Phage Strains Research Articles

Tertiary initiation of replication in bacteriophage T4. Deletion of the overlapping uvsY promoter/replication origin from the phage genome.

Tertiary initiation of bacteriophage T4 DNA replication is resistant to the RNA polymerase inhibitor rifampicin and apparently involved in the activity of recombination hot spots in the T4 genome (Kreuzer, K. N., and Alberts, B. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3345-3349). One of the origins that function by the tertiary mechanism maps at the promoter for gene uvs Y. A deletion and a linker-insertion mutation in the uvsY promoter/origin region were generated by in vitro manipulations and then placed into the T4 genome using the insertion/substitution system (Selick, H. E., Kreuzer, K. N., and Alberts, B. M. (1988) J. Biol. Chem. 263, 11336-11347). Both resulting phage strains are uvsY- mutants, but they differ in that one has a deletion of the minimal tertiary origin and the other does not. The effects of the uvsY mutations on tertiary origin activity were assayed by infecting tertiary origin plasmid-bearing Escherichia coli with the two phage mutants. The tertiary origin plasmids replicated extensively after infection by either uvsY- phage mutant, demonstrating that the uvsY protein is not required for tertiary initiation. The extent of plasmid replication was increased dramatically as a result of either mutation, indicating that the uvsY protein plays some negative role in either the initiation or subsequent processing of plasmid replicative intermediates. The phage strain with an origin deletion induced the replication of a tertiary origin plasmid with which it shared no homology. Therefore, plasmid-phage recombination is not required for the replication of tertiary origin plasmids. The replication of a tertiary origin plasmid is also shown to be independent of the phage genes uvsX, 59, and 46, but markedly reduced by mutations in the T4-induced topoisomerase.

A highly efficient second-step concentration technique for bacteriophages and enteric viruses using ammonium sulfate and Tween 80.

Addition of Tween 80 to a 1.5% solution of beef extract was found to enhance the elution of bacteriophages adsorbed to electronegative filters. When reconcentration of the eluate was attempted by ammonium sulfate precipitation, a floating layer containing most of the viruses was formed. This floating layer can be obtained with several nonionic detergents including Tween 80 and under a salt saturation of 55% with ammonium sulfate, potassium tartrate, and sodium phosphate. Virus recovery ranged from 91 to 103% and was obtained with several bacteriophage strains. With poliovirus type 1, coxsackievirus B-4, and rotavirus SA-11 the recoveries were 100, 20, and 80%, respectively, but toxicity to cell culture was encountered: after removal of the detergent by a second floating layer method the recovery was 32% for poliovirus. Compared with organic flocculation, this method also had both improved recovery for bacteriophages and protective properties for samples frozen at -70 degrees C.

An electron microscopic study of bacteriophages from marine waters

The morphology of 75 bacteriophage strains isolated from water samples collected in the North Sea or in the northern Atlantic was studied by electron microscopy. Only tailed phages were observed (bradley groups A, B, and C). According to structural similarities, the strains are ascribed to 12 groups, 5 of which comprise types of marine phages not reported before. Four of these 5 groups include phage types that have not been detected from any other source. Among the phages isolated from northern Atlantic water a high incidence was observed of strains the particles of which have long appendages. Certain types of the northern Atlantic phages investigated were derived only from samples collected either east or west of the Azores. This finding agrees with former observations pointing to the existence of different populations of closely related bacteria east and west, respectively, of the northern Mid-Atlantic Ridge.

Atypical isolates of Brucella abortus from Canada and the United States characterized as dye sensitive with M antigen dominant

A total of 41 Brucella isolates, examined by standard biotyping procedures, were found to be similar to Brucella abortus biovar 2 in dye sensitivity but had a dominant M antigen. Oxidative metabolic tests performed on 39 of the isolates confirmed them as B. abortus. Additional biochemical and bacteriophage susceptibility studies were performed on 35 of the isolates. The isolates had identical reactions in the various tests, except for one isolate which was resistant to lysis by all phage strains used. Two isolates were injected into guinea pigs and shown to be virulent. The isolates described in this study appear similar to atypical Brucella isolates previously reported in the United Kingdom and the United States and may form the basis of a new biovar, B. abortus biovar 10.

Pf1 Inovirus: Electron density distribution calculated by a maximum entropy algorithm from native fibre diffraction data to 3 Å resolution and single isomorphous replacement data to 5 Å resolution

We have calculated the electron density distribution of the Pf1 strain of filamentous bacteriophage by a maximum entropy method. In the calculation we included native X-ray fibre diffraction data extending to 3 Å resolution in the meridional direction on 60 layerlines that are resolved to 4 Å in the equatorial direction, and lower resolution data from a single isomorphous derivative iodinated on the Tyr25 residue. The electron density map indicates that the 46-residue protein subunit is a single, gently curved stretch of α-helix with its axis at an angle of about 20° to the axis of the virion. The α-helix subunit curves around the virion axis by about 1 6 turn, and decreases from about 27 Å radius to about 13 Å radius in the virion as the amino acid sequence of the subunit runs from the N terminus to the C terminus. Nearest-neighbour α-helical subunits are about 10 Å apart along their length, and the axis of each subunit makes an unexpected negative angle with its nearest neighbours in the virion. To confirm the validity of the maximum entropy calculation, we have varied the constraints on the calculation. All variations result in either a map that is close to the original map or a map that cannot be interpreted in terms of secondary structure: we find only one map that makes structural sense.

Variation within Pseudomonas syringae pv. philadelphi, the cause of a leaf spot of Philadelphus spp.

Roberts, S.J. 1985. Variation within Pseudomonas syringae pv. philadelphi, the cause of a leaf spot of Philadelphius spp. Journal of Applied Bacteriology59, 283–290In pathogenicity tests on Philadelphus and other plant species, belonging to ten genera in seven families, isolates of Pseudomonas syringae from leaf spots on Philadelphus spp. in England did not produce symptoms on any plants other than Philadelphus. It is therefore proposed that these isolates should be designated a distinct pathovar of Ps. syringae with the name Pseudomonas syringae pv. philadelphi. Isolates of this new pathovar varied in their reactions to 6 of 57 biochemical tests. In phage typing tests isolates also varied in their sensitivity to five of seven bacteriophage strains. Four of the six biochemical tests (aesculin hydrolysis, utilization of DL‐homoserine L‐leucine and sorbitol) and all five of the phages (P11, Pls, P2, A15, and A26) were used to separate the isolates into seven groups. These groups had some relation to their geographical origin, species of Philadelphus from which they were originally isolated, and relative virulence on P. coronarius and P. x purpureo‐maculatus. They may represent ecotypes of this new pathovar.

Assembly site of bacteriophage f1 corresponds to adhesion zones between the inner and outer membranes of the host cell.

Morphogenesis of the filamentous bacteriophage f1 occurred at adhesion zones between the inner and outer membranes of the host cell. Quantitation of adhesion zones in cells infected with mutant phage strains suggested that the phage gene I protein may be involved in the formation of adhesion zones for phage assembly.

Isolation of Actinomyces bacteriophage from human dental plaque.

Human dental plaque samples were screened for the presence of bacteriophage for Actinomyces viscosus and Streptococcus sanguis. None of the 336 samples yielded phage for S. sanguis, but 10 contained virulent actinomyces phage. A high host cell specificity was observed in that one phage isolate infected only A. viscosus T14V, eight phage isolates infected only A. viscosus MG-1, and one infected both strains. None was capable of productively infecting various other actinomyces strains that represented the six actinomyces coaggregation groups. Because phage-containing samples occurred randomly in this survey, no correlation between the individual collecting the samples, dental clinic, or type of patient and the presence of phage in the sample was noted. Examination of one of the samples that yielded phage for the presence of a natural host strain for that particular phage resulted in the isolation of two strains which were identified as A. viscosus serotype II and Actinomyces naeslundii serotype I. This is the first report of an A. naeslundii host strain and actinomyces bacteriophage of human dental plaque origin. The finding of both phage and host strains in the same dental plaque sample along with the observation of high host cell specificity by these phage provide indicators that support an active role for actinomyces bacteriophage in oral microbial ecology. The use of these freshly isolated phage as probes to study actinomyces coaggregation properties is discussed.

Function and structure of microvirid phage α3 genome. DNA sequence of H gene and properties of missense H mutant

The nucleotide sequence of wild-type α3 H gene and its surrounding region was determined and compared with those of ∅X174 and G4. The corresponding DNA regions in double mutants amJH22, amJH69 and amJH76 were also sequenced and their missense mutation sites located. A phage strain missH22 having a single missense mutation in gene H was constructed by replacing the J region of amJH22 in vitro with the wild-type DNA. Like amJH22, the missense mutant coded for H protein with aberrant electrophoretic mobility, but formed normal plaques on suppressor-deficient Escherichia coli. Heat stability, plating efficiency on certain hosts and rate of eclipse were higher in strain missH22 than in wild-type phage.

Host transfer RNA cleavage and reunion in T4-infected Escherichia coli CTr5x.

T4 mutants lacking polynucleotide kinase (pnk-) or RNA ligase (rli-) do not grow on E. coli CTr5x. During the abortive infections there accumulate host tRNA fragments that match into two species severed 3' to the anticodon. The CTr5x-specific fragments appear only transiently with wt phage, implicating the affected enzymes in phosphoryl group rearrangement and religation [David et al. (1982) Virol. 123, 480]. In a search for the vulnerable host tRNAs and putative religation products, tRNA ensembles from uninfected E. coli CTr5x or cells infected with various phage strains were fractionated and compared. A tRNA species absent from rli- infected cells but present in uninfected cells or late in wt infection was thus detected. RNase T1 finger prints of this species, isolated before or after wt infection, were compared with that of an in vitro ligated pair of CTr5x-specific fragments. The results indicated that this tRNA is cleaved upon infection and later on restored to it's original or to a very similar form, by polynucleotide kinase and RNA ligase reactions. It is suggested that depletion of such vulnerable host tRNA species underlies the restriction of pnk- or rli- phage on E. coli CTr5x.

In-vitro activity of methicillin against clinical isolates of Staphylococcus aureus.

Methicillin activity against 149 penicillin-resistant, methicillin-susceptible Staphylococcus aureus strains from bacteraemia cases with endocarditis (n = 89) or without endocarditis (n = 60), from the years 1976-1981, was studied with broth dilution and agar dilution. While no differences in methicillin susceptibility were found in relation to the origin of the strains, Staph. aureus of the phage type complex 94,96 showed significantly higher MIC and IC50 by agar dilution than strains of other phage groups/complexes. This difference probably has no clinical importance but is of epidemiological interest. Broth dilution MIC was generally one dilution higher than agar dilution MIC, possibly explained by methodological factors. The MBC/MIC ratios never exceeded two in any of the strains, indicating a lack of tolerance in these clinically important isolates.

Use of phage for the identification of Brucella canis and Brucella ovis cultures

The brucella phage strains R, R/O and R/C standardised at routine test dilution on their propagating strains were effective in identifying cultures of Brucella ovis and B canis and in differentiating these from other non-smooth brucella isolates.

Bacteriophages Lysing the Marine Fouling Bacteria

In order to obtain the fundamental data to control the formation of marine microbiofouling by bacteriophages, titanium and aluminum alloy plates were immersed at Okunouchi Bay in Seto Inland Sea, and the marine fouling bacteria attached were isolated and characterized. Then the bacteriophages lysing the marine fouling bacteria were located and characterized. The results obtained were as follows;(1) Nineteen strains of fouling bacteria were isolated from the surface of the test plates. As the result of their characterization, they belonged to the following genera; Pseudomonas (8 strains), Vibrio (5 strains), Flavobacterium (4 strains) and Moraxella-Acinetobacter (one strain). It was found that motile bacterial strains were dominant on the surface of the titanium plate, while non-motile bacterial strains were dominant on the surface of the aluminum alloy plate.(2) The bacteriophages lysing the marine fouling bacteria were isolated from seawater of Aki-Nada, Hiroshima Bay and other places. Thirteen of the 19 strains of the fouling bacteria were susceptible to the bacteriophage-lysates enriched from the seawater. All of the bacteriophage strains had hexagonal heads and their diameter were 54-58nm.

Taxonomic investigations of bacteriophage sensitive bacteria isolated from marine waters

Based on 28 criteria the taxonomy of 366 phage sensitive bacterial strains isolated from marine waters (Atlantic between European continental shelf and Sargasso Sea, Bay of Biscay, North Sea near Helgoland) was investigated. Seventy-eight phage-intensity strains derived from the same Atlantic Ocean regions as the sensitive ones were tested for comparison. While in the latter considerable diversity was observed, the results obtained with the phage-sensitive bacteria are characterized by stupendous uniformity. 362 of the 366 strains are assigned to the family Vibrionaceae, some 280 of which belong to the genusVibrio. As discussed, this taxonomic uniformity among the phage-sensitive bacteria is assumed to be an artifact mainly caused by the type of enrichment culture employed for the isolation of all but a few bacteriophage strains used and, to a lesser degree, by characteristics of the bacterial populations encountered.

Lytic and inhibition responses to bacteriophages among marine bacteria, with special reference to the origin of phage-host systems

The results of phage-host cross-reaction tests reported by Moebus & Nattkemper (1981) were re-examined using serially diluted bacteriophage suspensions to elicit the actual type of reaction between the bacteria and phage lysates tested. More than 1450 phage-host systems were studied at 25 °C incubation temperature. Among the nearly 300 phage strains used, 29 were identified as temperate ones. In about 65 % of the phage-host systems bacteriophage propagation was indicated by plaque formation. The remaining systems were characterized by the “inhibition” reaction of bacteria to phage lysates indicated by homogenously reduced bacterial growth within the test area without production of progeny phages. Since crude phage lysates had to be used, it remains obscure whether agents other than infective phage particles (defective ones or bacteriocins) caused this reaction. Among 269 systems of the inhibition type which were also tested at 5° and 15 °C, 54 were observed to propagate phages at one of or both the lower temperatures. Plaques produced at 15 °C with several phage-host systems were found to yield only few progeny phages which generally could not be propagated to produce high-titer phage stocks. With one system temperature-sensitive phage mutants were isolated. The probability of inhibition reactions occurring was found to be higher with phage-host systems isolated east of the Azores than with systems derived from the western Atlantic. With systems from the last mentioned area the proportion of inhibition versus lytic responses of bacteria to phages was observed to increase with the distance between the stations where both parts of the systems were derived. The latter findings are discussed in view of the assumption that bacterial and bacteriophage populations undergo genetic changes while being transported from west to east.

Morphogenesis of filamentous bacteriophage f1: Orientation of extrusion and production of polyphage

Crosslinking reagents were used to interrupt the process of filamentous phage morphogenesis and investigate the orientation in which nascent virions are extruded through the host cell membrane. Infected bacteria with emerging phage particles were crosslinked with glutaraldehyde. Immunoferritin-labeling studies on these emerging phage using anti-A protein IgG suggested that extrusion begins with the C protein end. To confirm this, phage extruding from infected bacteria were frozen using the reversible crosslinker dimethyl 3,3′-dithiobis-propionimidate and fragments of emerging phage were isolated by shearing. Protein analysis of these fragments showed them to be enriched in C protein relative to A protein, as predicted if phage extrusion begins with the C protein end. The production of multiple-length phage particles (polyphage) by nonpermissive bacterial hosts infected with amber mutant phage strains was also studied. Polyphage were produced upon infection with amber mutants in genes III, VI, VII, and IX which code for proteins found at the ends of the mature phage particle. No polyphage were produced by mutants in the other genes tested. Gene III amber mutants produce noninfective polyphage, but those produced by genes VII and IX are infective. Gene VI amber mutants appear to produce unstable, noninfective polyphage particles.

Some theoretical aspects of protein coevolution in the ecosystem “phage-bacteria” I. The problem

The possibility of prolonged coevolution of bacterial reception protein and viral adsorption one is questioned in this paper. A general deterministic model of ecosystem “phage-bacteria” is formulated. Analysis of the model accentuates the problem of clearing up conditions that provide steady regimes not of coexistence but coevolution, i.e. successive mutational changes of mutually “coadaptive” pairs of mutant strains of bacteria and phages. Heuristic validity of this model in a framework of some actual problems and paradoxes of molecular evolution theory is particularly emphasized.

High-frequency transduction of pBR322 by cytosine-substituted T4 bacteriophage: Evidence for encapsulation and transfer of head-to-tail plasmid concatemers

Transduction of plasmid pBR322 by cytosine-substituted T4 phages has been studied. Three T4 phage mutants which substitute cytosine for all of hydroxymethylcytosine residues in the DNA, were shown to transduce pBR322 at frequencies of 2 × 10 −2 to 4 × 10 −3 transductants per singly infected cell. Also, three T4 phage strains which partially substitute cytosine for hydroxymethylcytosine, transduced pBR322 at frequencies of 2 × 10 −3 to 2 × 10 −4. The transduction frequencies of pBR322 we attained are at least 10-fold higher than those reported by G. G. Wilson, K. Young, and G. J. Edlin (1979, Nature (London) 280, 80–82). We found that multiplicity of infection in preparation of the transducing phage is the most important factor affecting the frequency of pBR322 transduction. When a lysate made at a multiplicity of infection ranging from 0.5 to 0.05 was used as the donor phage, transduction frequency of pBR322 was 10- to 40-fold higher than that of high-m.o.i. lysate. The transduction frequency was not affected by either restriction systems or amber suppressors of the recipient cells. However, no pBR322-containing transductant was obtained when either recA or polA mutants were used as the recipients. DNA from T4dC phage containing pBR322-transducing particles was analyzed on agarose gel electrophoresis after cleavage with restriction endonucleases. It was suggested that the pBR322 DNA in the T4dC phage particles exists as head-to-tail concatemers.

Construction of gapped circular DNA from phage M13 by in vitro hybridization

Circular double-stranded phage M13 DNA-containing gaps of defined size and location were obtained in preparative amounts by in vitro hybridization of plus- and minus-strands derived from different phage strains.

The reaction of bacteriophage T6 with tryptophan: An improved description of T-even tail-fibre extension

Some strains of the T-even bacteriophages, T2, T4 and T6, require tryptophan for adsorption to host cells. It is shown that for T6 the presence of tryptophan is not an absolute requirement. Instead, the function of tryptophan is to shift the equilibrium between the “fibres in” and the “fibres out” conformation in favour of the latter condition. This leads to an improved mathematical formulation.