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Mechanism of coliphage M13 contraction: intermediate structures trapped at low temperatures.

The filamentous coliphage M13 can be transformed into a spherical particle (termed spheroid) by exposure to an interface of water and slightly polar but hydrophobic solvent such as chloroform-water at 24 degrees C. We report here that exposure of M13 filaments to a chloroform-water interface at 2 degrees C trapped the phage particles in forms morphologically intermediate to filaments and spheroids. These structures were rods 250 nm long and 15 nm wide, and each had a closed, slightly pointed end, an open flaired end, and a hollow central channel. The final contraction of these intermediates (termed I-forms) into spheroids was dependent upon both temperature and the presence of the solvent-water interface but was apparently independent of both the minor phage coat proteins and the virion DNA. Although stable in an aqueous environment, I-forms, in contrast to filaments, were readily disrupted by detergents, suggesting that the phage structure had been altered to a form more easily solubilized by membrane lipids. These solvent-induced changes might be related to the initial steps of phage penetration in vivo.

Chloroform↗

Genetic studies of coliphage 186. I. Genes associated with phage morphogenesis.

In coliphage 186, 22 essential genes were defined by complementation studies with amber mutants. Eighteen genes were associated with phage morphogenesis: 11 with phage tail formation, and 7 with phage head formation. The remaining four genes are discussed in the accompanying paper (S. M. Hocking and J. B. Egan, J. Virol. 44:1068-1071, 1982).

Alleles↗

Genetic studies of coliphage 186. II. Genes associated with phage replication and host cell lysis.

DNA synthesis in coliphage 186-infected cells was investigated. Phage 186 appeared to inhibit host DNA synthesis early in infection. The subsequent synthesis of phage 186 DNA was dependent on the product of 186 gene A. The product of gene B controlled both the production of late 186 proteins and the cessation of 186 DNA synthesis, and the products of genes O and P had no influence on 186 DNA synthesis. The product of gene P controlled host cell lysis, and the product of gene O may have some regulatory function.

Coliphages↗

Photodynamic action of proflavine on coliphage T3. I. Kinetics of inactivation.

The photodynamic inactivation of coliphage T3 was studied over a wide range of concentrations of the dye proflavine. With 2 x 10(7) phage/ml, two modes of inactivation were observed. Between 0.25 and 12 to 13 mug/ml, inactivation was biphasic. There was an initial first-order inactivation (Rx1) which became temporally associated with an apparently multiorder process (Rx2) at higher light doses. Dye concentrations above 12 to 13 mug/ml showed only two-target inactivation curves (Rx3), except at high dye concentrations where processes kinetically identical to Rx1 and Rx2 reappeared. Rx2 showed a normal rectangular hyperbolic saturation curve but Rx1 and Rx3 appeared to saturate prematurely. The saturation behavior of Rx1 and Rx2 was independent of phage concentration, but Rx3 was lost at phage titers above 2 x 10(7)/ml. No dark inactivation was seen with Rx1 and Rx2 subsequent to a period of illumination. With Rx3, an exponential dark inactivation was seen for at least 1 hr after a period of illumination. The dye-phage system equilibrated immediately, at any temperature, at proflavine concentrations where Rx1 and Rx2 occurred. With Rx3, prolonged equilibration times were necessary. Moreover, there was a temperature effect. The rate of inactivation at equilibrium was temperature-dependent, whereas the initial rate at which equilibrium was approached was essentially temperature-independent.

Acridines↗

Absence of competition between salmonella phage P22 and coliphage P1 for adsorption sites on a Salmonella typhosa - Escherichia coli hybrid strain.

Salmonella typhimurium mutants sensitive to coliphage P1 are resistant to salmonella phage P22 and lose their P1-sensitivity upon reversion to P22-sensitivity. A tryptophan-requiring Salmonella typhosa - Escherichia coli hybrid, which has the unique ability to serve as recipient in transduction mediated by both P22 and P1, was sued to determine if P22 and P1 adsorb at the same or overlapping sites: (i) The adsorption of each of P1 and P22 is similar when added individually or together to the hybrid at a saturating multiplicity of infection (moi). (ii) P1 grown on trp+ E. coli yields the maximum frequency of Trp+ abortive transductions at an moi of 6 with the trp hybrid recipient; the presence of increasing numbers of P22 grown on trp S. typhimurium does not decrease the number of Trp+ transduction from P1. (iii) A mixture of P1 (grown on trp+ E. coli) and P22 (grown on trp+ S. typhimurium) yields more abortive transductions than does P1 alone. Thus phages P1 and P22 adsorb to the hybrid cells on different sites.

Adsorption↗

Coliphage derived sialidase preferentially recognizes nonreducing end of polysialic acid.

Bacteriophages infecting Escherichia coli K1 strains generally have endotype polysialic acid-degrading enzymes. We studied the digestion mechanism of a sialidase associated with the coliphage 63D using polysialic acid radiolabeled at its nonreducing end or reducing end. It was found that this enzyme preferentially recognizes the nonreducing end of polysialic acid, suggesting that the 63D associated sialidase does not randomly digest its substrate but acts like an exotype glycosidase.

Coliphages↗

A randomized controlled trial assessing infectious disease risks from bathing in fresh recreational waters in relation to the concentration of Escherichia coli, intestinal enterococci, Clostridium perfringens, and somatic coliphages.

We performed epidemiologic studies at public freshwater bathing sites in Germany to provide a better scientific basis for the definition of recreational water quality standards. A total of 2,196 participants were recruited from the local population and randomized into bathers and nonbathers. Bathers were exposed for 10 min and had to immerse their head at least three times. Water samples for microbiological analysis were collected at 20-min intervals. Unbiased concentration-response effects with no-observed-adverse-effect levels (NOAELs) were demonstrated for three different definitions of gastroenteritis and four fecal indicator organisms. Relative risks for bathing in waters with levels above NOAELs compared with nonbathing ranged from 1.8 (95% CI, 1.2-2.6) to 4.6 (95% CI, 2.1-10.1), depending on the definition of gastroenteritis. The effect of swallowing water provided additional evidence for true dose-response relationships. Based on the NOAELs, the following guide values for water quality are suggested: 100 Escherichia coli, 25 intestinal enterococci, 10 somatic coliphages, or 10 Clostridium perfringens per 100 mL. Recreational water quality standards are intended to protect the health of those consumers who are not already immune or resistant to pathogens that may be associated with indicator organisms. In contrast to current World Health Organization recommendations, we concluded that standards should be based on rates of compliance with NOAELs rather than on attributable risks determined above NOAELs, because these risks depend mainly on the unpredictable susceptibility of the cohorts. Although in theory there is no threshold in real concentration-response relationships, we demonstrated that a NOAEL approach would be a more robust and practical solution to the complex problem of setting standards.

Clostridium perfringens↗

Immunological reaction of UV-induced radiation damage in coliphage DNA.

Denatured DNA of coliphage T1, poly-dT and dTpT were UV-irradiated (2537 A) and conjugated to methylated bovine serum albumin. These molecules act as haptens after injection into rabbits. Antibodies are predominantly formed against UV-induced cyclobutane-type of cis-synthymine dimers. They belong to the IgG-type of antibody molecules. Antigen-antibody complexes were detected by means of the CsCl-density gradient method. Denaturation of the DNA strongly influences the amount of specific antibodies bound to the antigen. In native DNA thymine dimers are masked concerning their reaction with antibodies. Photoproducts of UV-irradiated dTpT induce a specific production of antibody. Our results indicate that the cyclobutane type of thymine dimers is the relevant antigenic determinant group in UV-irradiated DNA.

Animals↗

Ultraviolet inactivation of feline calicivirus, human enteric viruses and coliphages.

Norwalk and Norwalk-like viruses (NLV) are major causes of food- and water-related disease in the United States. There is no host cell line in which the NLV can be tested for infectivity. Feline calicivirus (FCV) and NLV both belong to the family Caliciviridae. FCV can be assayed for infectivity in the Crandell Reese feline kidney cell line, so FCV serves as a surrogate for NLV. This study is the first report of UV inactivation of FCV and also of using the plaque technique, in contrast to the 50% tissue culture infectious dose end point technique, to determine the FCV infectivity titer. The infectivity titers (log10 plaque-forming units/mL) of UV-inactivated FCV, hepatitis A virus (HAV), poliovirus type 1 (PV1) and two small, round coliphages were plotted as a function of UV dose and analyzed by regression analysis and analysis of variance. These fitted straight-line curves represent exponential inactivation, so UV inactivation can be said to show "one-hit kinetics." The decimal inactivation doses of UV for FCV, HAV, PV1, MS2 and phiX174 were 47.85, 36.50, 24.10, 23.04 and 15.48 mW s/cm2, respectively. FCV appears to be the most UV resistant among the tested viruses.

Animals↗

Effects of temperature and moisture on coliphage PRD-1 survival in soil.

The goal of this study was to quantitatively assess the effects of temperature and soil moisture on the survival of coliphage PRD-1 in soil. PRD-1 was added to sandy loam soil at five different soil moisture levels. The soil seeded with PRD-1 was packed into sterile polyethylene jars and exposed to eight different temperatures in an oven. Samples were collected over 14 to 25 days depending on the temperature. The inactivation rate of PRD-1 increased linearly with increased temperature. The inactivation rate gradually decreased when the soil moisture level decreased from 20.9 to 8.9%. However, the inactivation rate increased when the soil moisture content reached 5.1%, suggesting the existence of an optimal soil moisture condition for PRD-1 survival. It is also possible that there is a threshold soil moisture level below which the inactivation of PRD-1 suddenly increases. Marked reductions in recoveries were observed as the soil moisture approached or fell below 5.0% as a result of evaporation. The increased inactivation of PRD-1 due to strong association with soil particles may have caused rapid reductions in recoveries. The evaporation process appeared to affect PRD-1 survival substantially at higher temperatures whereas little effect was observed at lower temperatures. A model developed from this study predicted PRD-1 survival in subsurface soil in field conditions with an average error of 11.0%.

Agriculture↗

[Coliphages inactivation using chitosan derivatives].

The effect of chitosan fragments with different degrees of polymerization and the chemical derivatives of chitosan differing in the number of amino groups and total molecule charge on phages T2, T4, and T7 was studied. The interaction of chitosan with bacteriophage particles inactivated them to the extent dependent on the chemical properties of chitosan and its concentration. Phage T2 was found to be most susceptible to inactivation by chitosan. The polycationic nature of chitosan plays an important role in the inactivation of phages. It is assumed that the abnormal rearrangement of the basal plate of phages, the loss of long tail fibers, and probably, modification of the receptor-recognizing phage proteins may be responsible for the inactivation of coliphages by chitosan.

Anti-Infective Agents↗

Physiological transition of a coliphage lambda DNA replication.

The "rolling-circle" replicative intermediate (sigma-type molecules) which is normally produced in the late stage of coliphage lambda DNA replication can be found during the first round of lambda DNA replication if cells infected with lambda replication mutant Ots28 are incubated at the nonpermissive temperature until the late stage of the latent period of lambda infection. After shifting to the permissive temperature, the vast majority of replicating forms are sigma-type rolling circle even during the first round of DNA replication. Concatemeric lambda DNA molecules, produced from these sigma-type intermediates, are efficiently packaged into progeny phage, indicating that in the first round of lambda DNA replication, double-branched theta-type molecules are not required for production of viable progeny phage.

Centrifugation, Density Gradient↗

Optimisation of the ISO-method on enumeration of somatic coliphages (draft ISO 10705-2).

As part of the EU project "Bacteriophages in Bathing Waters" (January 1996-June 1999) research was carried out to optimise the method for detection and enumeration of somatic coliphages in water as described in ISO/CD 10705-2 of August 1995. It was concluded that this draft ISO standard needed to be amended in certain aspects. For determining the viable count of the host culture WG5 Escherichia coli, a membrane filtration technique should be used instead of spread plate technique as the latter gives lower and less reproducible results. A freshly prepared inoculum culture of host strain WG5 should be used instead of a frozen inoculum culture as freezing of the inoculum culture is found to negatively influence the phage counts. The double agar layer method (DAL) is preferred to the single agar layer method (SAL) for performing the phage analysis as the DAL method gives higher phage counts than the SAL method.

Agar↗

Fate of coliphage in waste water treatment process and detection of phages carrying the Shiga toxin type 2 gene.

Abundances of phages specific to Escherichia coli in the wastewater treatment process were analyzed. Relatively abundant coliphages were detected in sewage influent. Phages in the influent were found both suspended in liquid phase and attached on the solid particles. Phage concentration was not reduced in the settling tank without chemical agglutination. Anaerobic followed by aerobic treatment of the sewage reduced concentration of suspended phages. Almost no phage was detected as a suspended form in the aerobic tank. Most of the phages were detected as attaching form and were excluded by aggregation with sludge. Using an experimental approach based on the detection of Shiga toxin 2 (Stx 2) gene by a phage enrichment culture followed by nested PCR, bacteriophages carrying Stx 2 gene were detected in the influent, settling tank, and anaerobic tank. It was revealed that the presence of phages carrying Stx 2 gene is common in sewage and these phages are effectively eliminated through sewage treatment process.

Coliphages↗

The receptor of an oyster juice-borne coliphage OJ367 in the outer membrane of Salmonella derby.

The objective of this study was to identify the receptor of OJ367, an oyster juice-borne bacteriophage, in Salmonella derby ATCC 6960. The crude receptor outer membrane (OM) fraction was prepared and examined from the total cell envelope (TCE) by differential extraction with N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES)-MgCl2 and then with Triton-HEPES-ethylenediamine tetra-acetic acid buffers. The OM proteins (Omps) were isolated by diethylaminoethyl column chromatography to screen for receptor activity. A 45-kDa protein belonging to a minor Omp species, with phage neutralization ability, was eluted in a homogeneous form. It was a non-peptidoglycan-associated protein which was digestible by trypsin. Lipopolysaccharide had no influence on its receptor activity when coexistent in the diethylaminoethyl column fractions. An S. derby mutant resistant to lysis by phage OJ367 was isolated. The mutant not only showed decreased receptor activity in vitro when its TCE was tested but had an altered Omp profile. This implied that the 45-kDa Omp is involved as a receptor in coliphage binding; however, this role is affected by the expression of other Omps.

Animals↗

[Viral flora (coliphages and human enteroviruses) found in river water after an urban district (Saint-Etienne). II. A virological and epidemiological survey (author's transl)].

A survey was carried out from March 1972 to February 1973 to identify viral flora found in the river Furan ater St. Etienne. The 54 samples examined revealed the following data: 1) more accurate results are obtained when viral concentration values are expressed in terms of m3/sec., taking into account the flow of the river and eliminating the seasonal dilution factor; 2) rates of enteroviruses remain constant throughout the year, in spite of a relatively rapid spontaneous inactivation of the viruses; 3) rates of coliphages vary considerably according to seasons, with a notable increase in summer; 4) the two previous data are unrelated; 5) 147 enterovirus strains were isolated, of which 44% were polioviruses; 6) virulent and attenuated types 2 and 3 polioviruses were found simultaneously at certain periods; 7) only virulent strains of type 1 poliovirus were isolated; 8) this type of survey may be useful in controlling the endemic residual poliomyelitis in the region of St. Etienne.

Coliphages↗

Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein.

We have purified and characterized a single-stranded DNA binding protein (N4 SSB) induced after coliphage N4 infection. It has a monomeric molecular weight of 31,000 and contains 10 tyrosine and 1-2 tryptophan amino acid residues. Its fluorescence spectrum is dominated by the tyrosine residues, and their fluorescence is quenched when the protein binds single-stranded DNA. Fluorescence quenching was used as an assay to quantitate binding of the protein to single-stranded nucleotides. The N4 single-stranded DNA binding protein binds cooperatively to single-stranded nucleic acids and binds single-stranded DNA more tightly than RNA. The binding involves displacement of cations from the DNA and anions from the protein. The apparent binding affinity is very salt-dependent, decreasing as much as 1,000-fold for a 10-fold increase in NaCl concentration. The degree of cooperativity (omega) is relatively independent of salt concentration. At 37 degrees C in 0.22 M NaCl, the protein has an intrinsic binding constant for M13 viral DNA of 3.8 x 10(4) M-1, a cooperativity factor omega of 300, and binding site size of 11 nucleotides per monomer. The protein lowers the melting point of poly(dA.dT).poly(dA-dT) by greater than 60 degrees C but cannot lower the melting transition or assist in the renaturation of natural DNA. N4 single-stranded DNA binding protein enhances the rate of DNA synthesis catalyzed by the N4 DNA polymerase by increasing the processivity of the N4 DNA polymerase and melting out hairpin structures that block polymerization.

Chromatography, Affinity↗