High Resolution Structure of the Mature Capsid of Ralstonia solanacearum Bacteriophage ϕRSA1 by Cryo-Electron Microscopy.

The ϕRSA1 bacteriophage has been isolated from Ralstonia solanacearum, a gram negative bacteria having a significant economic impact on many important crops. We solved the three-dimensional structure of the ϕRSA1 mature capsid to 3.9 Šresolution by cryo-electron microscopy. The capsid shell, that contains the 39 kbp of dsDNA genome, has an icosahedral symmetry characterized by an unusual triangulation number of T = 7, dextro. The ϕRSA1 capsid is composed solely of the polymerization of the major capsid protein, gp8, which exhibits the typical "Johnson" fold first characterized in E. coli bacteriophage HK97. As opposed to the latter, the ϕRSA1 mature capsid is not stabilized by covalent crosslinking between its subunits, nor by the addition of a decoration protein. We further describe the molecular interactions occurring between the subunits of the ϕRSA1 capsid and their relationships with the other known bacteriophages.

In vitro characterization of the site-specific recombination system based on genus Habenivirus ϕRSM small serine integrase.

The genus Habenivirus which includes Ralstonia virus ϕRSM encodes a site-specific integrase of a small serine recombinase belonging to the resolvase/invertase family. Here we describe the integrative/excisive recombination reactions mediated by ϕRSM integrase using in vitro assays. The products of attP/attB recombination, i.e. attL and attR, were exactly identical to those found in the prophage ϕRSM in R. solanacearum strains. The minimum size of attB required for integration was determined to be 37 bp, containing a 13 bp core and flanking sequences of 4 bp on the left and 20 bp on the right. ϕRSM integrative recombination proceeds efficiently in vitro in the absence of additional proteins or high-energy cofactors. Excision of a functional phage genome from a prophage fragment was demonstrated in vitro, demonstrating two-way activity of ϕRSM1 integrase. This is the first example of a small serine recombinase from the resolvase/invertase group that functions in integrative and excisive recombination for filamentous phages. This serine integrase could be used as a tool for several genome engineering applications.

The complete genomic sequence of the novel myovirus RP13 infecting Ralstonia solanacearum, the causative agent of bacterial wilt.

A novel lytic bacteriophage, Ralstonia phage RP13, was isolated from tomato fields in Pang Nga, Thailand. Electron microscopic observation showed it to have the features of a myovirus with a novel triangulation number (T = 21, dextro). The RP13 DNA appeared to be heavily modified. By applying RNA sequencing and RNA-sequence-mediated DNA sequencing, the whole genome of RP31 was determined to be 170,942 bp in length with a mean G+C content of 39.2%. A total of 277 ORFs were identified as structural, functional, or hypothetical genes in addition to four tRNA genes. Phylogenetic analysis suggested that RP13 is not closely related to any other known phages. Thus, we concluded that the RP13 is a novel phage infecting R. solanacearum strains and will be a useful biocontrol agent against bacterial wilt disease.

3D structure of three jumbo phage heads.

Jumbo phages are bacteriophages that carry more than 200 kbp of DNA. In this study we characterized two jumbo phages (ΦRSL2 and ΦXacN1) and one semi-jumbo phage (ΦRP13) at the structural level by cryo-electron microscopy. Focusing on their capsids, three-dimensional structures of the heads at resolutions ranging from 16 to 9 Å were calculated. Based on these structures we determined the geometrical basis on which the icosahedral capsids of these phages are constructed, which includes the accessory and decorative proteins that complement them. A triangulation number novel to Myoviridae (ΦRP13; T=21) was discovered as well as two others, which are more common for jumbo phages (T=27 and T=28). Based on one of the structures we also provide evidence that accessory or decorative proteins are not a prerequisite for maintaining the structural integrity of very large capsids.

Systemic method to isolate large bacteriophages for use in biocontrol of a wide-range of pathogenic bacteria.

Large phages are characterized by genomes around 200 kbp or more. They can infect wide host ranges of bacteria and maintain long-lasting infection. There is no standard method for selective isolation of large phages. In this study, we developed a systemic method to isolate large phages and succeeded in isolating 11 large phages, named Escherichia phage E1∼E11. Electron microscopy observations revealed typical Myoviridae phages with big capsids and long contractile tails. Genome sizes of the isolated phages were determined by pulsed-field gel electrophoresis and found to be in two groups, those around 200 kbp for E1, E2, E5, E6, E7, E9 and E10 phages, and others of approximately 450 kbp for E3, E4, E8 and E11 phages. The isolated large phages had wide host ranges: for example, E9 was effective against Shigella sonnei SH05001, Shigella bydii SH00007, Shigella flexneri SH00006, Salmonella enterica serovar Enteritidis SAL01078 and Escherichia coli C3000 (K-12 derivative), as well as its original host E. coli BL21. Screening of these jumbo phages was performed with non-pathogenic E. coli strains as hosts. Therefore, this method opens a way to isolate jumbo phages infecting wide ranges of pathogenic bacteria in a typical laboratory with standard laboratory strains as the hosts. The isolated large phages will be good candidates for biocontrol of various pathogens.

Correction to: Full genome sequence of a polyvalent bacteriophage infecting strains of Shigella, Salmonella, and Escherichia.

The original version of this article unfortunately contained a mistake.

Full genome sequence of a polyvalent bacteriophage infecting strains of Shigella, Salmonella, and Escherichia.

A novel lytic bacteriophage, Escherichia phage EcS1, was isolated from sewage samples collected in Higashi-Hiroshima, Japan. The complete genome sequence of EcS1 was determined using the Illumina Miseq System. The whole genome of EcS1 was found to be 175,437 bp in length with a mean G+C content of 37.8%. A total of 295 genes were identified as structural, functional, and hypothetical genes. BLAST analysis of the EcS1 genomic sequence revealed the highest identity (79%; query cover of 73-74%) to three T4-related phages that infect Serratia sp. ATCC 39006. Host range experiments revealed that EcS1 has lytic effects on three pathogenic strains of Shigella spp. and a pathogenic strain of Salmonella enterica as well as on E. coli strains. However, two strains of Serratia marcescens showed resistance to this phage. Phylogenetic trees for phage tail fiber protein sequences revealed that EcS1 is closely related to Enterobacteriaceae-infecting phages. Thus, EcS1 is a novel phage that infects several pathogenic strains of the family Enterobacteriaceae.

Xanthomonas citri jumbo phage XacN1 exhibits a wide host range and high complement of tRNA genes.

Xanthomonas virus (phage) XacN1 is a novel jumbo myovirus infecting Xanthomonas citri, the causative agent of Asian citrus canker. Its linear 384,670 bp double-stranded DNA genome encodes 592 proteins and presents the longest (66 kbp) direct terminal repeats (DTRs) among sequenced viral genomes. The DTRs harbor 56 tRNA genes, which correspond to all 20 amino acids and represent the largest number of tRNA genes reported in a viral genome. Codon usage analysis revealed a propensity for the phage encoded tRNAs to target codons that are highly used by the phage but less frequently by its host. The existence of these tRNA genes and seven additional translation-related genes as well as a chaperonin gene found in the XacN1 genome suggests a relative independence of phage replication on host molecular machinery, leading to a prediction of a wide host range for this jumbo phage. We confirmed the prediction by showing a wider host range of XacN1 than other X. citri phages in an infection test against a panel of host strains. Phylogenetic analyses revealed a clade of phages composed of XacN1 and ten other jumbo phages, indicating an evolutionary stable large genome size for this group of phages.

Chitin synthesis by Chlorella cells infected by chloroviruses: Enhancement by adopting a slow-growing virus and treatment with aphidicolin.

Chlorella viruses or chloroviruses contain a gene that encodes an enzyme that catalyzes chitin synthesis. This gene is expressed early in viral infections to produce chitin on the outside of the Chlorella cell wall. Interestingly, chitin synthesis by microalgal Chlorella cells in combination with chloroviruses represents a unique eco-friendly process for converting solar energy and CO2 into useful materials. However, during the final viral infection stage, the host cells are completely lysed, so chitin should be harvested before cells lyse. To increase chitin yields, slow-growing chlorovirus isolates were adopted and the viral replication process was modified with an inhibitor of DNA synthesis. The accumulation of chitin on the surface of Chlorella cells infected with one of nine chlorovirus isolates carrying the chitin synthase gene was compared with that of CVK2 (a standard virus)-infected cells. Chlorella cells infected with CVNF-1 (a slow-growing virus) accumulated chitin over the entire cell surface within 15 min post-infection (p.i.), and chitin continued to accumulate for up to 8 h p.i. before cells lysed. This was 2-fold longer than the chitin-accumulation period for cells infected with CVK2. The addition of aphidicolin delayed the progression of the virus replication cycle and extended the chitin-accumulation period of CVNF-1-infected cells to 12 h p.i. before cells lysed. Additionally, chitin production in the aphidicolin-treated CVNF-1-infected cells was approximately 6-fold higher than in CVK2-infected cells not treated with aphidicolin. Thus, chitin synthesis in a Chlorella-virus system may be prolonged by using slow-growing viral isolates treated with aphidicolin.

Lysogenic Conversion of the Phytopathogen Ralstonia solanacearum by the P2virus ϕRSY1.

A P2-like phage ϕRSY1 infecting the phytopathogen Ralstonia solanacearum was isolated and characterized. The 40-kb genome of ϕRSY1 showed high sequence similarity to the Ralstonia phage ϕRSA1 and the GMI1000 prophage ϕRSX. The major genomic differences between these phages were the different orientation of the int gene and the gene content close to the cosL. ϕRSY1 and ϕRSX use a 15-base 3' portion of the serine tRNA(GGA) gene as attB, while ϕRSA1 uses a 45-base 3' portion of the arginine tRNA(CCG) gene. The different orientation of int in the genomes means that the gene arrangements in the prophage states are reversed in ϕRSY1 and ϕRSA1. Several putative gene products of ϕRSY1 may affect the bacterium's fitness. ϕRSY1 contains an open reading frame (ORF) that seems to encode a protein similar to Vgr in the type VI secretion system of various bacterial species. ϕRSY1 lysogens showed phenotypic changes including enhanced twitching motility, large colony formation, and easy aggregation of cells, suggesting involvement of this ORF in the changes. In view of these phage gene arrangements, we surveyed prophages in the genomes of various R. solanacearum strains and found that the P2-like phages of R. solanacearum (14 phages) consist of two major groups: the ϕRSY1-type and the ϕRSA1-type. The relationships and evolution of these P2-like phages inferred from our data are discussed in detail.

Dynamic integration and excision of filamentous phage XacF1 in Xanthomonas citri pv. citri, the causative agent of citrus canker disease.

Inovirus XacF1 (7325 nucleotides) is integrated into the genome of Xanthomonas citri pv. citri (Xcc) strains at the host dif site (attB) by the host XerC/D recombination system. The XacF1 attP sequence is located within the coding region of ORF12, a possible phage regulator. After integration, this open reading frame (ORF) is split into two pieces on the host genome. We examined dynamic integration/excision of XacF1 in Xcc strain MAFF 301080 and found that the integration started at 4 h postinfection (p.i.) and peaked at 12 h p.i. Thereafter, the ratio of integrated to free forms remained constant, suggesting equilibrium of integration and excision of XacF1 in the host genome. However, the integrated state became very unstable following a 5'-deletion of ORF12 in XacF1, suggesting that ORF12 plays a key role in the integration cycle of XacF1 in Xcc strains.

Replications of Two Closely Related Groups of Jumbo Phages Show Different Level of Dependence on Host-encoded RNA Polymerase.

Ralstonia solanacearum phages ΦRP12 and ΦRP31 are jumbo phages isolated in Thailand. Here we show that they exhibit similar virion morphology, genome organization and host range. Genome comparisons as well as phylogenetic and proteomic tree analyses support that they belong to the group of ΦKZ-related phages, with their closest relatives being R. solanacearum phages ΦRSL2 and ΦRSF1. Compared with ΦRSL2 and ΦRSF1, ΦRP12 and ΦRP31 possess larger genomes (ca. 280 kbp, 25% larger). The replication of ΦRP12 and ΦRP31 was not affected by rifampicin treatment (20 µg/ml), suggesting that phage-encoded RNAPs function to start and complete the infection cycle of these phages without the need of host-encoded RNAPs. In contrast, ΦRSL2 and ΦRSF1, encoding the same set of RNAPs, did not produce progeny phages in the presence of rifampicin (5 µg/ml). This observation opens the possibility that some ΦRP12/ΦRP31 factors that are absent in ΦRSL2 and ΦRSF1 are involved in their host-independent transcription.

Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages.

Jumbo phages infecting Ralstonia solanacearum were isolated in Thailand (ϕRSL2) and Japan (ϕRSF1). They were similar regarding virion morphology, genomic arrangement, and host range. Phylogenetic and proteomic tree analyses demonstrate that the ϕRSL2 and ϕRSF1 belong to a group of evolutionary related phages, including Pseudomonas phages ϕKZ, 201ϕ2-1 and all previously described ϕKZ-related phages. Despite conserved genomic co-linearity between the ϕRSL2 and ϕRSF1, they differ in protein separation patterns. A major difference was seen in the detection of virion-associated-RNA polymerase subunits. All ß- and ß'-subunits were detected in ϕRSF1, but one ß'-subunit was undetected in ϕRSL2. Furthermore, ϕRSF1 infected host cells faster (latent period: 60 and 150min for ϕRSF1 and ϕRSL2, respectively) and more efficiently than ϕRSL2. Therefore, the difference in virion-associated-RNA polymerase may affect infection efficiency. Finally, we show that ϕRSF1 is able to inhibit bacterial wilt progression in tomato plants.

Genomic diversity of large-plaque-forming podoviruses infecting the phytopathogen Ralstonia solanacearum.

The genome organization, gene structure, and host range of five podoviruses that infect Ralstonia solanacearum, the causative agent of bacterial wilt disease were characterized. The phages fell into two distinctive groups based on the genome position of the RNA polymerase gene (i.e., T7-type and ϕKMV-type). One-step growth experiments revealed that ϕRSB2 (a T7-like phage) lysed host cells more efficiently with a shorter infection cycle (ca. 60 min corresponding to half the doubling time of the host) than ϕKMV-like phages such as ϕRSB1 (with an infection cycle of ca. 180 min). Co-infection experiments with ϕRSB1 and ϕRSB2 showed that ϕRSB2 always predominated in the phage progeny independent of host strains. Most phages had wide host-ranges and the phage particles usually did not attach to the resistant strains; when occasionally some did, the phage genome was injected into the resistant strain's cytoplasm, as revealed by fluorescence microscopy with SYBR Gold-labeled phage particles.

The involvement of the PilQ secretin of type IV pili in phage infection in Ralstonia solanacearum.

PilQ is a member of the secretin family of outer membrane proteins and specifically involved in type IV secretion. Here we report the effects of pilQ mutation in Ralstonia solanacearum on the host physiology including susceptibility to several phage types (Inoviridae, Podoviridae and Myoviridae). With three lines of cells, namely wild type, ΔpilQ and pilQ-complemented cells, the cell surface proteins, twitching motility and sensitivity to phages were compared. SDS-PAGE analysis revealed that the major TFP pilin (PilA) was specifically lost in pilQ mutants and was recovered in the complemented cells. Drastically inactivated twitching motility in pilQ mutants was recovered to the wild type level in the complemented cells. Several phages of different types including those of Inoviridae, Podoviridae, and Myoviridae that infect wild type cells could not form plaques on pilQ mutants but showed infectivity to pilQ-complemented cells. These results indicate that PilQ function is generally required for phage infection in R. solanacearum.

Identification of the mcpA and mcpM genes, encoding methyl-accepting proteins involved in amino acid and l-malate chemotaxis, and involvement of McpM-mediated chemotaxis in plant infection by Ralstonia pseudosolanacearum (formerly Ralstonia solanacearum phylotypes I and III).

Sequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in the Ralstonia pseudosolanacearum GMI1000 genome. PCR analysis and DNA sequencing showed that the highly motile R. pseudosolanacearum strain Ps29 possesses homologs of all 22 R. pseudosolanacearum GMI1000 mcp genes. We constructed a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29 by unmarked gene deletion. Screening of the mutant collection revealed that R. pseudosolanacearum Ps29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis to l-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designated mcpM and mcpA. While wild-type R. pseudosolanacearum strain Ps29 displayed attraction to 16 amino acids, the mcpA mutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructed mcpA and mcpM deletion mutants of highly virulent R. pseudosolanacearum strain MAFF106611 to investigate the contribution of chemotaxis to l-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, the mcpM mutant (but not the mcpA mutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis to l-malate, facilitates R. pseudosolanacearum motility to tomato roots in sand.

The filamentous phage XacF1 causes loss of virulence in Xanthomonas axonopodis pv. citri, the causative agent of citrus canker disease.

In this study, filamentous phage XacF1, which can infect Xanthomonas axonopodis pv. citri (Xac) strains, was isolated and characterized. Electron microscopy showed that XacF1 is a member of the family Inoviridae and is about 600 nm long. The genome of XacF1 is 7325 nucleotides in size, containing 13 predicted open reading frames (ORFs), some of which showed significant homology to Ff-like phage proteins such as ORF1 (pII), ORF2 (pV), ORF6 (pIII), and ORF8 (pVI). XacF1 showed a relatively wide host range, infecting seven out of 11 strains tested in this study. Frequently, XacF1 was found to be integrated into the genome of Xac strains. This integration occurred at the host dif site (attB) and was mediated by the host XerC/D recombination system. The attP sequence was identical to that of Xanthomonas phage Cf1c. Interestingly, infection by XacF1 phage caused several physiological changes to the bacterial host cells, including lower levels of extracellular polysaccharide production, reduced motility, slower growth rate, and a dramatic reduction in virulence. In particular, the reduction in virulence suggested possible utilization of XacF1 as a biological control agent against citrus canker disease.

Insights into the diversity of φRSM phages infecting strains of the phytopathogen Ralstonia solanacearum complex: regulation and evolution.

The filamentous φRSM phages (φRSM1 and φRSM3) have integration/excision capabilities in the phytopathogenic bacterium Ralstonia solanacearum. In the present study, we further investigated φRSM-like sequences present in the genomes of R. solanacearum strains belonging to the four major evolutionary lineages (phylotypes I-IV). Based on bioinformatics and comparative genomic analyses, we found that φRSM homologs are highly diverse in R. solanacearum complex strains. We detected an open reading frame (ORF)15 located upstream of the gene for φRSM integrase, which exhibited amino acid sequence similarity to phage repressor proteins. ORF15-encoded protein (a putative repressor) was found to encode a 104-residue polypeptide containing a DNA-binding (helix-turn-helix) domain and was expressed in R. solanacearum lysogenic strains. This suggested that φRSM3-ORF15 might be involved in the establishment and maintenance of a lysogenic state, as well as in phage immunity. Comparison of the putative repressor proteins and their binding sites within φRSM-related prophages provides insights into how these regulatory systems of filamentous phages have evolved and diverged in the R. solanacearum complex. In conclusion, φRSM phages represent a unique group of filamentous phages that are equipped with innate integration/excision (ORF14) and regulatory systems (ORF15).

Characterization of bacteriophages Cp1 and Cp2, the strain-typing agents for Xanthomonas axonopodis pv. citri.

The strains of Xanthomonas axonopodis pv. citri, the causative agent of citrus canker, are historically classified based on bacteriophage (phage) sensitivity. Nearly all X. axonopodis pv. citri strains isolated from different regions in Japan are lysed by either phage Cp1 or Cp2; Cp1-sensitive (Cp1(s)) strains have been observed to be resistant to Cp2 (Cp2(r)) and vice versa. In this study, genomic and molecular characterization was performed for the typing agents Cp1 and Cp2. Morphologically, Cp1 belongs to the Siphoviridae. Genomic analysis revealed that its genome comprises 43,870-bp double-stranded DNA (dsDNA), with 10-bp 3'-extruding cohesive ends, and contains 48 open reading frames. The genomic organization was similar to that of Xanthomonas phage phiL7, but it lacked a group I intron in the DNA polymerase gene. Cp2 resembles morphologically Escherichia coli T7-like phages of Podoviridae. The 42,963-bp linear dsDNA genome of Cp2 contained terminal repeats. The Cp2 genomic sequence has 40 open reading frames, many of which did not show detectable homologs in the current databases. By proteomic analysis, a gene cluster encoding structural proteins corresponding to the class III module of T7-like phages was identified on the Cp2 genome. Therefore, Cp1 and Cp2 were found to belong to completely different virus groups. In addition, we found that Cp1 and Cp2 use different molecules on the host cell surface as phage receptors and that host selection of X. axonopodis pv. citri strains by Cp1 and Cp2 is not determined at the initial stage by binding to receptors.

Cryo-electron microscopy three-dimensional structure of the jumbo phage ΦRSL1 infecting the phytopathogen Ralstonia solanacearum.

ϕRSL1 jumbo phage belongs to a new class of viruses within the Myoviridae family. Here, we report its three-dimensional structure determined by electron cryo microscopy. The icosahedral capsid, the tail helical portion, and the complete tail appendage were reconstructed separately to resolutions of 9 Å, 9 Å, and 28 Å, respectively. The head is rather complex and formed by at least five different proteins, whereas the major capsid proteins resemble those from HK97, despite low sequence conservation. The helical tail structure demonstrates its close relationship to T4 sheath proteins and provides evidence for an evolutionary link of the inner tail tube to the bacterial type VI secretion apparatus. Long fibers extend from the collar region, and their length is consistent with reaching the host cell surface upon tail contraction. Our structural analyses indicate that ϕRSL1 is an unusual member of the Myoviridae that employs conserved protein machines related to different phages and bacteria.