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PubMed1976

Inactivation and mutation of coliphage T4 by aliphatic nitrosamides and methanesulphonates: in vitro recovery of infectivity of T4 inactivated by isopropyl methanesulphonate.

The inactivation and mutation (to r phenotype) of extracellular coliphage T4 wild-type by the monofunctional alkylating agents N-methyl- and N-ethyl-N-nitrosourea and isopropyl methanesulphonate were investigated. The rate and extent of change in phage infectivity observed during the post-treatment period were found to correlate with what is known of the mechanisms by which these agents react in vitro. Loss of phage infectivity was found to occur during the period following treatment with these agents, but that resulting from treatment with isopropyl methanesulphonate was preceded, in the first 24 to 48 h, by a recovery of infectivity. This suggested that changes in phage infectivity occurring after treatment with monofunctional alkylating agents are resultant of various processes which diversely promote loss and recovery of infectivity. The mutagenicity of N-methyl-N-nitrosourea was similar to that of its ethyl homologue at a level of phage survival of 4 x 10-3, but less than that of isopropyl methanesulphonate. At a level of survival of 3 x 10-2 ethyl methanesulphonate was a mutagenic as its isopropyl homologue, but methyl methanesulphonate was only slightly if at all mutagenic. These results could not be correlated with the compounds' reaction mechanisms. The efficiency of isopropyl methanesulphonate (compared with its toxicity to phage) was found to decrease as the severity of the dose was increased.

Coliphages↗
PubMed1987

Characterization of phage 18, an unstable coliphage.

Phage 18, a noninducible coliphage, is quite unstable and therefore difficult to study. Newly developed very gentle lysis and mounting techniques yielded isolated virions for examination by electron microscopy. The phage has a contractile tail with a length of 130 nm and an isometric head with a capsid diameter of 50 nm. Phage 18 is similar in morphology to phage P2 but is heteroimmune to it. DNA extracted from a clear-plaque mutant of phage 18 was subjected to BamHI restriction endonuclease digestion and was found to be easily distinguishable from the published restriction patterns for P2, phage 299, or phage 186 DNA. The genome size was calculated to be 33.5 kb. Using the DNA melting point, phage 18 DNA (G+C) content was determined to be 55.0% and its buoyant density was determined to be 1.715.

Coliphages↗
PubMed1990

Secondary structure at the 3' terminal region of RNA coliphages: comparison with tRNA.

Secondary structure models for the 3' non-coding region of the four groups of coliphage RNA are proposed based on comparative sequence analysis and on previously published data on the sensitivity of nucleotides in MS2 RNA to chemical modification and enzymes. We report the following observations. (1) In contrast to the coding regions, the structure at the 3' terminus is characterized by stable regular helices. We note the occurrence of the loop sequences 5'-GUUCGC and 5'-CGAAAG, that are reported to confer exceptional stability to stem structures. These features are probably present to promote the segregation of mother and daughter strands during replication. (2) Comparison of homologous helices indicates that only those base pair substitutions are allowed that maintain the thermodynamic stability. (3) We have compared the structure of phage RNA with tRNA. Overall similarity is low, but one common element may exist. It is a quasi-continuous helix of 12 basepairs that could be the equivalent of the 12 basepair long coaxially stacked helix, formed by the T psi C arm and the aminoacyl acceptor arm in tRNA. As in tRNA, this structure element starts after the fourth nucleotide from the 3' end. (4) Phage RNA contains a large variable region of about 35 nucleotides bulging out from the quasi-continuous helix. We speculate that the variable loop in present-day tRNA could be the remnant of the variable region found in phage RNA. The variable region contains overlapping binding sites for the replicase enzyme and the maturation protein. This common binding site may serve as a switch from replication to packaging.

Anticodon↗
PubMed1991

Two different Escherichia coli capsular polysaccharide depolymerases each associated with one of the coliphage phi K5 and phi K20.

The Escherichia coli capsular polysaccharides (K antigens) K5 and K20 are known as primary receptors for the coliphage phi K5 and phi K20, respectively. A host range study of the phage revealed that E. coli K5 strains were not only lysed by phi K5 but also by phi K20, and furthermore that the E. coli K95 test strain was attacked by phi K5 in addition to K5 strains. In order to find out whether the phage can degrade the K antigens, the interaction of the phage with isolated polysaccharides was studied. It could be demonstrated that phi K5 was able to depolymerize the K5 and K95 polysaccharides and that phi K20 showed degrading activity towards the antigens K20 and K5. Obviously, each of the phages was associated with two different enzyme systems which enabled them to recognize and depolymerize chemically unrelated polysaccharides.

Acetylglucosamine↗
PubMed2004

Protein-protein interaction between monomers of coliphage HK022 excisionase.

Excisionase (Xis) is an accessory protein that is required for the site-specific excision reaction of the coliphages HK022 and lambda. Xis binds in a strong cooperative manner to two tandem binding sites (X1 and X2) located on the P arm of the attachment (att) sites on the phage genome. As a result of crosslinking experiments in vivo and in vitro of Xis-overexpressing cells, by gel filtration of purified Xis and by FRET analyses we show that Xis monomers of HK022 interact and form dimers that are not dependent on the single Cys residue of the protein and on the presence of DNA. The formation of the dimers may explain the strong binding cooperativity of Xis to its sites on DNA.

Chromatography, Gel↗
PubMed2004

The genome and proteome of coliphage T1.

The genome of enterobacterial phage T1 has been sequenced, revealing that its 50.7-kb terminally redundant, circularly permuted sequence contains 48,836 bp of nonredundant nucleotides. Seventy-seven open reading frames (ORFs) were identified, with a high percentage of small genes located at the termini of the genomes displaying no homology to existing phage or prophage proteins. Of the genes showing homologs (47%), we identified those involved in host DNA degradation (three endonucleases) and T1 replication (DNA helicase, primase, and single-stranded DNA-binding proteins) and recombination (RecE and Erf homologs). While the tail genes showed homology to those from temperate coliphage N15, the capsid biosynthetic genes were unique. Phage proteins were resolved by 2D gel electrophoresis, and mass spectrometry was used to identify several of the spots including the major head, portal, and tail proteins, thus verifying the annotation.

Amino Acid Sequence↗
PubMed1971

The physical measurement of radiation damage to coliphage T7 DNA.

Coliphage T7 was exposed to (60)Co gamma radiation while suspended in phosphate buffer or in phosphate buffer plus 0.001 M l-histidine. DNA was isolated from the phage by incubation with pronase, followed by extraction with cold phenol. The intrinsic viscosity of the DNA was measured as a function of radiation dose. The fraction of DNA molecules surviving radiation treatment with no double-strand breaks was measured from the radiation-induced heterogeneity of the DNA sedimentation boundary. From comparison of these measurements it is concluded that radiation introduces lesions other than double-strand breaks which affect the hydrodynamic properties of the DNA. In both buffer and buffer plus histidine the surviving fraction of intact virus genomes far exceeds the surviving fraction of plaque-forming units at any given dose. It was found that the decrease in intrinsic viscosity with dose is independent of the presence of histidine in the radiation medium. From this it is concluded that DNA damage is primarily due to a direct effect of radiation on the phage particle. The procedure necessary to isolate DNA from irradiated virus suggests that radiation produces covalent bonding of protein to the DNA.

Cobalt Isotopes↗
PubMed1990

DNA replication studies with coliphage 186. III. A single phage gene is required for phage 186 replication.

We have shown that the BglII to BamHI (79.6% to 95.8%) region of the coliphage 186 chromosome can direct 186-specific replication. DNA sequencing of the region revealed five presumptive genes, CP80, CP81, CP83, CP84 and CP87. Surprisingly, alleles of the previously defined replication gene, A, were localized in both CP84 and CP87. We have successfully constructed a 186 minichromosome using the single gene CP87, and determined that CP84 was not concerned with replication, neither of a minichromosome nor of the phage. Rather, the replication defect seen with amber mutants of CP84 reflects a polarity effect on the downstream expression of CP87. We have concluded that CP87 is the only phage gene necessary for 186 replication, and have called it gene A.

Amino Acid Sequence↗
PubMed2003

Seasonal change and fate of coliphages infected to Escherichia coli O157:H7 in a wastewater treatment plant.

Seasonal change of virulent phage infected to two E. coli O157:H7 strains (O:157-phage) in the influent of a domestic wastewater treatment plant in the central part of Japan and fate of O:157-phage in the plant were monitored almost monthly from March 2001 to February 2002. Coliphage infected to nonpathogenic E. coli O157:H7 ATCC43888 (43888-phage) was detected for 1 year. On the other hand, phage infected to pathogenic E. coli O157:H7 EDL933 (EDL-phage) was detected intermittently. Concentration of EDL-phage was almost one-tenth of that of 43888-phage. The progressive decrease in phage concentration with the treatment steps was observed. No phage was detected in the supernatant from the secondary settling tank and effluent. PCR amplification of the Stx 2 gene that encodes Shiga toxin (Stx) was observed when O:157-phage concentration in the influent was high x10(3) PFU/ml order. Concentration and percentage of suspended O:157-phage decreased with the progress of the wastewater treatment. 933W phage, which encodes Stx 2 gene, was more fragile and sensitive to chlorination than T4 phage. However, addition of 0.02 mg/l chlorine, in conformance with the required concentration of the plant, did not affect the viability of T4 and 933 W phages. On the other hand, 1mg/l chlorine inactivated the 933 W phage significantly.

Chlorine Compounds↗
PubMed1979

The influence of operating conditions of activated-sludge treatment on the behaviour of f2 coliphage.

The behaviour of f2 coliphage during activated-sludge treatment was influenced by the temperature, flow-through-time, concentration of mixed liquor suspended solids and the virus load. The most sensitive way to detect behavioural changes was to examine the regression coefficients for the rate of uptake or loss of virus by the mixed liquor solids. This type of analysis revealed, for instance, high values when the solids concentration was high and even greater values occurred when high inocula were used. At high temperature the rate of loss of virus titre after inoculation had stopped was greater than the rate of uptake of virus during inoculation although in all other conditions uptake occurred at a greater rate than the loss of virus. The coefficients were relatively low when the flow rate was increased, when the temperature was low or when the inoculum was small. The distribution of virus between the solids and liquid fractions of the mixed liquor varied somewhat for all conditions but was notably different when (a) the plant was incubated at 5 degrees C when there was much less virus in the solids fraction than usual, and (b) when the inoculum was low and a much higher proportion of virus was found in the solids. The efficiency with which virus was removed across the plant was the least-sensitive determinant of viral behaviour and the value was about the same for most treatment conditions. However, low or high inocula did result in some increased or decreased removal of virus, respectively.

Coliphages↗
PubMed1980

Inactivation of f2 coliphage in municipal effluent by the use of various disinfectants.

Bromine chloride, chlorine and peracetic acid inactivated f2 coliphages in effluent but in order to achieve 99.99% inactivation the three disinfectants were required at about 1, 10 and 100 mg/l respectively. The activity of chlorine was halved by the presence of added organic matter, whereas bromine chloride and peracetic acid were very little affected. When a second successive dose of virus was added to the reaction mixture, the virus was inactivated only by peracetic acid despite the fact that in the chlorine-treated effluent residual chlorine was detected. The addition of a second dose of disinfectant inactivated residual virus in the same way as the first dose.

Acetates↗
PubMed1973

A sequence of seventy-three nucleotides from the coliphage R17 genome.

1. A sequence of 73 nucleotides of the RNA genome from coliphage R17 was determined. It can be read through in only one translational frame. The fragment is not part of the coatprotein cistron (Min Jou et al., 1972), nor does it come from the untranslated sequences described previously (Steitz, 1969; Nichols, 1970; Cory et al., 1970; de Wachter et al., 1971; Contreras et al., 1971; Cory et al., 1972). It contains two sequences of 23 and 24 nucleotides, 22 of which are identical. This kind of reiteration is the first one found in bacteriophage nucleic acid. 2. Improved conditions were found and tested for blocking oligonucleotides with carbodi-imide and cleaving by ribonuclease A at cytidylate residues. 3. A synthetic medium is described which allows labelling in vivo with (32)P to give specific radioactivities higher than those obtained in the procedures used previously.

Animals↗
PubMed2001

A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2.

Sequence analysis of a 10-kb region of the genome of the marine cyanomyovirus S-PM2 reveals a homology to coliphage T4 that extends as a contiguous block from gene (g)18 to g23. The order of the S-PM2 genes in this region is similar to that of T4, but there are insertions and deletions of small ORFs of unknown function. In T4, g18 codes for the tail sheath, g19, the tail tube, g20, the head portal protein, g21, the prohead core protein, g22, a scaffolding protein, and g23, the major capsid protein. Thus, the entire module that determines the structural components of the phage head and contractile tail is conserved between T4 and this cyanophage. The significant differences in the morphology of these phages must reflect the considerable divergence of the amino acid sequence of their homologous virion proteins, which uniformly exceeds 50%. We suggest that their enormous diversity in the sea could be a result of genetic shuffling between disparate phages mediated by such commonly shared modules. These conserved sequences could facilitate genetic exchange by providing partially homologous substrates for recombination between otherwise divergent phage genomes. Such a mechanism would thus expand the pool of phage genes accessible by recombination to all those phages that share common modules.

Base Sequence↗
PubMed1972

Formation of hybrids between coliphage lambda and Salmonella phage P22 with a Salmonella typhimurium hybrid sensitive to these phages.

An unusual Salmonella typhimurium hybrid with sensitivity to coliphage lambda and salmonella phage P22 has been recovered from matings between an Escherichia coli K-12 Hfr donor and an S. typhimurium recipient. The hybrid is an excellent host for achieving genetic recombination between lambda and P22. Two broad classes of hybrid phages were isolated. The lambda-P22 hybrid class, which has the protein coat of lambda, contains at least the c region of P22. The P22-lambda hybrid class has the protein coat of P22 and has inherited at least the c marker of lambda.

Animals↗
PubMed1974

Expression of phage transcription in P2 lysogens infected with helper-dependent coliphage P4.

The DNA of helper-dependent coliphage P4 and the DNA of its helper-P2-show no detectable sequence homology as measured by DNA.DNA hybridization. The lack of cross-hybridization permits direct analysis of P4 as well as of P2 transcription in P4-infected P2 lysogens by RNA.DNA hybridization. P4-transactivated P2 transcription can be detected around 20 min after P4 infection of the P2 lysogen and the rate (per infected cell) of that transcription becomes equal to that of the P4 transcription at the end of the latent period of P4. Furthermore, P4 transcription appears to be stimulated by the presence of the helper. Conceivably, P2 codes for a stimulator of P4 transcription. Rifamycin has been used to investigate the role of the host RNA polymerase during P4 transactivation of P2 transcription. The results exclude the participation of a P4-coded RNA polymerase and indicate that the original host RNA polymerase is responsible for the bulk of P4 and P2 transcription during transactivation.

Chloramphenicol↗
PubMed1975

Acquisition of a determinant for chloramphenicol resistance by coliphage lambda.

A determinanat for chloramphenicol resistance, cam, initially detected on a resistance transfer factor (RTF) and since transferred to phage P1, may be acquired from P1 by coliphage lambda. Lambdapcam are obtained when a lambda prophage is induced in bacteria which also harbor P1 cam prophage. Lambdacam formation is not dependent upon host Rec or lambda Red recombination functions. Electron microscopic heteroduplex analysis shows that the cam locus in two lambdapcams is a 5% addition of DNA in the b2 region of lambda, not contiguous with att. The extent and nucloetide sequence of the DNA insertion in the two independent lambdapcam isolates appear to be the same though they are located at different sites within the b2 region. We conclude that the determinant for chloramphenicol resistance is contained on a unique piece of DNA which facilitates its insertion into a number of unrelated genomes.

Chloramphenicol↗
PubMed1978

Structure of nascent replicative form DNA of coliphage M13.

Nascent replicative form type II (RFII) DNA of coliphage M13 synthesized in an Escherichia coli mutant deficient in the 5' leads to 3' exonuclease associated uith DNA polymerase I contains ribonucleotides that are retained in the covalently closed RFI DNA sealed in vitro by the joint action of T5 phage DNA polymerase and T4 phage DNA ligase. These RFI molecules are labile to alkali and RNase H, unlike the RFI produced either in vivo or from RFII with E. coli DNA polymerase I and E. coli DNA ligase. The ribonucleotides are located at one site and predominantly in one strand of the nascent RF DNA. Furthermore, these molecules contain multiple small gaps, randomly located, and one large gap in the intracistronic region.

Chromosome Mapping↗
PubMed1980

Mechanisms of membrane assembly: effects of energy poisons on the conversion of soluble M13 coliphage procoat to membrane-bound coat protein.

The coat protein (gene 8 product) of coliphage M13 spans the host cell plasma membrane prior to its assembly into extruding virions. It is made as a soluble precursor, termed procoat, with an extra 23 NH2-terminal amino acid residues. We have examined the effect of metabolic poisons on the assembly of procoat into the plasma membrane and its proteolytic conversion to coat protein. Protein synthesis and proline uptake were measured to assess the effect of each poison on cellular high-energy phosphate and on the transmembrane protonmotive force, respectively. Arsenate, which abolished protein synthesis but did not affect proline uptake, had no measurable effect on the conversion of procoat to coat protein. In contrast, the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) blocked conversion of procoat to coat protein. Neither CCCP nor arsenate inhibited the ability of a detergent-solubilized and highly purified preparation of leader peptidase to convert procoat to coat protein in the presence of detergents. The procoat that accumulated in the presence of CCCP was membrane bound. A spontaneous mutant that grows in the presence of CCCP showed (i) CCCP-resistant proline uptake in whole cells, (ii) CCCP-resistant uptake in inner membrane vesicles, and (iii) CCCP-resistant conversion of procoat protein to coat protein. These data suggest that an electrochemical gradient is at least indirectly necessary for the proper assembly of procoat into the cellular membrane.

Arsenates↗