Halobacterium halobium phage øH

Phage øH, a novel virus of the archaebacterium Halobacterium halobium, resembles in size and morphology two other Halobacterium phages. One-step growth curves show a 5.5 h eclipse, a latent period of 7 h, and an apparent burst size of 170. Phage øH contains linear, double-stranded DNA which has a molecular weight of 39 x 10⁶ and a GC content of 65%. A packaging model accounting for the partial circular permutation and terminal redundancy of øH DNA is suggested. Partial homology of øH DNA with the DNA of H. halobium, predominantly with the AT-rich satellite DNA, was observed. The presence of minor restriction fragments of øH DNA which could be removed by purification of phage from single plaques suggests the existence of phage variants with rearranged DNA. A strain of H. halobium containing øH DNA was isolated which is resistant to infection by phage øH.     

Marine phage genomics: what have we learned?

Marine phages are the most abundant and diverse form of life on the planet, and their genomes have been described as the largest untapped reservoir of genomic information. To date, however, the complete genome sequences of only 17 marine phage are known. Nevertheless, these genomes have revealed some interesting features, including the presence of photosynthetic genes in cyanophage and common patterns of genomic organization. Intriguing findings are also being made from studies of the uncultivated marine viral community genome ('metavirome'). The greatest challenge in interpreting the biology of these phages, and for making comparisons with their terrestrial counterparts, is the high proportion of unidentifiable open reading frames (approximately 60%). Future studies are likely to focus on sequencing more marine phage genomes from disparate hosts and diverse environments and on further basic studies of the biology of existing marine phages.     

UV-sensitization of phage ?X-174 by 5-bromouracil

Summary Experimental evidence for the sensitization of buffer-suspended phage X-174 by incorporation of 5-bromouracil against UV-light of 2,537 Å is given. The significance of missing desensitization by radical-scavenging compounds like cysteamine in single-stranded DNA is discussed. It is concluded tentatively that host-cell and/or phage depending reactivation processes are involved in sensitization and desensitization of BU-DNA.     

Secondary responsein vitro to antigen ΦX 174 phage

In a model secondary reactionin vitro, a correlation was demonstrated between the size of the antigen dose used for the prestimulation of spleen tissue donors and the type of antibodies formed in the anamnestic reaction. After a small dose of antigen ΦX 174, the antibody response three days after prestimulation was of the 19 S (IgM) type, but later secondary contactin vitro (after four months) did not produce a 19 S anamnestic reaction. After large primary doses of antigen, a short interval between primary and secondary contact led to the formation of 19 and 7 S type antibodies, while after a long interval only 7 S (IgG) type antibodies were formed. The results are discussed in relation to differences in the size of the antigen dose needed to induce short-term 19 S and long-term 7 S immunological memory.     

Modulation effect of filamentous phage on α-synuclein aggregation

Conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases, including Parkinson’s disease (PD). Here we describe the modulating effect of filamentous phages on aggregation of α-synuclein (AS) in vitro and in a PD cellular model. Filamentous phages, well understood at both structural and genetic levels, have a nanotubular appearance, showing conformational similarities to amyloid fibrils. Since filamentous phages can infect only bacteria and have no tropism to mammalian cells, we utilized the f88 system to present a peptide containing a cyclic RGD (arg-gly-asp), which enabled phage internalization into the cells. Detection of intracellular AS oligomers, in differentiated SH-SY5Y cells, stably transfected with wild type AS gene, was performed using Western blot and ELISA measurements. Data presented here show reduced levels of AS soluble aggregates in phage treated cells compared to non-treated cells, suggesting new therapeutics for PD.     

Ionic switch controls the DNA state in phage λ

We have recently found that DNA packaged in phage λ undergoes a disordering transition triggered by temperature, which results in increased genome mobility. This solid-to-fluid like DNA transition markedly increases the number of infectious λ particles facilitating infection. However, the structural transition strongly depends on temperature and ionic conditions in the surrounding medium. Using titration microcalorimetry combined with solution X-ray scattering, we mapped both energetic and structural changes associated with transition of the encapsidated λ-DNA. Packaged DNA needs to reach a critical stress level in order for transition to occur. We varied the stress on DNA in the capsid by changing the temperature, packaged DNA length and ionic conditions. We found striking evidence that the intracapsid DNA transition is ‘switched on’ at the ionic conditions mimicking those in vivo and also at the physiologic temperature of infection at 37°C. This ion regulated on-off switch of packaged DNA mobility in turn affects viral replication. These results suggest a remarkable adaptation of phage λ to the environment of its host bacteria in the human gut. The metastable DNA state in the capsid provides a new paradigm for the physical evolution of viruses.     

Comparative genomics of Bacillus thuringiensis phage 0305φ8-36: defining patterns of descent in a novel ancient phage lineage

Background

Members of the familyIridoviridae can cause severe diseases resulting in significant economic and environmental losses. Very little is known about how iridoviruses cause disease in their host. In the present study, we describe the re-analysis of theIridoviridae family of complex DNA viruses using a variety of comparative genomic tools to yield a greater consensus among the annotated sequences of its members.

Results

A series of genomic sequence comparisons were made among, and between theRanavirus andMegalocytivirus genera in order to identify novel conserved ORFs. Of these two genera, theMegalocytivirus genomes required the greatest number of altered annotations. Prior to our re-analysis, the Megalocytivirus species orange-spotted grouper iridovirus and rock bream iridovirus shared 99% sequence identity, but only 82 out of 118 potential ORFs were annotated; in contrast, we predict that these species share an identical complement of genes. These annotation changes allowed the redefinition of the group of core genes shared by all iridoviruses. Seven new core genes were identified, bringing the total number to 26.

Conclusion

Our re-analysis of genomes within theIridoviridae family provides a unifying framework to understand the biology of these viruses. Further re-defining the core set of iridovirus genes will continue to lead us to a better understanding of the phylogenetic relationships between individual iridoviruses as well as giving us a much deeper understanding of iridovirus replication. In addition, this analysis will provide a better framework for characterizing and annotating currently unclassified iridoviruses.     

Damages induced in λ phage DNA by enzyme-generated triplet acetone

Exposure of λ phage to triplet acetone, generated via the oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only under alkaline conditions, and so true breaks do not occur. Also, no sites sensitive to UV-endonuclease from Micrococcus leteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed.     

An origin of DNA replication from streptomycete phage ΦU1

A DNA fragment from phage ΦU1 containing an origin of DNA replication was identified. This fragment, designatedori, was able to support the maintenance inStreptomyces lividans of a plasmid lacking a functional Gram-positiveori. The sequence of the minimalori fragment was determined and analyzed. The minimal fragment conferring replication origin function contained a number of direct and inverted repeats. The absence of an open reading frame in thisori fragment indicates that host factors alone were sufficient to initiate replication atori.     

Mapping protein–ligand interactions using whole genome phage display libraries

The function of many genes cannot be deduced from sequence similarity, and biochemical methods are usually required. Whole genome sequences can be thought of as not only a set of genes but also collections of functional domains. These domains can be studied by affinity methods whereby identification of the ligand can provide information on biochemical function. To take advantage of this method, one must express all functional domains in a form suitable for affinity studies. Phage display technology provides a means for accomplishing this. The pJuFo phage display system, based on the interaction between the leucine zippers Jun and Fos, has been modified and used to create a genomic phage display library from Escherichia coli MG1655. The system has been tested by using the library to map the dominant binding epitopes for an anti-RecA protein polyclonal antibody sera. This methodology provides a general biochemical approach to functional analysis of protein–ligand interactions on a genome-wide basis.     

Cloning and characterization of the immunity region of phage ?80

Summary The immunity region of phage 80 has been localized. It codes for at least three proteins: a protein of 34 kDa which has the biological properties of the phage repressor, and two other proteins of 9 kDa and 18 kDa which are the first proteins on the rightward operon. These two proteins are negatively regulated by the 34 kDa protein at a divergent promoter site. By position analogy with phage , but not by its biological activity, the 9 kDa protein could be the cro roduct. The 18 kDa protein is able to block totally UV induction of phage 80.     

Ultraviolet inactivation and photoreactivation of the cholera phage ‘Kappa’

Summary The lysogenic cholera phage, Kappa is some ten to twenty folds more resistant to UV (254 nm) than are most of the T. phages ofE. coli, or the cholera phage PL 163/10, or the hostV. cholerae strain H218 Sm^r, the 37% (D ₃₇) and 10% (D ₁₀) survival doses being 255.8 J/m² and 633.6 J/m² respectively. The UV-irradiated Kappa phages could be photoreactivated in the hostV. cholerae strain H218 Sm^r to a maximum extent of 40%. The removal of the number of lethal hits per phage by the survival-enhancement treatment (photoreactivation) with time followed an exponential relation, the constant probability of removal of lethal hit per unit time being 2.8 × 10^–2 min^–1. The UV-irradiated phages could also be Weigle reactivated in the host strain H218 Sm^r by a small degree, the maximum reactivation factor (ratio of survivals in UV-irradiated and non-irradiated hosts) being 1.50.     

Osmotic pressure: resisting or promoting DNA ejection from phage?

Recent in vitro experiments have shown that DNA ejection from bacteriophage can be partially stopped by surrounding osmotic pressure when ejected DNA is digested by DNase I in the course of ejection. In this work, we argue by a combination of experimental techniques (osmotic suppression without DNase I monitored by UV absorbance, pulse-field electrophoresis, and cryo-transmission electron microscopy visualization) and simple scaling modeling that intact genome (i.e., undigested) ejection in a crowded environment is, on the contrary, enhanced or eventually complete with the help of a pulling force resulting from DNA condensation induced by the osmotic stress itself. This demonstrates that in vivo, the osmotically stressed cell cytoplasm will promote phage DNA ejection rather than resist it. The further addition of DNA-binding proteins under crowding conditions is shown to enhance the extent of ejection. We also found some optimal crowding conditions for which DNA content remaining in the capsid upon ejection is maximum, which correlates well with the optimal conditions of maximum DNA packaging efficiency into viral capsids observed almost 20 years ago. Biological consequences of this finding are discussed.