Search

Cronobacter sakazakii (C. sakazakii) is an important pathogen contaminating dairy products (e.g., milk pow-der) and causes high mortality in infants. Bacteriophage as a potential biocontrol agent is a good alternative method for the control of this pathogen in dairy production and its environment. Thus, it is important to complete the C. sakazakii phage library by isolating and characterizing the broad host range of bacteriophage against C. sakazakii for control use. In this study, C. sakazakii strains from different sources were used as hosts to isolate and purify phages from human stool and sewage samples by double-layer plates. The biological characteristics, antibacterial properties, and genomes of these phages were then studied. Finally, ten virulent phages (EspYZU01–EspYZU10) infecting C. sakazakii were isolated and identified as belonging to the Myoviridae, Podoviridae, Tectivirus, and Stylovinidae families. Phage EspYZU08 presented the broadest host range and could infect all the five host strains of C. sakazakii. All 10 phages retained their infectivity at 50°C and pH 5–9. Both genomes of EspYZU05 and EspYZU08 were double-stranded DNAs with sizes of 41723 bp and 145582 bp, G+C contents of 55.69% and 46.75%, and open reading frames of 47 and 103, respectively. No toxins and antibiotic resistance genes were detected in both EspYZU05 and EspYZU08. Phage EspYZU08 and phage cocktail-3 (EspYZU01 + EspYZU03 + EspYZU08 + EspYZU09 + EspYZU10) presented excellent antibacterial efficacy for C. sakazakii in liquid broth and milk at 4°C, 25°C, and 37°C, suggesting that the phages in this study have great potential for the development of biocontrol agents against C. sakazakii in dairy and its processing environment.

Tectiviruses infecting the Bacillus cereus group represent part of the bacterial “plasmid repertoire” as they behave as linear plasmids during their lysogenic cycle. Several novel tectiviruses have been recently found infecting diverse strains belonging the B. cereus lineage. Here, we report and analyze the complete genome sequences of phages Sato and Sole. The linear dsDNA genome of Sato spans 14,852 bp with 32 coding DNA sequences (CDSs), whereas the one of Sole has 14,444 bp comprising 30 CDSs. Both phage genomes contain inverted terminal repeats and no tRNAs. Genomic comparisons and phylogenetic analyses placed these two phages within the genus Betatectivirus in the family Tectiviridae. Additional comparative genomic analyses indicated that the “gene regulation-genome replication” module of phages Sato and Sole is more diverse than previously observed among other fully sequenced betatectiviruses, displaying very low sequence similarities and containing some ORFans. Interestingly, the ssDNA binding protein encoded in this genomic module in phages Sato and Sole has very little amino acid similarity with those of reference betatectiviruses. Phylogenetic analyses showed that both Sato and Sole represent novel tectivirus species, thus we propose to include them as two novel species in the genus Betatectivirus.

Bacillus thuringiensis is the most used biopesticide in agriculture. Its entomopathogenic capacity stems from the possession of plasmid-borne insecticidal crystal genes (cry), traditionally used as discriminant taxonomic feature for that species. As such, crystal and plasmid identification are key to the characterization of this species. To date, about 600 B. thuringiensis genomes have been reported, but less than 5% have been completed, while the other draft genomes are incomplete, hindering full plasmid delineation. Here we present the complete genome of Bacillus thuringiensis HER1410, a strain closely related to B. thuringiensis entomocidus and a known host for a variety of Bacillus phages. The combination of short and long-read techniques allowed fully resolving the genome and delineation of three plasmids. This enabled the accurate detection of an unusual location of a unique cry gene, cry1Ba4, located in a genomic island near the chromosome replication origin. Two megaplasmids, pLUSID1 and pLUSID2 could be delineated: pLUSID1 (368 kb), a likely conjugative plasmid involved in virulence, and pLUSID2 (156 kb) potentially related to the sporulation process. A smaller plasmidial prophage pLUSID3, with a dual lifestyle whose integration within the chromosome causes the disruption of a flagellar key component. Finally, phylogenetic analysis placed this strain within a clade comprising members from the B. thuringiensis serovar thuringiensis and other serovars and with B. cereus s. s. in agreement with the intermingled taxonomy of B. cereus sensu lato group., This research was funded by Fundación Ramón Areces (XIX Concurso Nacional de Investigación en Ciencias de la Vida y la Materia) and institutional grants from Fundación Ramón Areces and Banco de Santander to the Centro de Biología Molecular Severo Ochoa. A. L. is holder of a PhD fellowship [FPU15/05797] from the Spanish Ministry of Science, Innovation and Universities. C. L. is supported by a PhD fellowship from FWO Vlaanderen (1S64720N).

With the objective to isolate phages infecting wine bacterial spoilers, we designed a method for the isolation and purification of phages infecting grape-associated bacteria. The method proved successful to isolate GC1 tectivirus infecting the acetic acid bacterium Gluconobacter cerinus. The isolated phage represents a new genus within the Tectiviridae, named “Gammatectivirus”. Using a traditional technique for the concentration of phage particles involving several steps of centrifugation, further insights in the ultrastructure of GC1 could be observed by cryo electron microscopy, saving time and effort. The simple workflow presented may be applied to other viruses infecting bacteria inhabiting other vegetal niches.

Tectiviridae are composed of tailless bacteriophages with an icosahedral capsid and an inner membrane enclosing a double-stranded 15 kb linear DNA genome. Five of the seven previously studied Tectivirus isolates infect bacteria from Bacillus cereus sensu lato group (Betatectivirus), one distantly related member (PRD1) infect Enterobactericeae (Alpatectivirus) and one recently discovered virus infect Gluconobacter cerinus (Gammatectivirus). Here we expand the host spectrum of Betatectivirus elements to four additional genera (Streptococcus, Exiguobacterium, Clostridium and Brevibacillus) and to more distantly related Bacillus species (B. pumilus and B. flexus) by studying the genomes of fourteen novel tectiviral elements. Overall, the genomes show significant conservation in gene synteny and in modules responsible for genome replication and formation of the virion core (including DNA packaging). Notable variation exists in regions encoding host attachment and lysis along with the surrounding area of a site in which mutations are known to alter phage life cycle.

The oleaginous bacterium Rhodococcus opacus PD630 is metabolically diverse and can be cultivated on various renewable resources to serve as a sustainable triacylglycerol (TAG) feedstock for biodiesel production. Current methods for TAG extraction are costly, but infection of cultures by lytic bacteriophages (phages) may be a viable approach for achieving release of intracellular lipid from oleaginous bacteria such as R. opacus. This study reports the novel tectiviral phage Toil capable of releasing intracellular contents including a fluorescent protein marker and TAGs into the supernatant after phage infection of R. opacus PD631, a domesticated derivative of strain PD630. Phage Toil is placed in the Tectiviridae by its morphology, the presence of a lipid membrane, its genome architecture and the presence of terminal covalently-linked proteins. Toil is the first tectivirus capable of infecting a member of the Actinobacteria. Microscopy shows that infected cells do not undergo sudden lysis but instead maintain their original shape for several hours, with the cellular morphology gradually deteriorating. Approximately 30% of intracellular TAGs could be recovered from the culture supernatants of Toil-infected PD631 cells. Phage Toil has potential to be used as an agent in extraction of TAGs from oleaginous bacterium R. opacus. Importance: This study reported the first tectivirus (Phage Toil) capable of infecting a member of the Actinobacteria. In this study, we showed that Phage Toil can infect oleaginous bacterium Rhodococcus opacus to release intracellular contents such as a fluorescent protein marker and TAG lipid granules, which can serve as a starting material for biodiesel production. This study demonstrates a new method to extract TAGs by using this phage. Additionally, Phage Toil can be a new model phage to advance knowledge regarding phage infection mechanisms in Rhodococcus and other mycolic acid-containing bacteria such as Mycobacterium.

Streptomyces phages WheeHeim and Forthebois are two novel members of the Tectiviridae family. These phages were isolated on cultures of the plant pathogen Streptomyces scabiei, known for its worldwide economic impact on potato crops. Transmission electron microscopy showed viral particles with double-layered icosahedral capsids, and frequent instances of protruding nanotubes harboring a collar-like structure. Mass-spectrometry confirmed the presence of lipids in the virion, and serial purification of colonies from turbid plaques and immunity testing revealed that both phages are temperate. Streptomyces phages WheeHeim and Forthebois have linear dsDNA chromosomes (18,266 bp and 18,251 bp long, respectively) with the characteristic two-segment architecture of the Tectiviridae. Both genomes encode homologs of the canonical tectiviral proteins (major capsid protein, packaging ATPase and DNA polymerase), as well as PRD1-type virion-associated transglycosylase and membrane DNA delivery proteins. Comparative genomics and phylogenetic analyses firmly establish that these two phages, together with Rhodococcus phage Toil, form a new genus within the Tectiviridae, which we have tentatively named Deltatectivirus. The identification of a cohesive clade of Actinobacteria-infecting tectiviruses with conserved genome structure but with scant sequence similarity to members of other tectiviral genera confirms that the Tectiviridae are an ancient lineage infecting a broad range of bacterial hosts.

Bacteriophage PR772, a member of the Tectiviridae family, has a 70 nm diameter icosahedral protein capsid that encapsulates a lipid membrane, dsDNA, and various internal proteins. An icosahedrally averaged CryoEM reconstruction of the wild-type virion and a localized reconstruction of the vertex region reveal the composition and the structure of the vertex complex along with new protein conformations that play a vital role in maintaining the capsid architecture of the virion. The overall resolution of the virion is 2.75 angstrom, while the resolution of the protein capsid is 2.3 angstrom. The conventional penta-symmetron formed by the capsomeres is replaced by a large vertex complex in the pseudo T = 25 capsid. All the vertices contain the host-recognition protein, P5; two of these vertices show the presence of the receptor-binding protein, P2. The 3D structure of the vertex complex shows interactions with the viral membrane, indicating a possible mechanism for viral infection.

The family Tectiviridae comprises a group of tailless, icosahedral, membranecontaining bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well-characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria. In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing knowledge about the viral proteome, we carried out a comprehensive yeast two-hybrid analysis of all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprised 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggest that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, with the latter finding also being supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving nonstructural proteins, such as the DNA-binding protein P1 and the genome terminal protein (P4), which was confirmed by coimmunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle., Spanish Economy and Competitiveness and Education Ministries, respectively; An institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa

Tectiviridae is a family of tailless bacteriophages with Gram-negative and Gram-positive hosts. The family model PRD1 and its close relatives all infect a broad range of enterobacteria by recognizing a plasmid-encoded conjugal transfer complex as a receptor. In contrast, tectiviruses with Gram-positive hosts are highly specific to only a few hosts within the same bacterial species. The cellular determinants that account for the observed specificity remain unknown. Here we present the genome sequence of Wip1, a tectivirus that infects the pathogen Bacillus anthracis . The Wip1 genome is related to other tectiviruses with Gram-positive hosts, notably, AP50, but displays some interesting differences in its genome organization. We identified Wip1 candidate genes for the viral spike complex, the structure located at the capsid vertices and involved in host receptor binding. Phage adsorption and inhibition tests were combined with immunofluorescence microscopy to show that the Wip1 gene product p23 is a receptor binding protein. His-p23 also formed a stable complex with p24, a Wip1 protein of unknown function, suggesting that the latter is involved with p23 in host cell recognition. The narrow host range of phage Wip1 and the identification of p23 as a receptor binding protein offer a new range of suitable tools for the rapid identification of B. anthracis .

International audience; The Gluconobacter phage GC1 is a novel member of the Tectiviridae family isolated from a juice sample collected during dry white wine making. The bacteriophage infects Gluconobacter cerinus, an acetic acid bacterium which represents a spoilage microorganism during wine making, mainly because it is able to produce ethyl alcohol and transform it into acetic acid. Transmission electron microscopy revealed tail-less icosahedral particles with a diameter of ~78 nm. The linear double-stranded DNA genome of GC1 (16,523 base pairs) contains terminal inverted repeats and carries 36 open reading frames, only a handful of which could be functionally annotated. These encode for the key proteins involved in DNA replication (protein-primed family B DNA polymerase) as well as in virion structure and assembly (major capsid protein, genome packaging ATPase (adenosine triphosphatase) and several minor capsid proteins). GC1 is the first tectivirus infecting an alphaproteobacterial host and is thus far the only temperate tectivirus of gram-negative bacteria. Based on distinctive sequence and life-style features, we propose that GC1 represents a new genus within the Tectiviridae, which we tentatively named "Gammatectivirus". Furthermore, GC1 helps to bridge the gap in the sequence space between alphatectiviruses and betatectiviruses.

Summary Bacteriophage PRD1 is a tailless membrane-containing double-stranded (ds) DNA virus infecting a variety of Gram-negative bacteria. In order to affect cell lysis, like most dsDNA phages, PRD1 uses the holin-endolysin system. In this study, we identified two accessory lysis genes, XXXVI and XXXVII, coding for proteins P36 and P37, respectively. Using genetic complementation assays, we show that protein pair P36/P37 is a functional and interchangeable analogue of the Rz/Rz1 of bacteriophage λ. Utilizing molecular biology, electrochemical as well as various microscopic techniques, we characterized the lysis phenotypes of PRD1 host cells infected with mutant viruses. Our results indicate that proteins P36 and P37 confer a competitive advantage to the phage by securing the efficient disruption of the infected cell and consequent release of the phage progeny under less favourable growth conditions. In concordance with prior data and the results obtained in this study, we propose a model explaining the role of Rz/Rz1-like proteins in the lysis process: Rz/Rz1 complexes transform the mechanical stress caused by the holin lesion at the CM to the OM leading to its disintegration. Finally, identification of the Rz/Rz1-like genes in PRD1 suggests that tailless icosahedral phages are involved in genetic trade with tailed bacteriophages.

The assembly of bacteriophage PRD1 proceeds via formation of empty procapsids containing an internal lipid membrane, into which the linear double-stranded DNA genome is subsequently packaged. The packaging ATPase P9 and other putative packaging proteins have been shown to be located at a unique vertex of the PRD1 capsid. Here, we describe the isolation and characterization of a suppressor-sensitive PRD1 mutant deficient in the unique vertex protein P6. Protein P6 was found to be an essential part of the PRD1 packaging machinery; its absence leads to greatly reduced packaging efficiency. Lack of P6 was not found to affect particle assembly, because in the P6-deficient mutant infection, wild-type (wt) amounts of particles were produced, although most were empty. P6 was determined not to be a specificity factor, as the few filled particles seen in the P6-deficient infection contained only PRD1-specific DNA. The presence of P6 was not necessary for retention of DNA in the capsid once packaging had occurred, and P6-deficient DNA-containing particles were found to be stable and infectious, albeit not as infectious as wt PRD1 virions. A packaging model for bacteriophage PRD1, based on previous results and those obtained in this study, is presented.

PRD1, an icosahedra-shaped, 62 nm (diameter), double-stranded DNA bacteriophage with an internal membrane, has emerged as an important model virus for studying the manner in which microorganisms are transported through a variety of groundwater environments. The popularity of this phage for use in transport studies involving geologic media is due, in part, to its relative stability over a range of temperatures and low degree of attachment in aquifer sediments. Laboratory and field investigations employing PRD1 are leading to a better understanding of viral attachment and transport behaviors in saturated geologic media and to improved methods for describing mathematically subsurface microbial transport at environmentally significant field scales. Radioisotopic labeling of PRD1 is facilitating additional information about the nature of viral interactions with solid surfaces in geologic media, the importance of iron oxide surfaces, and allowing differentiation between inactivation and attachment in field-scale tracer tests.

A simple method to isolate, screen and select phage-resistant mutants of Bacillus thuringiensis was developed. The traditional double-layer agar method was improved by a combination of the spotting assay using a lytic phage, to generate the bacterial-resistant mutants, with an inverted spotting assay (ISA), to rapidly screen the candidate-resistant mutants.

Tectiviridae is a family of tailless bacteriophages with Gram-negative and Gram-positive hosts. The family model PRD1 and its close relatives all infect a broad range of enterobacteria by recognizing a plasmid-encoded conjugal transfer complex as a receptor. In contrast, tectiviruses with Gram-positive hosts are highly specific to only a few hosts within the same bacterial species. The cellular determinants that account for the observed specificity remain unknown. Here we present the genome sequence of Wip1, a tectivirus that infects the pathogen Bacillus anthracis. The Wip1 genome is related to other tectiviruses with Gram-positive hosts, notably, AP50, but displays some interesting differences in its genome organization. We identified Wip1 candidate genes for the viral spike complex, the structure located at the capsid vertices and involved in host receptor binding. Phage adsorption and inhibition tests were combined with immunofluorescence microscopy to show that the Wip1 gene product p23 is a receptor binding protein. His-p23 also formed a stable complex with p24, a Wip1 protein of unknown function, suggesting that the latter is involved with p23 in host cell recognition. The narrow host range of phage Wip1 and the identification of p23 as a receptor binding protein offer a new range of suitable tools for the rapid identification of B. anthracis. peerReviewed

Virus transport through groundwater is limited by attachment to mineral surfaces and inactivation. Current virus transport models do not consider the implications of the reversibility of virus attachment to minerals. To explore the reversibility of virus attachment to mineral surfaces, we attached PRD1, a bacteriophage considered to be a good model of enteric viruses, to quartz and ferric oxyhydroxide-coated quartz surfaces over a range of pH values in equilibrium “static columns.” Following attachment, we detached the viruses by replacing the pore solution with solutions of equal and higher pH. The extent of virus attachment followed an attachment “edge” that occurred at a pH value about 2.5–3.5 pH units above the pHiep of the mineral surfaces. Viruses attached below this edge were irreversibly attached until the pH of the detachment solution exceeded the pH value of the attachment edge. Viruses attached above this edge were reversibly attached. Derjaguin-Landau-Verwey-Overbeek (DLVO) potential energy calculations showed that the attachment edge occurred at the pH at which the potential energy of the primary minimum was near zero, implying that the position of the primary minimum (attractive or repulsive) controlled the equilibrium distribution of the viruses. The results suggest that the reversibility of virus attachment must be considered in virus transport models for accurate predictions of virus travel time.

Two bacteriophages, MS-2 and PRD-1, were added to water to an initial concentration of 10 5 -10 6 pfu mL -1 in a trench-to-trench lateral groundwater flow and tracer migration experiment in the upper 5.5 m of a weathered and fractured clay-rich till. Phage were detected in water from seepage collectors set in the wall of a downgradient (gradient=0.24) trench, located 4 m from the source trench, between 1 and 2 days after the start of the injection. Peak phage concentrations in the collectors were typically 10 2 -10 4 pfu mL -1 , and detectable phage (>0.1 pfu mL -1 ) persisted for up to 5 days. In contrast, the travel time for bromide in the same trenches was several months

PRD1 is a dsDNA virus that infects Escherichia coli and Salmonella typhimurium. The genome is a linear molecule with 5' covalently linked terminal protein. The virus has a lipid membrane inside the protein coat. We describe the large-scale purification of the virus using a zonal rotor and the yields and quality of the virus and its subviral assemblies for subsequent biophysical measurements.

The genome sequence of a Bacillus anthracis -specific clear plaque mutant phage, AP50c, contains 31 open reading frames spanning 14,398 bp, has two mutations compared to wild-type AP50t, and has a colinear genome architecture highly similar to that of gram-positive Tectiviridae phages. Spontaneous AP50c-resistant B. anthracis mutants exhibit a mucoid colony phenotype.

Bacteriophage PRD1 infecting Escherichia coli and Salmonella typhimurium translocates its membrane from the host plasma membrane to the virus particle. One obligatory component in this process is the major capsid protein. In this investigation we describe characteristics of the homomultimeric major and minor capsid proteins including the sequences of the corresponding genes. The minor capsid protein was found to contain a short collagen-like region (Gly-X-Y)6. This is the first time this motif has been reported for a prokaryotic protein.

This article describes the structure and assembly of bacteriophage PRD1, a lipid-containing virus able to infect Escherichia coli . This phage, with an approximate diameter of 65 nm, is composed of an outer protein shell surrounding a lipid-protein membrane which, in turn, encloses the nucleic acid. The phage genome consists of a single linear dsDNA molecule of about 15 kb that has a protein covalently linked to each of its 5′ ends. This protein is used as a primer in DNA replication. During assembly membrane proteins are inserted into the host cytoplasmic membrane while major capsid protein multimers are found in the cytoplasm. Capsid multimers, assisted by two nonstructural assembly factors, are capable of translocating the virus-specific membrane resulting in the formation of cytoplasmic empty particles. Subsequent DNA packaging leads to the formation of infectious virus.

Static and dynamic batch experiments were conducted to study the effects of temperature and the presence of sand on the inactivation of bacteriophage MS2 and PRD1. The experimental data suggested that the inactivation process can be satisfactorily represented by a pseudo-first-order expression with time-dependent rate coefficients. The time-dependent rate coefficients were used to determine pertinent thermodynamic properties required for the analysis of the molecular processes involved in the inactivation of each bacteriophage. A combination of high temperature and the presence of sand appears to produce the greatest disruption to the surrounding protein coat of MS2. However, the lower activation energies for PRD1 indicate a weaker dependence of the inactivation rate on temperature. Instead, the presence of air-liquid and air-solid interfaces appears to produce the greatest damage to specific viral components that are related to infection. These results indicate the importance of using thermodynamic parameters based on the time-dependent inactivation model to better predict the inactivation of viruses in groundwater.

The structure of the membrane-containing bacteriophage PRD1 has been determined by X-ray crystallography at about 4 A resolution. Here we describe the structure and location of proteins P3, P16, P30 and P31. Different structural proteins seem to have specialist roles in controlling virus assembly. The linearly extended P30 appears to nucleate the formation of the icosahedral facets (composed of trimers of the major capsid protein, P3) and acts as a molecular tape-measure, defining the size of the virus and cementing the facets together. Pentamers of P31 form the vertex base, interlocking with subunits of P3 and interacting with the membrane protein P16. The architectural similarities with adenovirus and one of the largest known virus particles PBCV-1 support the notion that the mechanism of assembly of PRD1 is scaleable and applies across the major viral lineage formed by these viruses.

Field and laboratory experiments were conducted to investigate inactivation of viruses attached to mineral surfaces. In a natural gradient transport field experiment, bacteriophage PRD1, radiolabeled with 32P, was injected into a ferric oxyhydroxide-coated sand aquifer with bromide and linear alkylbenzene sulfonates. In a zone of the aquifer contaminated by secondary sewage infiltration, small fractions of infective and 32P-labeled PRD1 broke through with the bromide tracer,followed bythe slow release of 84% of the 32P activity and only 0.011% of the infective PRD1. In the laboratory experiments, the inactivation of PRD1, labeled with 35S (protein capsid), and MS2, dual radiolabeled with 35S (protein capsid) and 32P (nucleic acid), was monitored in the presence of groundwater and sediment from the contaminated zone of the field site. Release of infective viruses decreased at a much faster rate than release of the radiolabels, indicating that attached viruses were undergoing surface inactivation. Disparities between 32P and 35S release suggest that the inactivated viruses were released in a disintegrated state. Comparison of estimated solution and surface inactivation rates indicates solution inactivation is approximately 3 times as fast as surface inactivation. The actual rate of surface inactivation may be substantially underestimated owing to slow release of inactivated viruses.

Publisher Summary The widely used phage isolation method, described in Adams, includes a chloroform treatment step. As a result, bacterial viruses have been considered to be nucleic acid-protein assemblies. The omission of the organic solvent treatment from the isolation process resulted in the isolation of phages that also had a membrane component. The Tectiviridae are one of the most common phage groups containing a biological membrane. The Tectiviridae are divided into two subgroups, those infecting gram-positive and those infecting gram-negative hosts. The type organism for the gram-negative subgroup is PRD1, the other isolates being PR3, PR4, PR5, L17, and PR772. The best studied members of this virus group are PRD1 and PR4, and information given in this review is based mostly on the results obtained with these two virus systems. A comprehensive description on the current knowledge on PRD1 and related viruses are given in this chapter. Two characteristics, in particular, have drawn attention to the PRD1-type viruses: (1) the virion has an outer rigid protein capsid, inside of which resides a membrane that follows the internal capsid surface. (2) the viral genome is a linear dsDNA molecule that has a covalently linked protein at the 5' ends and inverted terminal repeat (ITR) sequences at both termini.

We describe a novel ion exchange chromatographic technique suitable for large-scale preparation of viruses and other biomacromolecular assemblies in highly purified form. The method, which utilizes anion exchange on either of two commercially available cellulose cartridges, is applied to the Escherichia coli bacteriophage PRD1. Viral particles eluted from both QMA and DEAE cartridges retain infectivity and exhibit greater homogeneity of composition, as judged by gel electrophoresis and electron microscopy, than particles purified by rate zonal sucrose gradient centrifugation. The ion exchange protocols are rapid, requiring less than 15 min elution time, and permit retrieval of the purified viral particles at high concentration in aqueous media without centrifugal pelleting. The present method is particularly well suited to the preparation of milligram to decigram quantities of virus, sufficient for many biophysical structural analyses, including investigations by solution spectroscopic and crystal diffraction techniques. The feasibility and advantages of the ion exchange chromatographic procedure are demonstrated by application of laser Raman spectroscopy to ion exchange purified PRD1 virions and subviral assemblies.

PRD1 is a broad-host-range virus that infects Escherichia coli cells. It has a linear double-stranded DNA genome that replicates by a protein-primed mechanism. The virus particle is composed of a protein coat enclosing a lipid membrane. On the basis of this structure, PRD1 is being used as a membrane biosynthesis and structure model. In this investigation, we constructed the transcription map of the 15-kb-long phage genome. This was achieved by a computer search of putative promoters, which were then tested for activity by primer extension and for the capability to promote the synthesis of chloramphenicol acetyltransferase.

Phage PR4 contains a lipid bilayer within the phage protein capsid. The phospholipids of the bilayer are derived from those of the host. We report that phage morphogenesis selects against the unusually bulky phospholipids synthesized by Escherichia coli grown in the presence of various sugar alcohols. These data indicate that assembly of the PR4 lipid bilayer is a selective process rather than the bulk appropriation of host membrane lipids. We also demonstrate that phage PR4 morphogenesis is compatible with the incorporation of several abnormal lipids, monoacylglycerol, diacylglycerol, and phosphatidylinositol into the phage particle.

Trimeric capsomeres of protein P3 (395 aa) are the main component of the phage PRD1 capsid, which encloses a lipid protein vesicle containing the vital dsDNA genome. In this study characterize a panel of monoclonal antibodies (MAb) against P3. The epitopes recognized by the MAbs are analyzed by immunoprecipitation of intact virions or of released P3 trimers, and by Western blotting using a series of C-terminally truncated P3 molecules. Nine of the MAbs recognize epitopes on the virion surface, whereas five require unmasking of epitopes by disruption of the virions. Several of the MAbs are capable of neutralizing the virus; this is most probably due to virus aggregation by the antibodies. Analysis of the C-terminal truncations (the 6 Western blot-positive MAbs were used) delineates three major antigenic regions of the protein. The epitope of MAb 3T74 is included in the 66 N-terminal amino acids, and is not accessible on the virion surface, suggesting that the N-terminus is internally located in the capsid. MAbs 3N81 and 3R2 recognize epitopes In the region of amino acids 159-168, which is part of the first predicted β-barrel structure of P3. The third antigenic region is in the second predicted β-barrel, between amine acids 217-242, where the epitopes of 3N180, 3P4, and 3T5 map. The trimerization of P3 was found to be independent of the non-structural assembly factor proteins P10 and P17. Functional studies of the truncated proteins reveal that molecules comprising of 294 or more residues from the P3 N-terminus are capable of trimer formation.

Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Using singlestranded oligonucleotide templates carrying the sequence corresponding to the 25 first bases of the 3é end of PRD1 DNA, and Mg2+ as the activating metal ion of the phage DNA polymerase, we show that the fourth base from the 3é end of the template directs, by base complementarity, the dNMP to be linked to the phage terminal protein (TP) in the initiation reaction. This result suggests that phage PRD1 maintains its 3é end DNA sequences via a sliding-back mechanism. The single-stranded DNA templates could not be replicated by the PRD1 DNA polymerase, much in contrast to the natural TP-DNA. Nevertheless, the analysis of the transition products obtained with TP-DNA and origin containing oligonucleotides suggests that sliding-back occurs stepwise, the fourth base being the directing position during the entire process., This work was supported by a grant from the Direcci6n General de Investigaci6n Cientffica y Tecnica (to J.C.), by a grant from the Finnish Academy of Sciences (to D.H.B.), by grant 5RO1 GM27242-13 from the NationalInstitutes of Health (to M.S.), by grant PB90-0091 from Direcci6n General de Investigaci6n Cientffica y T&cnica (to M.S.), and by an institutional grant from Fundacion Ramon Areces (to Centro de Biologia Molecular).

Bacteriopbage PRD1, a double-stranded DNA virus infecting Escherichia coli, has a membrane inside the protein capsid. Chemical cross-linking and scanning transmission electron microscopy showed that the multimeric major coat protein (P3) exists in a trimeric form. Cross-linking revealed, in addition, that protein P11, located between the protein coat and the membrane, exists also as a homotrimer. Minor protein P7 was associated with the major coat protein P3. Under nonreducing conditions the infectivity proteins P16 and P18 formed homomultimeric complexes which were dissociated upon addition of 2-mercaptoethanol.

PRD1 is a double-stranded DNA virus infecting Escherichia coli and Salmonella typhimurium. It has an icosahedral outer protein capsid which encloses the viral membrane, inside of which resides the phage genome. In this investigation we demonstrate the detergent resistance of the intact virus particles. The membrane of empty DNA-free particles, however, is very sensitive to detergent action. We assume that their sensitivity is due to the access of detergents through a portal structure to the virus interior. Using the anionic detergent sodium dodecyl sulfate, it is possible to obtain a shell structure composed of the major coat protein P3 alone. The treatment of empty particles with the milder nonionic detergents n -octyl β-D-glucopyranoside and Triton X-100 yielded P3 particles which retained the membrane-associated proteins P7 and P11. Deoxycholic acid treatment yielded shells of intermediate composition between those obtained with the nonionic detergents and sodium dodecyl sulfate.

The double-stranded DNA bacteriophage PRD1 replicates in Escherichia coli and Salmonella typhimurium. It has an outer protein coat surrounding a membrane. The phage lipids are derived from the host, but the membrane proteins are of phage origin. In this investigation we studied the effects of heat, pH, and sodium dodecyl sulfate on the integrity of phage particles. Heat and high pH result in the release of the main coat protein, P3, as trimers, whereas treatment of phage particles with detergent results in the solubilization of the membrane. Our results enable a distinction to be made between the phage structural proteins that are embedded in the lipid bilayer and those that appear to be more loosely associated with the membrane.

IncP plasmid RP1 Tra regions are needed to assemble the receptor for lipid-containing double-stranded DNA bacteriophage PRD1 on the cell surface. Using radioactively labeled phage and electron microscopic techniques, we showed that the surfaces of Salmonella typhimurium(RP1) and Escherichia coli(RP1) cells contained approximately 50 and 20 PRD1 binding sites, respectively. Expression of the receptor was growth phase dependent and was highest at late logarithmic or early stationary phase. The PRD1-resistant RP1 transposon mutants isolated were all Tra-, and the transposons were located in both the Tra1 and Tra2 regions.