RESUMO
Pseudomonas fluorescens WH6 secretes a germination-arrest factor (GAF) that we have identified previously as 4-formylaminooxyvinylglycine. GAF irreversibly inhibits germination of the seeds of numerous grassy weeds and selectively inhibits growth of the bacterial plant pathogen Erwinia amylovora. WH6-3, a mutant that has lost the ability to produce GAF, contains a Tn5 insertion in prtR, a gene that has been described previously in some strains of P. fluorescens as encoding a transmembrane regulator. As in these other pseudomonads, in WH6, prtR occurs immediately downstream of prtI, which encodes a protein homologous to extracytoplasmic function (ECF) sigma factors. These two genes have been proposed to function as a dicistronic operon. In this study, we demonstrated that deletion of prtI in WT WH6 had no effect on GAF production. However, deletion of prtI in the WH6-3 mutant overcame the effects of the Tn5 insertion in prtR and restored GAF production in the resulting double mutant. Complementation of the double prtIR mutant with prtI suppressed GAF production. This overall pattern of prtIR regulation was also observed for the activity of an AprX protease. Furthermore, reverse transcription quantitative real-time PCR analysis demonstrated that alterations in GAF production were mirrored by changes in the transcription of two putative GAF biosynthetic genes. Thus, we concluded that PrtI exerted a negative regulatory effect on GAF production, although the mechanism has not yet been determined. In addition, evidence was obtained that the transcription of prtI and prtR in WH6 may be more complex than predicted by existing models.
Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas fluorescens/metabolismo , Fator sigma/metabolismo , Proteínas de Bactérias/genética , Óperon , Pseudomonas fluorescens/genética , Fator sigma/genéticaRESUMO
BACKGROUND: Pseudomonas fluorescens is a genetically and physiologically diverse species of bacteria present in many habitats and in association with plants. This species of bacteria produces a large array of secondary metabolites with potential as natural products. P. fluorescens isolate WH6 produces Germination-Arrest Factor (GAF), a predicted small peptide or amino acid analog with herbicidal activity that specifically inhibits germination of seeds of graminaceous species. RESULTS: We used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for P. fluorescens WH6. We employed automated, manual, and experimental methods to further improve the draft genome sequence. From this assembly of 6.27 megabases, we predicted 5876 genes, of which 3115 were core to P. fluorescens and 1567 were unique to WH6. Comparative genomic studies of WH6 revealed high similarity in synteny and orthology of genes with P. fluorescens SBW25. A phylogenomic study also placed WH6 in the same lineage as SBW25. In a previous non-saturating mutagenesis screen we identified two genes necessary for GAF activity in WH6. Mapping of their flanking sequences revealed genes that encode a candidate anti-sigma factor and an aminotransferase. Finally, we discovered several candidate virulence and host-association mechanisms, one of which appears to be a complete type III secretion system. CONCLUSIONS: The improved high-quality draft genome sequence of WH6 contributes towards resolving the P. fluorescens species, providing additional impetus for establishing two separate lineages in P. fluorescens. Despite the high levels of orthology and synteny to SBW25, WH6 still had a substantial number of unique genes and represents another source for the discovery of genes with implications in affecting plant growth and health. Two genes are demonstrably necessary for GAF and further characterization of their proteins is important for developing natural products as control measure against grassy weeds. Finally, WH6 is the first isolate of P. fluorescens reported to encode a complete T3SS. This gives us the opportunity to explore the role of what has traditionally been thought of as a virulence mechanism for non-pathogenic interactions with plants.
Assuntos
Proteínas de Bactérias/biossíntese , Genoma Bacteriano/genética , Pseudomonas fluorescens/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Mapeamento Cromossômico , DNA Circular/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Genes Reguladores/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação/genética , Filogenia , Pseudomonas fluorescens/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Sintenia/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The etiology of pallidosis, a disease of strawberry identified more than 45 years ago, remains unknown. We report a putative agent of the disease, a virus belonging to the Crinivirus genus of the Closterovirideae family. A sensitive reverse transcription-polymerase chain reaction (RTPCR) test has been developed. Polyclonal antibodies that can be used to detect the virus in petiole tissue blots were developed using a recombinant virus coat protein. The nucleotide sequences of regions of the viral genome that encode the heat shock protein 70 homolog and the major coat protein were obtained. Alignments of the major coat protein show that the virus isolated from strawberry plants positive for pallidosis is most closely related to Cucumber yellows virus (syn. Beet pseudo-yellows virus) and Cucurbit yellow stunt disorder virus, members of the Crinivirus genus.
