RESUMO
The genus Habenivirus which includes Ralstonia virus ÏRSM 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 ÏRSM 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 ÏRSM 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. ÏRSM 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 ÏRSM1 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.
Assuntos
Bacteriófagos/genética , Inoviridae/genética , Integrases/genética , Recombinação Genética/genética , Serina/genética , Proteínas Virais/genéticaRESUMO
The original version of this article unfortunately contained a mistake.
RESUMO
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.
Assuntos
Bacteriófagos/isolamento & purificação , Escherichia coli/virologia , Genoma Viral , Salmonella/virologia , Shigella/virologia , Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/fisiologia , Composição de Bases , Sequência de Bases , Especificidade de Hospedeiro , Japão , Fases de Leitura Aberta , FilogeniaRESUMO
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.
Assuntos
Bacteriófagos/fisiologia , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Ralstonia solanacearum/citologia , Ralstonia solanacearum/virologia , Internalização do VírusRESUMO
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).
Assuntos
Variação Genética , Genoma Viral/genética , Inovirus/genética , Ralstonia solanacearum/virologia , Proteínas Virais/genética , Sequência de Aminoácidos , Sítios de Ligação Microbiológicos , Sequência de Bases , Sítios de Ligação , Biologia Computacional , DNA Viral/genética , Evolução Molecular , Inovirus/fisiologia , Integrases/genética , Lisogenia , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Prófagos/genética , RNA Viral/genética , Alinhamento de Sequência , Deleção de Sequência , Proteínas Virais/metabolismoRESUMO
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.
Assuntos
Bacteriófagos/genética , DNA Viral/genética , Genoma Viral , Xanthomonas axonopodis/virologia , Bacteriófagos/fisiologia , Bacteriófagos/ultraestrutura , DNA Viral/química , Ordem dos Genes , Dados de Sequência Molecular , Fases de Leitura Aberta , Receptores Virais , Análise de Sequência de DNA , Siphoviridae/genética , Siphoviridae/ultraestrutura , Ligação ViralRESUMO
Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured-cells (BY-2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY-2 cells harboring a vSPO-cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N-terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA.
Assuntos
Enzimas/metabolismo , Glucuronosiltransferase/genética , Ácido Hialurônico/biossíntese , Nicotiana/enzimologia , Sequência de Bases , Parede Celular , Células Cultivadas , Primers do DNA , Hialuronan Sintases , Ácido Hialurônico/metabolismo , Organelas/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Frações Subcelulares/enzimologia , Nicotiana/citologiaRESUMO
Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. ÏRSS1 is a filamentous phage that infects R. solanacearum strains. Upon infection, it alters the physiological state and the behavior of host cells. Here, we show that R. solanacearum infected by ÏRSS1 becomes more virulent on host plants. Some virulence and pathogenicity factors, such as extracellular polysaccharide (EPS) synthesis and twitching motility, increased in the bacterial host cells infected with ÏRSS1, resulting in early wilting. Tomato plants inoculated with ÏRSS1-infected bacteria wilted 2 to 3 days earlier than those inoculated with wild-type bacteria. Infection with ÏRSS1 induced early expression of phcA, the global virulence regulator. phcA expression was detected in ÏRSS1-infected cells at cell density as low as 10(4) CFU/ml. Filamentous phages are assembled on the host cell surface and many phage particles accumulate on the cell surface. These surface-associated phage particles (phage proteins) may change the cell surface nature (hydrophobicity) to give high local cell densities. ÏRSS1 infection also enhanced PilA and type IV pilin production, resulting in increased twitching motility.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Doenças das Plantas/microbiologia , Polissacarídeos Bacterianos/metabolismo , Ralstonia solanacearum/patogenicidade , Solanum lycopersicum/microbiologia , Proteínas de Bactérias/genética , Celulase/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Ralstonia solanacearum/genética , Ralstonia solanacearum/fisiologia , Ralstonia solanacearum/virologia , Fatores de Tempo , Fatores de Transcrição/genética , Proteínas Virais/metabolismo , Virulência , Fatores de VirulênciaRESUMO
φRSM1 and φRSM3 (φRSM phages) are filamentous phages (inoviruses) that infect Ralstonia solanacearum, the causative agent of bacterial wilt. Infection by φRSM phages causes several cultural and physiological changes to host cells, especially loss of virulence. In this study, we characterized changes related to the virulence in φRSM3-infected cells, including (i) reduced twitching motility and reduced amounts of type IV pili (Tfp), (ii) lower levels of ß-1,4-endoglucanase (Egl) activity and extracellular polysaccharides (EPS) production, and (iii) reduced expression of certain genes (egl, pehC, phcA, phcB, pilT, and hrpB). The significantly lower levels of phcA and phcB expression in φRSM3-infected cells suggested that functional PhcA was insufficient to activate many virulence genes. Tomato plants injected with φRSM3-infected cells of different R. solanacearum strains did not show wilting symptoms. The virulence and virulence factors were restored when φRSM3-encoded orf15, the gene for a putative repressor-like protein, was disrupted. Expression levels of phcA as well as other virulence-related genes in φRSM3-ΔORF15-infected cells were comparable with those in wild-type cells, suggesting that orf15 of φRSM3 may repress phcA and, consequently, result in loss of virulence.
Assuntos
Genes Virais/genética , Inovirus/fisiologia , Doenças das Plantas/microbiologia , Ralstonia solanacearum/patogenicidade , Solanum lycopersicum/microbiologia , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Inovirus/genética , Polissacarídeos Bacterianos/metabolismo , Ralstonia solanacearum/genética , Ralstonia solanacearum/fisiologia , Ralstonia solanacearum/virologia , Virulência/genéticaRESUMO
The wide host range of Ralstonia solanacearum, causal agent of bacterial wilt, and its ability to survive for long periods in the environment restrict the effectiveness of cultural and chemical control measures. The use of phages for disease control is a fast-expanding trend of plant protection with great potential to replace chemical measures. The filamentous phage ÏRSM3 that infects R. solanacearum strains and inactivates virulence on plants is a potential agent for controlling bacterial wilt in tomato. We demonstrated that inoculation of ÏRSM3-infected cells into tomato plants did not cause bacterial wilt. Instead, ÏRSM3-infected cells enhanced the expression of pathogenesis-related (PR) genes, including PR-1a, PR-2b, and PR7, in tomato plants. Moreover, pretreatment with ÏRSM-infected cells protect tomato plants from infection by virulent R. solanacearum strains. The effective dose of ÏRSM3-infected cells for disease prevention was determined to be approximately 105 CFU/ml. Because the ÏRSM3-infected cells can grow and continue to produce infectious phage particles under appropriate conditions, ÏRSM phages may serve as an efficient tool to control bacterial wilt in crops.
RESUMO
Ralstonia solanacearum is a Gram-negative bacterium and the causative agent of bacterial wilt in many important crops. We treated R. solanacearum with three lytic phages: ÏRSA1, ÏRSB1, and ÏRSL1. Infection with ÏRSA1 and ÏRSB1, either alone or in combination with the other phages, resulted in a rapid decrease in the host bacterial cell density. Cells that were resistant to infection by these phages became evident approximately 30 h after phage addition to the culture. On the other hand, cells infected solely with ÏRSL1 in a batch culture were maintained at a lower cell density (1/3 of control) over a long period. Pretreatment of tomato seedlings with ÏRSL1 drastically limited penetration, growth, and movement of root-inoculated bacterial cells. All ÏRSL1-treated tomato plants showed no symptoms of wilting during the experimental period, whereas all untreated plants had wilted by 18 days postinfection. ÏRSL1 was shown to be relatively stable in soil, especially at higher temperatures (37 to 50°C). Active ÏRSL1 particles were recovered from the roots of treated plants and from soil 4 months postinfection. Based on these observations, we propose an alternative biocontrol method using a unique phage, such as ÏRSL1, instead of a phage cocktail with highly virulent phages. Using this method, ÏRSL1 killed some but not all bacterial cells. The coexistence of bacterial cells and the phage resulted in effective prevention of wilting.
Assuntos
Bacteriófagos/crescimento & desenvolvimento , Controle Biológico de Vetores/métodos , Doenças das Plantas/prevenção & controle , Ralstonia solanacearum/crescimento & desenvolvimento , Ralstonia solanacearum/virologia , Bacteriólise , Solanum lycopersicum/microbiologia , Viabilidade Microbiana , Doenças das Plantas/microbiologiaRESUMO
PhiRSB1 is a wide-host-range, T7-like bacteriophage that infects and efficiently lyses the phytopathogenic bacterium Ralstonia solanacearum. The phiRSB1 genome comprises 43,079 bp of double-stranded DNA (61.7% G+C) with 325-bp terminal repeats and contains 47 open reading frames. Strong activity of tandem early promoters and wide specificity of phage promoters of phiRSB1 were demonstrated.
Assuntos
Bacteriófagos/genética , Ralstonia solanacearum/virologia , Bacteriófago T7/classificação , Bacteriófago T7/genética , Bacteriófagos/classificação , Bacteriófagos/ultraestrutura , DNA Viral/genética , Genoma Viral , Dados de Sequência Molecular , Fases de Leitura Aberta , Podoviridae/classificação , Podoviridae/genética , Regiões Promotoras Genéticas , Transcrição Gênica , Ensaio de Placa Viral , Proteínas Virais/genética , Vírion/genéticaRESUMO
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 E1â¼E11. 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.
Assuntos
Bactérias/patogenicidade , Bactérias/virologia , Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologia , Controle de Infecções/métodos , Antibiose/fisiologia , Bacteriófagos/genética , Eletroforese em Gel de Campo Pulsado , Escherichia coli/patogenicidade , Especificidade de Hospedeiro , Myoviridae/fisiologiaRESUMO
PhiRSA1 is a wide-host-range bacteriophage isolated from Ralstonia solanacearum. In this study, the complete nucleotide sequence of the phiRSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5'-extruding cohesive ends (cos) and contained 51 open reading frames (ORFs). Two-thirds of the phiRSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A phiRSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these phiRSA1 genes as 5' TGTTGT-(X)13-ACAACA. The genomic sequence similarity between phiRSA1 and related phages phi52237 and phiCTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. phiRSA1 was found to be integrated into at least three different strains of R. solanacearum, and the chromosomal integration site (attB) was identified as the 3' portion of the arginine tRNA(CCG) gene. In the light of the phiRSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of R. solanacearum strain GMI1000 was characterized as a phiRSA1-related prophage (designated phiRSX). phiRSX was found to be integrated at the serine tRNA (GGA) gene as an att site, and its size was determined to be 40,713 bp. phiRSX ORFs shared very high amino acid identity with their phiRSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.
Assuntos
Bacteriófagos/genética , DNA Viral/genética , Genoma Viral , Prófagos/genética , Ralstonia solanacearum/virologia , Bacteriófagos/classificação , Bacteriófagos/patogenicidade , Primers do DNA , DNA Viral/isolamento & purificação , Genoma de Planta , Hibridização In Situ , Lisogenia , Dados de Sequência Molecular , Fases de Leitura Aberta , Ralstonia solanacearum/classificação , Ralstonia solanacearum/crescimento & desenvolvimento , Ralstonia solanacearum/patogenicidade , Nicotiana/microbiologia , VirulênciaRESUMO
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.
Assuntos
Afidicolina/farmacologia , Quitina/metabolismo , Chlorella/metabolismo , Chlorella/virologia , Phycodnaviridae/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Chlorella/efeitos dos fármacos , Phycodnaviridae/efeitos dos fármacos , Phycodnaviridae/crescimento & desenvolvimento , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologiaRESUMO
A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage phiRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the phiRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. These results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies.
Assuntos
Bacteriófagos/genética , Genes Reporter/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Ralstonia solanacearum/isolamento & purificação , Ralstonia solanacearum/metabolismo , Solanum lycopersicum/microbiologia , Transfecção/métodos , DNA Viral/genética , Proteínas de Fluorescência Verde/genética , Plasmídeos/genética , Espectrometria de Fluorescência/métodosRESUMO
A P2-like phage ÏRSY1 infecting the phytopathogen Ralstonia solanacearum was isolated and characterized. The 40-kb genome of ÏRSY1 showed high sequence similarity to the Ralstonia phage ÏRSA1 and the GMI1000 prophage ÏRSX. The major genomic differences between these phages were the different orientation of the int gene and the gene content close to the cosL. ÏRSY1 and ÏRSX use a 15-base 3' portion of the serine tRNA(GGA) gene as attB, while ÏRSA1 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 ÏRSY1 and ÏRSA1. Several putative gene products of ÏRSY1 may affect the bacterium's fitness. ÏRSY1 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. ÏRSY1 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 ÏRSY1-type and the ÏRSA1-type. The relationships and evolution of these P2-like phages inferred from our data are discussed in detail.
RESUMO
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.
RESUMO
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.
RESUMO
To elucidate the contribution of LINE-like retrotransposon Zepp elements to the formation and maintenance of chromosomal telomeres, newly formed minichromosomes in irradiated Chlorella vulgaris cells were isolated and structurally characterized. A minichromosome (miniV4) of approximately 700 kb in size contained a Zepp cluster taking the place of the telomeric repeats on one terminus, whereas the other end of this chromosome consisted of canonical telomeric repeats. The Zepp copies in this cluster were in a tandem array with their poly(A) tails towards the centromere. Another minichromosome Y32 ( approximately 400 kb in size) was shown to have several copies of Zepp elements on both termini. On the right arm terminus, two copies of Zepp were found in a tandem array with poly(A) tracts facing towards the chromosomal end. The poly(A) tail and the 3'-end of approximately 400 bp of the distal copy were replaced by the telomeric repeats. On the 5'-side of the proximal copy was another Zepp element in the reverse orientation. These newly formed telomeric structures are very similar to those previously found in the left arm of chromosome I and the terminus of an unidentified chromosome and support the model of Zepp-mediated restoration and maintenance of Chlorella telomeres.