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PubMed1991

Secondary structure of coliphage Q beta RNA. Analysis by electron microscopy.

The secondary structure of genomic RNA from the coliphage Q beta has been examined by electron microscopy in the presence of varying concentrations of spermidine using the Kleinschmidt spreading technique. The size and position of structural features that cover 70% of the viral genome have been mapped. The structural features that are visualized by electron microscopy in Q beta RNA are large. They range in size from 170 to 1600 nucleotides. A loop containing approximately 450 nucleotides is located at the 5' end of the RNA. It includes the initiation region for the viral maturation protein. A large hairpin containing approximately 1600 nucleotides is located in the center of the molecule. It is multibranched and includes most of the viral coat gene, the readthrough region of the A1 gene, and approximately one third of the viral replicase gene. Within the central hairpin, the initiation region for the viral replicase gene pairs with a region within the distal third of the viral coat gene. This structure may participate in the regulation of translational initiation of the viral replicase gene. Two structural variants of the central hairpin were observed. One of them brings the internal S and M viral replicase binding regions into juxtaposition. These observations suggest that the central hairpin may also participate in the regulation of translation of the viral coat gene. The secondary structures that are observed in Q beta RNA differ significantly from structures that we described previously in the genomic RNA of coliphage MS2 but are similar to structures we observed by electron microscopy in the related group B coliphage SP.

Coliphages↗

PubMed1992

Improved method for coliphage detection based on beta-galactosidase induction.

An improved method for coliphage detection based on the induction of beta-galactosidase in Escherichia coli is described. Upon infection by coliphages, the cells are lysed and a stable indolyl product that is dark blue becomes visible within each plaque. The improved method is compared to the proposed coliphage detection procedure described in Standard Methods for the Examination of Water and Wastewater.

Coliphages↗

PubMed2002

F-specific RNA coliphages: occurrence, types, and survival in natural waters.

A small, well-defined watershed was investigated over a 2-year period to determine the prevalence of F-specific RNA coliphage (F + RNA) serotypes as indicators of animal fecal contamination. Sampling sites collected runoff from areas of urban and agricultural land use patterns. F-specific coliphages were concentrated from 2-L freshwater samples by polyethylene glycol precipitation, isolated using the double agar layer (DAL) method, confirmed as F + RNA by RNAse suppression, and serotyped. A subset of serotyped F + RNA were confirmed by genotyping. To determine relative survival, 10 confirmed F + RNA field isolates and 5 prototypic F + RNA were spiked into surface water and incubated at 25 degrees C for 36 days. F-specific coliphage isolation was strongly associated with rainfall events and was infrequent from primarily animal impacted surface waters. Field isolates were predoffiinantly Type I F + RNA (81%) and raw sewage isolates were predominantly Type III F + RNA (57%). Genotyping from either the watershed or raw sewage samples never positively identified Type IV F + RNA. Results from laboratory studies showed that F + RNA differ in their survival in water and that Type IV strains were the least persistent. Type III F + RNA were found to be reliably related to the release of uncontrolled human fecal material in the watershed, but the results of this study suggest that further study is required before utilizing for fecal source identification in natural waters.

Agriculture↗

PubMed1993

Sensitivity of naturally occurring coliphages to type I and type II restriction and modification.

Protection against lethal infections by bacteriophage may seem the most likely role of restriction-modification (R-M) systems in bacteria and the reason for their evolution. There are, however, phenomena which question this phage-mediated selection hypothesis for the maintenance of extant R-M systems. Most prominent among these are the mechanisms phage have to avoid or otherwise limit the effects of the restriction endonucleases produced by their host bacteria. To evaluate the importance of these antirestriction mechanisms in Escherichia coli, we have examined the sensitivity of coliphage from natural and laboratory sources to a series of type I and II R-M systems. The results of our study indicate that, in vivo, restriction endonucleases have no effect on a substantial fraction of naturally occurring coliphage. The absence of restriction sites appears to be the most common reason why these phage are unaffected by type II restriction endonucleases, but other antirestriction mechanisms also operate. On the other hand, the frequency of naturally occurring coliphage sensitive to restriction appears sufficiently great for phage-mediated selection to be a viable hypothesis for the maintenance of R-M in E. coli and its accessory elements.

Biological Evolution↗

PubMed1981

Recovery of coliphages from wastewater effluents and polluted lake water by the magnetite-organic flocculation method.

A magnetite-organic flocculation method was developed for the concentration of coliphages from wastewater effluents and polluted lake water. A high percent (68 to 100%) recovery of coliphages from sewage effluents was achieved by this procedure. Coliphage recovery from Lake Alice, a sewage-contaminated lake, showed phage concentrations ranging from 2.3 X 10(2) to 1.9 X 10(3) plaque-forming units per liter. This method is simple and inexpensive and may be carried out under field conditions.

Coliphages↗

PubMed1983

Concentration of coliphage from water and sewage with charge-modified filter aid.

Methods of detecting and concentrating animal viruses from large volumes of water and wastewater have experienced rapid development in recent years, but only a few methods are available for the concentration of bacteriophages. The present study describes the use of a charge-modified (Zeta Plus) filter aid (AMF Cuno, Meriden, Conn.) for the concentration of coliphages from large volumes of water and sewage. Coliphages MS-2 and f2 were efficiently adsorbed from water and sewage to the positively charged filter aid. Elution was accomplished with 4% beef extract--0.5 M NaCl adjusted to pH 9.5. The recovery of f2 from 10- to 20-liter volumes of tap water ranged between 11 and 70%, and the recovery of MS-2 ranged between 43 and 70%. The efficiency of recovery of naturally occurring coliphages from secondarily treated sewage ranged between 16 and 44%. This technique appears to be promising because it requires low-cost equipment (47-mm polypropylene filter housing), is easy to handle, and can filter large volumes of water (greater than or equal to 20 liters) with good recoveries. Filtrations can be conducted at the ambient pH of the water, and the unit cost per filtration (i.e., the cost of filter aid) comes to less than three cents per sampling. The technique could be useful in evaluation of viral water quality, study of ecology and occurrence of phages in natural waters, and isolation of rare phages from natural waters.

Coliphages↗

PubMed1994

Effect of distance from the polluting focus on relative concentrations of Bacteroides fragilis phages and coliphages in mussels.

Concentrations of fecal bacteria, somatic and F-specific coliphages, and phages infecting Bacteroides fragilis in naturally occurring black mussels (Mytilus edulis) were determined. Mussels were collected over a 7-month period at four sampling sites with different levels of fecal pollution. Concentrations of both fecal bacteria and bacteriophages in mussel meat paralleled the concentration of fecal bacteria in the overlying waters. Mussels bioaccumulated efficiently, although with different efficiencies, all of the microorganisms studied. Ratios comparing the levels of microorganisms in mussels were determined. These ratios changed in mussels collected at the different sites. They suggest that bacteriophages infecting B. fragilis and somatic coliphages have the lowest decay rates among the microorganisms studied, with the exception of Clostridium perfringens. On the contrary, concentrations of F-specific coliphages showed a greater rate of decay than the other bacteriophages at sites more distant from the focus of contamination. Additionally, levels of enteroviruses were studied in a number of samples, and in these samples, the B. fragilis bacteriophages clearly outnumbered the enteroviruses. The results of this study indicate that, under the environmental conditions studied, the fate of phages infecting B. fragilis released into the marine environment resembles that of human viruses more than any other microorganism examined.

Animals↗

PubMed1974

Synthesis of indicator strains and density of ribonucleic acid-containing coliphages in sewage.

Escherichia coli strains freshly isolated from natural sources are inefficient indicators of coliphages present in sewage. Four E. coli strains recently isolated from clinical specimens were mutagenized to obtain lac(-) mutants. Such mutants were infected with an F'lac(+) sex factor of E. coli K-12. Pairs of isogenic lac(-) and lac(-)/F'lac(+) strains were used as indicators of coliphages present in sewage, and it was found that such strains can be effectively used for a direct and almost selective enumeration of F-specific coliphage contents of sewage samples. Serological tests were applied to a number of F-specific phages isolated. All the isolates that were tested fell into two distinguishable antigenic classes: members of one class being related to ribonucleic acid (RNA) phage MS2 and those of the other being related to another RNA phage, namely, Qbeta. MS2-related phages have been found to be more widely distributed than the Qbeta related phages. Most habitats sampled were found to yield only one or the other kind of phage. Single-stranded deoxyribonucleic acid-containing F-specific phages were not detectable by the methods employed by us.

Anti-Bacterial Agents↗

PubMed1976

Role of lipopolysaccharide in adsorption of coliphage T4D to Escherichia coli B.

Coliphage T4D was strongly adsorbed to intact lipopolysaccharides and alkaline and lipase-treated lipopolysaccharides from cells of Escherichia coli B, but was not so adsorbed to heat-treated cells. In contrast, coliphage T2h was not adsorbed to lipopolysaccharides and the heat-treated cells. Acid hydrolysate of lipopolysaccharides strongly inhibited the adsorption of phage T4D to acetone and ether-treated cells. The adsorption of phage T4D to the acetone and ether-treated cells was markedly inhibited by authentic D-glucosamine, N-acetyl-D-glucosamine, alpha-methyl-N-acetyl-D-glucosaminide, alpha-methyl-D-glucoside, and D-maltose. Authentic D-glucose and E,L-2,6-diaminopimelic acid also showed similar activity. These compounds did not affect the adsorption of phage T2h to the acetone- and ether-treated cells. Concanavalin A and wheat-germ agglutinin inhibited phage T4D adsorption to the acetone and ether-treated cells probably by blocking the phage-receptor sites on the cell wall. The blocking by concanavalin A and by wheat-germ agglutinin was reversed by alpha-methyl-D-glucoside and by alpha-methyl-N-acetyl-D-glucosaminide, respectively. Results suggested the possibility that coliphage T4D requires N-acetyl-D-glucosaminyl-glucose or glucosyl-D-glucosamine residues of the core of lipopolysaccharides for the initial attachment to the cell wall of Escherichia coli B.

Acetone↗

PubMed1994

[Evaluation of the hygienic and sanitary quality of water from shallow wells situated in an urban area: a comparison between the use of coliphages and bacterial indicators of fecal pollution].

One hundred and four water samples from eight private shallow wells situated in the urban area of Jaboticabal city, State of S. Paulo, Brazil, were submitted to coliphage, total coliform, fecal coliform and fecal streptococcus counts, for the purpose of discovering their hygienic and sanitary quality and of verifying the correlations between the coliphage numbers and the fecal pollution indicator bacteria. Ninety-six (92.3%) of the samples were not up to the microbiological potability standards. This result demonstrates the unsatisfactory hygienic and sanitary quality of the water samples. The results show also the absence of correlations among coliphages, and the fecal pollution indicator bacteria.

Brazil↗

PubMed1981

Dispersal and fate of coliphages in the River Saar.

The dispersal of coliphages was analysed quantitatively in longitudinal profiles of the partially canalized River Saar. The numbers of coliphages ranged between 0 and 2380 PFU (plaque-forming units)/ml in the water and between 0 and 2550 PFU/g in the sediments, depending on the degree of fecal pollution. In canalized parts the phages were found mainly in the sediments, whereas in non-canalized parts they prevailed in the water phase (Fig. 3). Determinations performed at municipal sewage water outfalls revealed that in the canalized part most of the phages precipitated to the sediment on a water course of only about 250 m (Fig. 5). In the non-canalized part of the River Saar, sediments were not contaminated to this extent, since phages rather remained in the water, due to high current velocities (Fig. 5). According to these results coliphages are, just like some fecal bacteria, closely associated with particles. They are rapidly inactivated in the sediment and water (inactivation rates: 0.12 to 0.66 d-1).

Clostridium perfringens↗

PubMed1998

Yeast positive-stranded virus-like RNA replicons. 20 S and 23 S RNA terminal nucleotide sequences and 3' end secondary structures resemble those of RNA coliphages.

Saccharomyces cerevisiae strains carry single-stranded RNAs called 20 S RNA and 23 S RNA. These RNAs and their double-stranded counterparts, W and T dsRNAs, have been cloned and sequenced. A few nucleotides at both ends, however, remained unknown. These RNAs do not encode coat proteins but their own RNA-dependent RNA polymerases that share a high degree of conservation to each other. The polymerases are also similar to the replicases of RNA coliphages, such as Qbeta. Here we have determined the nucleotide sequences of W and T dsRNAs at both ends using reverse transcriptase polymerase chain reaction-generated cDNA clones. We confirmed the terminal sequences by primer-extension and RNase protection experiments. Furthermore, these analyses demonstrated that W and T dsRNAs and their single-stranded RNA counterparts (i) are linear molecules, (ii) have identical nucleotide sequences at their ends, and (iii) have no poly(A) tails at their 3' ends. Both 20 S and 23 S RNAs have GGGGC at the 5' ends and the complementary 5-nucleotides sequence, GCCCC-OH, at their 3' ends. S1 and V1 secondary structure-mapping of the 3' ends of 20 S and 23 S RNAs shows the presence of a stem-loop structure that partially overlaps with the conserved 3' end sequence. Nucleotide sequences and stem-loop structures similar to those described here have been found at the 3' ends of RNA coliphages. These data, together with the similarity of the RNA-dependent RNA polymerases encoded among these RNAs and RNA coliphages, suggest that 20 S and 23 S RNAs are plus-strand single-stranded virus-like RNA replicons in yeast.

Cloning, Molecular↗

PubMed1982

Adsorption of coliphages T1 and T7 to clay minerals.

Coliphages T1 and T7 of Escherichia coli were absorbed by kaolinite (K) and montmorillonite (M). Maximum adsorption of T7 (96%) to M was greater than that of T1 (84%), but the adsorption of both coliphages to K was the same (99%). Positively charged sites (i.e., anion exchange sites) on the clays appeared to be primarily responsible for the adsorption of T1 to K but only partially responsible for the adsorption of T1 to M; equilibrium adsorption isotherms of T1 to K and M did not show a correlation between adsorption and the cation exchange capacity of the clays, and the reduction in adsorption caused by sodium metaphosphate (a polyanion that interacts with positively charged sites on clay) was more pronounced with K than with M. The equilibrium adsorption isotherms of T7 to K and M suggested a correlation between adsorption and the cation exchange capacity of the clays. However, studies with sodium metaphosphate indicated that T7 also adsorbed to positively charged sites on the clays, especially on K. Adsorption of the coliphages to positively charged sites was greater with K than with M, probably because the ratio of positively charged sites to negatively charged sites was greater on K than on M.

Adsorption↗

PubMed2003

The genome sequence of Yersinia pestis bacteriophage phiA1122 reveals an intimate history with the coliphage T3 and T7 genomes.

The genome sequence of bacteriophage phiA1122 has been determined. phiA1122 grows on almost all isolates of Yersinia pestis and is used by the Centers for Disease Control and Prevention as a diagnostic agent for the causative agent of plague. phiA1122 is very closely related to coliphage T7; the two genomes are colinear, and the genome-wide level of nucleotide identity is about 89%. However, a quarter of the phiA1122 genome, one that includes about half of the morphogenetic and maturation functions, is significantly more closely related to coliphage T3 than to T7. It is proposed that the yersiniophage phiA1122 recombined with a close relative of the Y. enterocolitica phage phiYeO3-12 to yield progeny phages, one of which became the classic T3 coliphage of Demerec and Fano (M. Demerec and U. Fano, Genetics 30:119-136, 1945).

Amino Acid Sequence↗

PubMed1993

A two-dimensional model at the nucleotide level for the central hairpin of coliphage Q beta RNA.

We describe the construction and testing of a structural model at the nucleotide level for conformation CH of the central hairpin of genomic RNA from coliphage Q beta. The model was developed with the computer program MFOLD using both optimal and suboptimal predictions. Structural information obtained by electron microscopic analysis of Kleinschmidt spreadings of Q beta RNA was used to guide the modeling. The model was tested in solution with three enzymatic probes: RNase T1, RNase T2, and RNase V1, as well as four chemical probes: dimethylsulfate, diethylpyrocarbonate, kethoxal and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT). The structural analyses in solution are consistent with the predicted structural model. The model is also supported by comparative structural analysis with the related coliphage SP. The model provides a structural basis for published biochemical and genetic studies implicating large, long-range structural features in the co-regulation of viral coat and replicase expression. In addition, we show that the read-through region of the viral protein A1 forms a separate structural domain, and we suggest that it functions as a nucleation site that participates in the folding and refolding of the molecule during replication and translation. In addition to the central hairpin, we have analyzed the structure of the viral coat initiation region. Our studies show that the entire region consists of small local hairpins and that 26 nucleotides immediately surrounding the coat initiation codon are single-stranded.

Base Sequence↗

PubMed1998

Removal of waterborne human enteric viruses and coliphages with oxidized coal.

Human enteric viruses and coliphages have been detected in water that has undergone what is generally considered adequate treatment, including chlorination. Because small numbers of virus particles are needed for the initiation of a productive virus infection, the presence of any number of virus particles in water resources will always be of concern. In this investigation the ability of oxidized coal to remove viruses from water was investigated. The oxicoal product was found to be able to remove not only coliphages, but also various pathogenic human viruses from seeded water sources. Removal was dependent upon the type of virus, the period of exposure, and the concentration of oxidized coal.

Coal↗

PubMed1976

Inactivation of the T7 coliphage by monofunctional alkylating agents. Action of phage adsorption and injection of its DNA.

Alkylation by ethyl or methyl methanesulfonate to an extent that inactivates more than 99.5% of T7 coliphages has no effect on phage adsorption on Escherichia coli B cells, but decreases the amount of phage DNA injected into the host cells. Depurination interferes with the injection of the phage DNA. Failure to inject the whole phage genome thus appears to be a cause of the immediate as well as of the delayed inactivation of the T7 coliphage treated by monofunctional alkylating agents; the hypothesis that it is the only cause of inactivation, although not very likely, cannot be excluded at the present time.

Binding Sites↗

PubMed2001

Rapid coliphage detection assay.

A rapid coliphage detection assay was developed, based on the phage-induced release of beta-galactosidase from cells of Escherichia coli. The assay could detect as few as five coliphage per sample without an overnight incubation period. The range of acceptable assay parameters was identified.

Coliphages↗