protein is inactive in T4-infected cells because it is unable to catalyze reactions involving single stranded DNA containing glucosyl-hydroxylmethyl-deoxycytidine. We found, however, that with modified and unmodified deoxycytidine containing DNAs, uvsX protein and recA protein catalyze in vitro reactions related to DNA recombination, but in T4-infected cells recA protein fails to promote strand transfer of DNA which contains unmodified deoxycytidine.Abbreviations dC-DNA deoxycytidine containing DNA - dC-T4 T4 phage containing dC-DNA - dHMC-DNA glucosyl-hydroxymethyl-deoxycytidine containing DNA - dsDNA double stranded DNA - gp gene product - ssDNA single stranded DNA     

Der Einfluß der Infektionsmultiplizität auf die Lysogenisierung im System Salmonella typhimurium — Phage P 22

Summary The increase of lysogenization in phage infected cells has been investigated with increasing multiplicities of infection in the system Salmonella thyphimurium-phage P 22. The increase of infection resp. lysis and lysogenization with multiplicity follows first order reaction kinetics as concluded from multiplicities<0.3. Under the experimental conditions employed, the probability per phage is 0.57 for lysogenization and 0.43 for lysis. If multiplicity is>0.3 and cells are infected with more than one phage, the lysogenizations increase according to one hit kinetics, whereas the lysis of cells decreases. It is concluded, that lytic reactions in multicomplexes, which can be initiated independently by every one of the infecting phage particles will be suppressed by lysogenic reactions initiated by other independently infecting phages of the complex. Our experiments suggest, that immunity of the prelysogenic condition is the process responsible for the suppression of the lytic reaction. Therefore, in multicomplexes the immunity induced by one of the infecting phages is superimposed upon the one hit lytic infection causing the percentage of lysogenization increasing with multiplicity.     

PCR preparation of DNA inserts from lambda and plasmid vectors for RFLP mapping

A simplified method is described for preparing insert DNA for labelling reactions to be used in Southern hybridization. This method works with sequences cloned into both plasmid and lambda phage, and eliminates many of the steps leading to the labelling reaction. Small quantities of hostE. coli or lambda phage carrying a probe sequence are lysed and amplified via the polymerase chain reaction using standard sequencing primers. Unincorporated nucleotides are removed by ethanol precipitation or gel purification and insert DNA is ready for radio-labelling. This method reduces the time and expense associated with conventional insert preparation, and greatly simplifies the use of sequences cloned into lambda phage.     

Some properties ofStreptococcus thermophilus bacteriophages

The morphology, host range, structural proteins and serological properties ofStreptococcus thermophilus phages isolated from Finnish cheese plants were investigated. The results show that all the nine phages belong morphologically to Ackermann’s group B1. The host—phage reactions and plating efficiency justify the division of these phages into four specificity groups. Most of the phages showed an absolute host specificity as to their plating efficiency but were not strictly specific in the adsorption to different hosts. The electrophoretic profiles of the structural proteins appeared nearly identical. Ten to eleven well separated proteins could be detected. The antiserum raised against one of the phages contained antibodies with different neutralization capacity depending on the phage. Using an immunoblotting technique, four structural proteins were detected that could bind phage antibodies.     

Isolation and characterization ofEscherichia coli O157:H7 strains in China

Five strains ofEscherichia coli O157:H7 were isolated from 486 stool specimens collected in 1986, 1987, and 1988 from patients with diarrhea in Xuzhou City, Jiangsu Province, China; 21 of the specimens were from patients with bloody diarrhea. The biochemical reactions of all five strains were almost identical with those of the well-knownE. coli O157:H7 strain 933. All of the strains were found to carry a 60 Md plasmid and two small plasmids. The plasmid DNA Hind 111 restriction patterns were identical. The strains were lysed byE. coli typing phage E1, E2, and E3, but not by E4 or E5. Data suggested that it might belong to a single phage or plasmid group. All strains produced vero toxin and caused diarrhea and death in infant rabbits and mice.     

Partial purification of an activity from human cells that promotes homologous pairing and the formation of heteroduplex DNA in the presence of ATP

Summary An activity that can promote homologous pairing and strand transfer between suitable DNA substrates has been partially purified from human skin fibroblasts and from Hela cells. The strand transfer reaction was investigated with DNA substrates consisting of single-stranded circular and duplex linear phage DNA. It requires ATP, and under optimal conditions yields heteroduplex molecules containing one strand from each parental DNA substrate. The reactions appears to be of the same general nature as those mediated by RecA proteins of Escherichia coli and the Rec1 protein of Ustilago maydis.     

Lysogenicity as a tool for bacteriophage typing ofSalmonella Paratyphi B

Summary and Conclusions The serological identity of the phage produced by a spontaneously lysogenic strain ofS. paratyphi B depends on the bacterial type of that particular strain. The frequency of the phenomenon of spontaneous lysogenicity is such, in the case ofS. paratyphi B, that it can be utilised as a tool for typing.It is clear, however, that the possibilities of the method are limited. Firstly, types exist that do not as a rule produce bacteriophages. Secondly, alysogenic strains may exist of types that as a rule are lysogenic.Therefore the method can be utilised only together with, and as a check upon, a system of phage reactions.In Holland the types Kampen and Leeuwarden are frequent types, that can readily be recognised by the identity of the phages they produce. This is sufficient to justify the use of the method.The relation shown between true lysogenicity and bacterial types may be of theoretical interest. Perhaps it will be possible in this way to relate every existing phage to a bacterial type. The theoretical aspects will, however, be discussed elsewhere.     

Growth of coliphage BF23 on rough strains of Salmonella typhimurium: the bfe locus

Summary Coliphage BF23 develops in Salmonella typhimurium rough strains. The phage is neither restricted nor modified by S. typhimurium. The growth patterns of the phage were slightly different in S. typhimurium than in Escherichia coli, although phage propagated on S. typhimurium is identical to the phage propagated in E. coli by several criteria used. Mutants of S. typhimurium resistant to BF23 were isolated and found to map (by P22-and Plmediated transduction) in the same position as bfe mutants of E. coli. The order of genes was: metB-argC-bfe-rif-purD-metA.Phage BF23 does not form plaques on smooth S. typhimurium strains, since the phage fails to adsorb irreversibly to smooth cells. Nevertheless, on solid agar, the phage prevents growth of many (but not all) smooth strains. Moreover, UV-and alkali-inactivated phage BF23, although unable to form plaques on sensitive hosts, retains the ability to prevent growth of the host on solid medium. This ability is sensitive to protease and resistant to DNAse and RNase. Heat treatment of the phage causes rapid loss of the cell-growth-preventing-ability whereas the ability to form plaques is lost much more slowly. These results lead to a proposal that phage BF23 virions carry a colicin-like factor that kills sensitive cells.     

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

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

Influence of chitosan derivatives on the development of phage infection in theBacillus thuringiensis culture

The influence of chitosan fragments with different degrees of polymerization and some chemical chitosan derivatives on the infectionof Bacillus thuringiensis by phage 1–97 A was studied. It was shown that chitosan inhibits phage infection and inactivates phage particles. The extent of inhibition of phage infection inversely depended on the degree of polymerization of chitosan fragments. On the contrary, the extent of inactivation of phage virulence was proportional to the degree of polymerization. Chitosan derivatives did not inhibit the growth of bacilli. Deaminated chitosan derivatives at a concentration of 100 μg/ml efficiently inhibited phage reproduction, exhibiting no correlation between the degree of deamination and antiviral activity. The anionic derivative chitosan sulfate andN-succinate-6-O-sulfate did not inactivate the phage, did not influence bacterial growth, and did not inhibit the process of viral infection.     

Bacteriophage predation promotes serovar diversification in Listeria monocytogenes

Listeria monocytogenes is a bacterial pathogen classified into distinct serovars (SVs) based on somatic and flagellar antigens. To correlate phenotype with genetic variation, we analyzed the wall teichoic acid (WTA) glycosylation genes of SV 1/2, 3 and 7 strains, which differ in decoration of the ribitol‐phosphate backbone with N‐acetylglucosamine (GlcNAc) and/or rhamnose. Inactivation of lmo1080 or the dTDP‐l ‐rhamnose biosynthesis genes rmlACBD (lmo1081–1084) resulted in loss of rhamnose, whereas disruption of lmo1079 led to GlcNAc deficiency. We found that all SV 3 and 7 strains actually originate from a SV 1/2 background, as a result of small mutations in WTA rhamnosylation and/or GlcNAcylation genes. Genetic complementation of different SV 3 and 7 isolates using intact alleles fully restored a characteristic SV 1/2 WTA carbohydrate pattern, including antisera reactions and phage adsorption. Intriguingly, phage‐resistant L. monocytogenes EGDe (SV 1/2a) isolates featured the same glycosylation gene mutations and were serotyped as SV 3 or 7 respectively. Again, genetic complementation restored both carbohydrate antigens and phage susceptibility. Taken together, our data demonstrate that L. monocytogenes SV 3 and 7 originate from point mutations in glycosylation genes, and we show that phage predation represents a major driving force for serovar diversification and evolution of L. monocytogenes.     

On the site specific recombination of phage 16-3 of Rhizobium meliloti: identification of genetic elements and att recombinations

Summary The genome segment carrying the activities int and xis, responsible for integration and excision of phage 16-3, have been identified and cloned. Mutants were isolated, permitting the investigation of int, xis and att sites (attP, attR, attB) in trans arrangements. The efficiency and role of int- and xis-promoted reactions and of homologous recombination in the formation of lysogenic cells are established. The possible use of the cloned int-attP chromosomal segment in the manipulation of Rhizobium meliloti is discussed.     

Observations on Nonconverting Phage,c-n71, Obtained from a Nontoxigenic Strain of Clostridium botulinum Type C

A nontoxigenic mutant (C-N71) obtained from a toxigenic strain of Clostridium botulinum type C, Stockholm, with nitrosoguanidine treatment was found to be lysogenic by the lysis test. Although the filtrate of a passaged lysate of this nontoxigenic but lysogenic strain, C-N71, lysed cells of the nontoxigenic strain C-AO2 equally as well as the converting phage c-st obtained from the strain C-Stockholm, it did not convert C-AO2 to the toxigenic state. The lysis spectrum of this filtrate was the same as that of the c-st phage. The ability of the filtrate to lyse the indicator cells, C-AO2, was destroyed neither by trypsin nor DNase but was inactivated by heat treatment at 80 C for 10 min. This suggested that the agent which caused lysis was not boticin but probably a phage. An electron micrograph of the complete phage, c-n71, which was similar in morphology to that of the c-st phage was obtained from the filtrate of strain C-N71. Anti-c-n71 phage rabbit serum neutralized both the lytic and the converting activities of the c-st phage. These findings strongly suggest that the c-n71 phage is a mutant of the c-st phage which lacks the gene controlling production of botulinum type C toxin.     

Molecular characterization of new group A streptococcal bacteriophages containing the gene for streptococcal erythrogenic toxin A (speA)

Summary Bacteriophage T12 is the prototype phage carrying the streptococcal erythrogenic toxin A (speA) gene. To examine more closely the phages involved in lysogenic conversion, we examined 300 group A streptococcal strains, and identified and isolated two new phages that carry the speA gene. The molecular sizes of these phage genomes were between 32 and 40 kb, similar to that of phage T12 (35 kb). However, as ascertained by restriction analysis, the physical maps of the new phage genomes were different from phage T12 and from each other. Hybridization analysis also showed that all of these phages were only partially related to one another and the speA gene was always located close to the phage attachment site. Additionally, colony hybridization showed that whereas phage T12 or one of its close relatives is the most common phage associated with the group A streptococci, phage 49 has a much stronger association with the speA gene. A defective phage was also found following pulsed field gel electrophoresis of total phage DNA. This phage appears to be a resident of strain T25₃c and is found only following induction of a T25₃c lysogen. Restriction enzyme analysis of the isolated defective phage DNA suggests that it is the source of the submolar amounts of DNA previously found in association with phage T12 digestion patterns. Additionally, the defective phage may serve as the site of integration of the speA gene-carrying phages described above.     

Iterative Cycles of In Vitro Protein Selection for DNA Polymerase Activity

Abstract

Isolation of genes encoding catalysts for defined chemical reactions should be facilitated by selection of proteins for catalysis from molecular repertoires. Display of proteins on phage allows the coupling between a protein and its gene. Furthermore, if the reaction product can be linked to the phage enzyme catalyzing the reaction, affinity chromatography for the product yields the protein catalyzing the reaction and its gene. One main advantage of this selection method is that it can be in principle generalized to most chemical reactions.

Here, iterative in vitro selections for polymerase activity were used to isolate a single phage-polymerase among more than 10⁸ phage particles. Three to five selection cycles were required depending on the fraction of infectious phage-polymerases in the initial phage population. This is the first report quantifying the enrichment over iterative selection rounds for the isolation of rare catalysts displayed on phage.     

Influence of the Chitosan Oligomer on the Phage Particles and Reproduction of Phage 1-97A in the Culture of Bacillus thuringiensis

The causes of bacteriophage 1-97A inactivation by the chitosan oligomer with a polymerization degree of 15 and the influence of the oligomer on the phage reproduction in the culture of Bacillus thuringiensissubsp. galleriae, strain 1-97, were studied. The study of the inactivation kinetics showed that, in 1 h, virtually all chitosan was bound to the phage particles, causing, as evidenced by electron microscopy, DNA release from the phage head, destruction of the phage particles, and agglutination of the phage particles or of their tails in the region of the basal plate. High-polymeric chitosan caused more pronounced destruction of the phage particles than the oligomer. It was established that chitosan prevented the production of complete phage particles. One of the mechanisms of such an influence may be the production in the presence of chitosan of phage particles devoid of DNA.     

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

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

The Oligomerization of Phage T4 DNA-(Adenine-N6)-Methyltransferase and Its Effect on the Catalytic Characteristics of the Enzyme

The structural and catalytic properties of the phage T4 DNA-(adenine-N ⁶)-methyltransferase (EC 2.1.1.72) were studied at different enzyme–substrate concentration ratios by chemical crosslinking of the protein subunits and by measuring the presteady state kinetics of the reactions. Various structural states of the methyltransferase were correlated with its catalytic activity, and it was shown that the oligomeric forms of the enzyme are catalytically active but are characterized by the reaction parameters different from those of the monomer.