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PubMed1989

Further characterization of a bacteriophage recovered from an avian strain of Chlamydia psittaci.

The genome of a 22 nm icosahedral phage which infects some avian Chlamydia psittaci strains recovered from domestic ducks has been characterized as a ss circular DNA molecule of about 4850 bases. The replicative form of this genome was isolated from purified chlamydial organisms. A restriction endonuclease cleavage site map of the genome was constructed from dsDNA synthesized in vitro from ss phage DNA and EcoRI fragments were then cloned into pUC9. The phage genome was detected only by Southern blot hybridization in C. psittaci which was productively infected with phage; no evidence was found for the integration of phage DNA into the chlamydial chromosome. Three viral polypeptides, of approximate Mr values 75K, 30K and 16.5K were identified when phage was analysed by SDS-PAGE. This virus, which we have designated Chp 1, is either an aberrant member of the Microviridae or the first member of a new bacteriophage family.

Animals↗

PubMed1983

Sewage coliphages studied by electron microscopy.

Sewage was enriched with 35 Escherichia coli strains, and sediments of enrichment cultures were studied in the electron microscope. They contained up to 10 varieties of morphologically different particles. T-even-type phages predominated in 14 samples. Thirteen phages were enriched, representing the families Myoviridae (seven), Styloviridae (two), Podoviridae (three), and Microviridae (one). Twelve of these corresponded to known enterobacterial phage species, namely, 121, K19, FC3-9, O1, 9266, T2, 16-19, kappa, beta 4, N4, T7, and phi X174. Cubic RNA phages and filamentous phages were not detected. Types 121 and 9266 have previously been observed only in Romania and South Africa. Identification by morphology is usually simple. Our investigative technique is qualitative and will not detect all phages present. Most enrichment strains are polyvalent, and electron microscopy is always required for phage identification. In a general way, electron microscopy seems to be the method of choice for investigation of phage geography and ecology.

Coliphages↗

PubMed1994

Differential Effect of Tetrazolium Dyes upon Bacteriophage Plaque Assay Titers.

This study examined whether the practice of incorporating either tetrazolium red or tetrazolium violet dye into plaque assay medium deleteriously influences plaque assay titers. Representative members of six different virus families were studied: Cystoviridae (varphi6), Leviviridae (MS2), Microviridae (varphiX174), Myoviridae (T2), Podoviridae (P22), and Siphoviridae (Denver, T1, and VD13). Each of the members of the Podoviridae and Siphoviridae families appeared to be suppressed by either one or both dyes at a 300-mug/ml concentration. The chosen representatives of the other bacteriophage families were not suppressed by either dye at a 300-mug/ml concentration. Subsequent trials revealed no suppression of Podoviridae or Siphoviridae plaque assay titers when members of these virus families were tested with the same two dyes at the lower concentrations of 150 and 50 mug/ml. Interestingly, the bacteriophage families whose members were affected by the dyes have additional commonality in that they are the two bacteriophage families whose members possess both double-stranded DNA genomes and noncontractile tails.

Journal Article↗

PubMed2005

Identification of an interacting coat-external scaffolding protein domain required for both the initiation of phiX174 procapsid morphogenesis and the completion of DNA packaging.

The phiX174 external scaffolding protein D mediates the assembly of coat protein pentamers into procapsids. There are four external scaffolding subunits per coat protein. Organized as pairs of asymmetric dimers, the arrangement is unrelated to quasi-equivalence. The external scaffolding protein contains seven alpha-helices. The protein's core, alpha-helices 2 to 6, mediates the vast majority of intra- and interdimer contacts and is strongly conserved in all Microviridae (canonical members are phiX174, G4, and alpha3) external scaffolding proteins. On the other hand, the primary sequences of the first alpha-helices have diverged. The results of previous studies with alpha3/phiX174 chimeric external scaffolding proteins suggest that alpha-helix 1 may act as a substrate specificity domain, mediating the initial coat scaffolding protein recognition in a species-specific manner. However, the low sequence conservation between the two phages impeded genetic analyses. In an effort to elucidate a more mechanistic model, chimeric external scaffolding proteins were constructed between the more closely related phages G4 and phiX174. The results of biochemical analyses indicate that the chimeric external scaffolding protein inhibits two morphogenetic steps: the initiation of procapsid formation and DNA packaging. phiX174 mutants that can efficiently utilize the chimeric protein were isolated and characterized. The substitutions appear to suppress both morphogenetic defects and are located in threefold-related coat protein sequences that most likely form the pores in the viral procapsid. These results identify coat-external scaffolding domains needed to initiate procapsid formation and provide more evidence, albeit indirect, that the pores are the site of DNA entry during the packaging reaction.

Amino Acid Sequence↗

PubMed2004

Comparison of bacteriophages for use in iodine inactivation: batch and continuous flow studies.

Inactivation rates in batch studies for four commonly used surrogate bacteriophages were measured in stable aqueous iodine solutions for the purpose of determining which was the most suited to evaluate iodine disinfection efficacy in batch and continuous flow conditions. Two types of group Leviviridae bacteriophages were used, Type I (MS2) and Type II (GA), along with group Microviridae, Phi-X174, and group Tectiviridae, PRD1. Inactivation was compared at iodine doses of 1.0-1.5 mg l2/l. MS2 was the most susceptible to iodine inactivation of the four phages tested. Inactivation of naked, icosahedral bacteriophages, MS2 and Phi-X174 demonstrated removals to below detection limits (>99.99%) in less than 10 min. Lipid-containing PRD1 and F+ssRNA GA bacteriophages demonstrated the greatest iodine resistance in batch experiments with an average of 1.82 logs of inactivation (98.5%) after 60 min and 1.05 logs of inactivation (91.1%) after 30 min respectively. Similarly, in continuous flow studies through pentaiodide quaternary ammonium strong base resin, MS2, GA and Phi-X174 were more strongly inactivated than PRD1. The lipid component of PRD1 is thought to enhance resistance to iodine over non-lipid-containing bacteriophages by protecting easily oxidized groups on the protein capsid, but further research is needed before proving this hypothesis. The results from this research may provide a surrogate standard for more rigorous and developed research into the mode of iodine disinfection and its inactivation kinetics.

Anion Exchange Resins↗

PubMed2004

Genetic and functional analyses of the øX174 DNA binding protein: the effects of substitutions for amino acid residues that spatially organize the two DNA binding domains.

The øX174 DNA binding protein contains two DNA binding domains, containing a series of DNA binding basic amino acids, separated by a proline-rich linker region. Within each DNA binding domain, there is a conserved glycine residue. Glycine and proline residues were mutated and the effects on virion structure were examined. Substitutions for glycine residues yield particles with similar properties to previously characterized mutants with substitutions for DNA binding residues. Both sets of mutations share a common extragenic second-site suppressor, suggesting that the defects caused by the mutant proteins are mechanistically similar. Hence, glycine residues may optimize DNA-protein contacts. The defects conferred by substitutions for proline residues appear to be fundamentally different. The properties of the mutant particles along with the atomic structure of the virion suggest that the proline residues may act to guide the packaged DNA to the adjacent fivefold related asymmetric unit, thus preventing a chaotic packaging arrangement.

Amino Acid Sequence↗

PubMed2003

Bacteriophage observations and evolution.

Bacteriophages are classified into one order and 13 families. Over 5100 phages have been examined in the electron microscope since 1959. At least 4950 phages (96%) are tailed. They constitute the order Caudovirales and three families. Siphoviridae or phages with long, noncontractile tails predominate (61% of tailed phages). Polyhedral, filamentous, and pleomorphic phages comprise less than 4% of bacterial viruses. Bacteriophages occur in over 140 bacterial or archaeal genera. Their distribution reflects their origin and bacterial phylogeny. Bacteriophages are polyphyletic, arose repeatedly in different hosts, and constitute 11 lines of descent. Tailed phages appear as monophyletic and as the oldest known virus group.

Bacteriophages↗

PubMed2003

Bacterial host strains that support replication of somatic coliphages.

Somatic coliphages detected by Escherichia coli strain WG5 have been proposed as potential indicators of water quality. Their potential replication in the water environment is considered a drawback for their use as indicators. However, the contribution of replication outside the gut to the total numbers has never been quantified. It has not been determined either the fraction of bacterial strains that might support replication of phages detected by strain WG5 in the water environment. We examined the sensitivity of 291 host strains to 25 phages by streaking slants of the presumptive host strain onto an agar layer that contains bacteriophages, which gives a total of 7275 combinations (sensitivity tests). Only a 3.02% of the tests showed sensitivity. Additionally, six environmental strains were used as hosts to count phages in sewage and seawater. Phages isolated on these strains were used to infect strain WG5. The environmental strains detected 1 log10 fewer phages than strain WG5 in sewage and seawater. The fraction of phages that were detected by the six strains and that also infected strain WG5 ranged from < 0.07% to < 2.0% of the total amount of bacteriophages detected by strain WG5 in the same samples. Our results confirm that less than 3% of naturally occurring hosts support replication of phages infecting E. coli. We conclude that the contribution of replication to the number of somatic coliphages detected in the aquatic environment is negligible.

Coliphages↗