Phage S144, A New Polyvalent Phage Infecting Salmonella spp. and Cronobacter sakazakii.

Phages are generally considered species- or even strain-specific, yet polyvalent phages are able to infect bacteria from different genera. Here, we characterize the novel polyvalent phage S144, a member of the Loughboroughvirus genus. By screening 211 Enterobacteriaceae strains, we found that phage S144 forms plaques on specific serovars of Salmonella enterica subsp. enterica and on Cronobacter sakazakii. Analysis of phage resistant mutants suggests that the O-antigen of lipopolysaccharide is the phage receptor in both bacterial genera. The S144 genome consists of 53,628 bp and encodes 80 open reading frames (ORFs), but no tRNA genes. In total, 32 ORFs coding for structural proteins were confirmed by ESI-MS/MS analysis, whereas 45 gene products were functionally annotated within DNA metabolism, packaging, nucleotide biosynthesis and phage morphogenesis. Transmission electron microscopy showed that phage S144 is a myovirus, with a prolate head and short tail fibers. The putative S144 tail fiber structure is, overall, similar to the tail fiber of phage Mu and the C-terminus shows amino acid similarity to tail fibers of otherwise unrelated phages infecting Cronobacter. Since all phages in the Loughboroughvirus genus encode tail fibers similar to S144, we suggest that phages in this genus infect Cronobacter sakazakii and are polyvalent.

ICTV Virus Taxonomy Profile: Corticoviridae.

The Corticoviridae is a family of icosahedral, internal-membrane-containing viruses with double-stranded circular DNA genomes of approximately 10 kb. Only one species, Pseudoalteromonas virus PM2, has been recognized. Pseudoalteromonas virus PM2 infects Gram-negative bacteria and was isolated from seawater in 1968. Pseudoalteromonas virus PM2 is the first bacterial virus in which the presence of lipids in the virion has been demonstrated. Viral lipids are acquired selectively during virion assembly from the host cytoplasmic membrane. The outer protein capsid is an icosahedron with a pseudo T=21 symmetry. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Corticoviridae, which is available at www.ictv.global/report/corticoviridae.

Mechanisms of DNA damage by photoexcited 9-methyl-ß-carbolines.

It has been well documented that ß-carboline alkaloids, particularly the 9-methyl derivatives, are efficient photosensitizers. However, structure-activity relationships are missing and the photochemical mechanisms involved in the DNA photodamage still remain unknown. In the present work, we examined the capability of three 9-methyl-ß-carbolines (9-methyl-norharmane, 9-methyl-harmane and 9-methyl-harmine) to induce DNA damage upon UVA excitation at physiological pH. The type and extent of the damage was analyzed together with the photophysical and binding properties of the ß-carboline derivatives investigated. The results indicate that even at neutral pH most of the DNA damage is generated from the protonated form of the excited ß-carbolines in a type-I reaction. Oxidized purine residues are produced in high excess over oxidized pyrimidines, single-strand breaks and sites of base loss. In addition, the excited neutral form of the ß-carbolines is responsible for significant generation of cyclobutane pyrimidine dimers (CPDs) by triplet-triplet-energy transfer. In the case of 9-methyl-norharmane, the yield of CPDs is increased in D2O, probably due to less rapid protonation in the deuterated solvent.

Genetics for Pseudoalteromonas provides tools to manipulate marine bacterial virus PM2.

The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 (Corticoviridae) has been limited so far. The isolation of an autonomously replicating DNA element of Pseudoalteromonas haloplanktis TAC125 and construction of a shuttle vector replicating in both Escherichia coli and Pseudoalteromonas enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2 sus2 mutant deficient in the structural protein P10. To infect and replicate, PM2 delivers its 10-kbp genome across the cell envelopes of two gram-negative Pseudoalteromonas species. The events leading to the internalization of the circular supercoiled dsDNA are puzzling. In a poorly understood process that follows receptor recognition, the virion capsid disassembles and the internal membrane fuses with the host outer membrane. While beginning to unravel the mechanism of this process, we found that protein P10 plays an essential role in the host cell penetration.

Putative prophages related to lytic tailless marine dsDNA phage PM2 are widespread in the genomes of aquatic bacteria.

BACKGROUND: The origin and evolution of viruses is currently a heavily discussed issue. One element in this discussion is the innate viral "self" concept, which suggests that viral structures and functions can be divided into two categories. The first category consists of genetic determinants that are inherited from a viral ancestor and encode the viral "self". The second group consists of another set of structures and functions, the "nonself", which is interchangeable between different viruses and can be obtained via lateral gene transfer. Comparing the structures and sequences of the "self" elements, we have proposed that viruses can be grouped into lineages regardless of which domain of life (bacteria, archaea, eukarya) they infect. It has also been suggested that viruses are ancient and possibly predate modern cells. RESULTS: Here we identified thirteen putative prophages (viral genomes integrated into bacterial chromosome) closely related to the virulent icosahedral tailless lipid-containing bacteriophage PM2. Using the comparative genomics approach, we present evidence to support the viral "self" hypothesis and divide genes of the bacteriophage PM2 and related prophages into "self" and "nonself" categories. CONCLUSION: We show here that the previously proposed most conserved viral "self" determinants, the major coat protein and the packaging ATPase, were the only proteins that could be recognized in all detected corticoviral elements. We also argue here that the genes needed for viral genome replication, as well as for host cell lysis, belong to the "nonself" category of genes.Furthermore, we suggest that abundance of PM2-like viruses in the aquatic environment as well as their importance in the ecology of aquatic microorganisms might have been underestimated.

A novel lysis system in PM2, a lipid-containing marine double-stranded DNA bacteriophage.

In this study we investigated the lysis system of the lipid-containing double-stranded DNA bacteriophage PM2 infecting Gram-negative marine Pseudoalteromonas species. We analysed wt and lysis-deficient phage-induced changes in the host physiology and ascribed functions to two PM2 gene products (gp) involved in lysis. We show that bacteriophage PM2 uses a novel system to disrupt the infected cell. The novelty is based on the following findings: (i) gp k is needed for the permeabilization of the cytoplasmic membrane and appears to play the role of a typical holin. However, its unique primary structure [53 aa, 1 transmembrane domain (TMD)] places it into a new class of holins. (ii) We have proposed that, unlike other bacteriophages studied, PM2 relies on lytic factors of the cellular origin for digestion of the peptidoglycan. (iii) gp l (51 aa, no TMDs) is needed for disruption of the outer membrane, which is highly rigidified by the divalent cations abundant in the marine environment. The gp l has no precedent in other phage lytic systems studied so far. However, the presence of open reading frame l-like genes in genomes of other bacterial viruses suggests that the same system might be used by other phages and is not unique to PM2.

[A novel method of transfection of marine bacteria Pseudoalteromonas espejiana with deoxyribonucleic acid of bacteriophage PM2].

DNA of bacteriophage PM2 is a convenient test object for studying DNA-damaging genotoxic agents. The extent of DNA damage can be estimated by the ability of damaged DNA for transfection of host cells, marine bacterium Pseudoalteromonas espejiana (Pae), str. BAL-31. The efficiency of transfection of Pae lines maintained for long periods without freezing was found to be very low upon the use of a widely accepted transfection method developed by van der Schans et al. (1971). Such cultures grown in a medium with 10 mM Ca2+ standard for Pae contained cell aggregates and exopolymer material. Pae was found to be capable of growing in a medium without the calcium supplement in the presence of chelator EGTA (low-calcium medium, LCM). After growth in LCM, cells did not aggregate, cultures lacked the activity of nuclease BAL, and transfection efficiency of cells grown in LCM drastically increased. Based on these results, a novel procedure of transfection with an efficiency of 2 x 10(4)-2 x 10(5) infectious centers per microgram of PM2 DNA was developed.

Genome characterization of lipid-containing marine bacteriophage PM2 by transposon insertion mutagenesis.

Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed- and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.

Biochemical quantitation of PM2 phage DNA as a substrate for endonuclease assay.

Bacteriophage PM2 has a closed circular form of double stranded DNA as a genome. This DNA from the phage is a useful source for nick-circle endonuclease assay in the fmol range. Due to difficulties in the maintenance of viral infectivity, storage conditions of the phage should be considered for the purification of PM2 DNA. The proper condition for a short-term storage of less than 2 months is to keep the PM2 phage at 4 degrees C; whereas the proper condition for a long-term storage of the PM2 phage for over 2 months is to keep it under liquid nitrogen in 7.5% glycerol. The optimal conditions for a high yield of phage progeny were also considered with the goal to achieve a successful PM2 DNA preparation. A MOI(Multiplicity Of Infection) of 0.03, in which the OD600 of the host bacteria was between 0.3 and 0.5, turned out to be optimal for the mass production of PM2 phage with a burst size of about 214. Considerations of PM2 genome size, and the concentrations and radiospecific activities of purified PM2 DNA, are required to measure the endonuclease activity in the fmol range. This study reports the proper quantitation of radioactivity and the yield of purified DNA based on these conditions.

New preparation of PM2 phage DNA and an endonuclease assay for a single-strand break.

PM2 is a bacteriophage which has closed circular double-stranded DNA as a genome, which is the sole source for endonuclease assay for a single strand break in the fmol range. Therefore, it is important to isolate PM2 DNA with low control nicks for the endonuclease assay. Usually, the isolation method of phage DNA is to use ultracentrifugation which takes at least 4 days. In this report, a fast and effective method which takes only 2 days was developed to purify DNA using polyethylene glycol (PEG) 8000 and the yields of phage DNA isolated by these two methods were compared. The method using PEG 8000 increased the yield of PM2 DNA from 31.2% to 45.2%, and decreased the nick from 17.1% to 13.1%. Recently, the complete PM2 DNA genome sequence of 10,079 bp was published. The exact number of nucleotides of PM2 DNA is important for the correct enzyme assay which measures nicks generated by an endonuclease. The correct calculation of endonuclease activity of rpS3 for nick-circle assay was performed to measure single-strand breaks in this report.

Transcription of bacteriophage PM2 involves phage-encoded regulators of heterologous origin.

Bacteriophage PM2 is the only described member of the Corticoviridae family. It is an icosahedral dsDNA virus with a membrane residing underneath the protein coat. PM2 infects some gram-negative Pseudoalteromonas spp. In the present study, we mapped the viral promoters and showed that the PM2 genome consists of three operons. Four new virus genes were assigned based on their function in transcription. Proteins P15 and P16 are shown to repress early transcription, and proteins P13 and P14 are shown to activate late transcription events. The early regulatory region, containing genes for proteins P15 and P16, as well as the newly identified early promoter region in PM2, has significant sequence similarity with the Pseudoalteromonas pAS28 plasmid. P14, the transcription activator for the structural genes, has a zinc finger motif homologous to archaeal and eukaryotic TFIIS-type regulatory factors.

Bacteriophage PM2 has a protein capsid surrounding a spherical proteinaceous lipid core.

The marine double-stranded DNA (dsDNA) bacteriophage PM2, studied since 1968, is the type organism of the family Corticoviridae, infecting two gram-negative Pseudoalteromonas species. The virion contains a membrane underneath an icosahedral protein capsid composed of two structural proteins. The purified major capsid protein, P2, appears as a trimer, and the receptor binding protein, P1, appears as a monomer. The C-terminal part of P1 is distal and is responsible for receptor binding activity. The rest of the structural proteins are associated with the internal phospholipid membrane enclosing the viral genome. This internal particle is designated the lipid core. The overall structural organization of phage PM2 resembles that of dsDNA bacteriophage PRD1, the type organism of the family TECTIVIRIDAE:

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.

tert-Butoxyl radicals generate mainly 7,8-dihydro-8-oxoguanine in DNA.

Like hydroxyl radicals, alkoxyl radicals have been implicated in the generation of cellular oxidative DNA damage under physiological conditions; however, their genotoxic potential has not yet been established. We have analyzed the DNA damage induced by a photochemical source of tert-butoxyl radicals, the water soluble peroxy ester [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), using various repair endonucleases as probes. The irradiation (UV(360)) of BCBT in the presence of bacteriophage PM2 DNA was found to generate a DNA damage profile that consisted mostly of base modifications sensitive to the repair endonuclease Fpg protein. Approximately 90% of the modifications were identified as 7,8-dihydro-8-oxoguanine (8-oxoGua) residues by HPLC/ECD analysis. Oxidative pyrimidine modifications (sensitive to endonuclease III), sites of base loss (AP sites) and single-strand breaks were only minor modifications. Experiments with various scavengers and quenchers indicated that the DNA damage by BCBT+UV(360) was caused by tert-butoxyl radicals as the ultimate reactive species. The mutagenicity associated with the induced damage was analyzed in the gpt gene of plasmid pSV2gpt, which was exposed to BCBT+UV(360) and subsequently transfected into Escherichia coli. The results were in agreement with the specific generation of 8-oxoGua. Nearly all point mutations (20 out of 21) were found to be GC-->TA transversions known to be characteristic for 8-oxoGua. In conclusion, alkoxyl radicals generated from BCBT+UV(360) induce 8-oxoGua in DNA with a higher selectivity than any other reactive oxygen species analyzed so far.

Oxidative DNA base damage induced by singlet oxygen and photosensitization: recognition by repair endonucleases and mutagenicity.

We have analyzed the recognition by various repair endonucleases of DNA base modifications induced by three oxidants, viz. [4-(tert-butyldioxycarbonyl)benzyl]triethylammonium chloride (BCBT), a photochemical source of tert-butoxyl radicals, disodium salt of 1,4-etheno-2,3-benzodioxin-1,4-dipropanoic acid (NDPO(2)), a chemical source of singlet oxygen, and riboflavin, a type-I photosensitizer. The base modifications induced by BCBT, which were previously shown to be mostly 7,8-dihydro-8-oxoguanine (8-oxoGua) residues, were recognized by Fpg and Ogg1 proteins, but not by endonuclease IIII, Ntg1 and Ntg2 proteins. In the case of singlet oxygen induced damage, 8-oxoGua accounted for only 35% of the base modifications recognized by Fpg protein. The remaining Fpg-sensitive modifications were not recognized by Ogg1 protein and relatively poor by endonuclease III, but they were relatively good substrates of Ntg1 and Ntg2. In the case of the damage induced by photoexcited riboflavin, the fraction of Fpg-sensitive base modifications identified as 8-oxoGua was only 23%. In contrast to the damage induced by singlet oxygen, the remaining lesions were not only recognized by Ntg1 and Ntg2 proteins and (relatively poor) by endonuclease III, but also by Ogg1 protein. The analysis of the mutations observed after transfection of modified plasmid pSV2gpt into Escherichia coli revealed that all agents induced near exclusively GC-->TA and GC-->CG transversions, the numbers of which were correlated with the numbers of 8-oxoGua residues and Ntg-sensitive modifications, respectively. In conclusion, both singlet oxygen and the type-I photosensitizer riboflavin induce predominantly oxidative guanine modifications other than 8-oxoGua, which most probably give rise to GC-->CG transversions and in which eukaryotic cells are substrates of Ntg1 and Ntg2 proteins.

The complete genome sequence of PM2, the first lipid-containing bacterial virus To Be isolated.

Bacteriophage PM2 was isolated from the Pacific Ocean off the coast of Chile in the late 1960s. It was a new virus type, later classified as Corticoviridae, and also the first bacterial virus for which it was demonstrated that lipids are part of the virion structure. Here we report the determination and analysis of the 10, 079-bp circular dsDNA genome sequence. Noteworthy discoveries are the replication initiation system, which is related to the rolling circle mechanism described for phages such as φX174 and P2, and a 1.2-kb sequence that is similar to the maintenance region of a plasmid found in a marine Pseudoalteromonas sp. strain A28.

Modulation of metal-induced genotoxicity by Maillard reaction products isolated from coffee.

PM2 bacteriophage DNA was exposed to non-dialysable Maillard reaction products (MRPs) isolated from brewed (Br), boiled (Bo) and instant (I) coffee brew extracts in a Fe2+ catalysed Fenton reaction at four pH conditions (i.e. 7.5, 4.0, 3.2, 2.6). MRPs were incubated with DNA either directly with Fe2+, or following a short preincubation period conducted with Fe2+ in an atmosphere of oxygen or argon. Damage to supercoiled DNA resulting in strand scissions as characterized by both nicked circular and linear forms were found to occur either with coffee MRPs or Fe2+ alone, in a dose-dependent manner at all pH conditions tested. At low MRP concentrations, damage to DNA with respect to Fe2+ was lowered only when MRPs were preincubated with Fe2+ in argon or oxygen before incubating with DNA. The addition of MRPs and Fe2+ to DNA without preincubation, had no effects in protecting DNA damage. This finding showed that a preincubation step is necessary for MRPs to chelate Fe2+ in order to mitigate the Fenton reaction. In contrast, the protective effects against Fe2+-induced DNA breakage by MRPs were lost at high coffee MRP concentrations, irrespective of the incubation method used. Increasingly higher concentrations of MRPs in combination with Fe2+ actually enhanced the breakage of DNA with respect to the control. These results indicate that MRPs at high concentrations do not improve Fe2+ ion chelation, but rather accelerate the DNA breakage by possibly changing the redox state of the transition element.

DNA strand break induction and enhanced cytotoxicity of propyl gallate in the presence of copper(II).

The antioxidant propyl gallate (PG) induced single strand breaks in PM2 DNA at concentrations higher than 0.25 microM when it was combined with copper concentrations at 5 microM and above. In combination with 100 microM CuCl2, extensive double strand breakage was also observed. Neither PG alone nor CuCl2 showed any strand breaking properties. DNA strand breakage was inhibited by addition of catalase or the Cu(I) chelator neocuproine, indicating the involvement of H2O2 and a Cu(II)/Cu(I) redox cycle in the DNA damage. DNA damage of PG/Cu(II) was also observed in human fibroblasts. Using the alkaline elution technique concentrations of 0.15-0.5 mM PG induced DNA strand breaks in combination with 2.5 mM CuCl2, while the single substances did not show any effect. At these concentrations cell viability measured by the MTT assay was not reduced by more than 10%; however, cell growth was inhibited by PG in combination with Cu(II). This growth inhibition was apparently due to the DNA damage incurred by PG/Cu(II). The synergistic interaction between PG and Cu(II) is probably caused by a redox reaction between both compounds, whereby reactive species such as ROS are formed, which are responsible for the observed genotoxic and cytotoxic effects. Our results demonstrate that the antioxidative and cytoprotective properties of propyl gallate may change to prooxidative, cytotoxic and genotoxic properties in the presence of Cu(II).

Transcription of bacteriophage PM2.

Transcription of bacteriophage PM2 after infection of Alteromonas espejiana BAL-31 was examined. A wave of PM2 late transcription was observed 22 to 44 min after infection. This transcription was chloramphenicol- and rifampicin-sensitive. Regions of PM2 DNA transcribed with high efficiency were determined by DNA--RNA hybridization and Southern's technique.