No abstract

BackgroundPseudomonas 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.ResultsWe 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.ConclusionsThe 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.

A serious disease was observed in black raspberry (Rubus occidentalis) in Oregon in the last decade. Plants showing mosaic symptoms declined rapidly and, in many cases, died after several years. Double-stranded RNA extraction from symptomatic black raspberry revealed the presence of two high molecular weight bands which were cloned and sequenced. Sequence analysis disclosed the presence of a novel virus that was tentatively named Black raspberry decline-associated virus (BRDaV). The complete sequences of the two genomic RNAs, excluding the 3' poly-adenosine tails, were 7,581 and 6,364 nucleotides, respectively. The genome organization was identical to that of Strawberry mottle virus, a member of the genus Sadwavirus. The C terminus of the RNA 1 poly-protein is unique within the genus Sadwavirus, with homology to AlkB-like domains, suggesting a role in repair of alkylation damage. A reverse-transcriptase polymerase chain reaction test was designed for the detection of BRDaV from Rubus tissue, and tests revealed that BRDaV was associated consistently with the observed decline symptoms. While this publication was under review, it came to our attention that scientists at the Scottish Crop Research Institute had molecular data on Black raspberry necrosis virus (BRNV), a virus that shared many biological properties with BRDaV. After exchange of data, we concluded that BRDaV is a strain of BRNV, a previously described yet unsequenced virus. The North American strain was vectored nonpersistently by the large raspberry aphid and the green peach aphid. Phylogenetic analysis indicates that BRNV belongs to the genus Sadwavirus.

Aims:  The germination‐arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and identified as 4‐formylaminooxyvinylglycine, specifically inhibits the germination of a wide range of grassy weeds. This study was undertaken to determine whether GAF has antimicrobial activity in addition to its inhibitory effects on grass seed germination. Methods and Results:  Culture filtrate from Ps. fluorescens WH6 had little or no effect on 17 species of bacteria grown in Petri dish lawns, but the in vitro growth of Erwinia amylovora, the causal agent of the disease of orchard crops known as fire blight, was strongly inhibited by the filtrate. The anti‐Erwinia activity of WH6 culture filtrate was shown to be due to its GAF content, and a commercially available oxyvinylglycine, 4‐aminoethoxyvinylglycine (AVG), exhibited anti‐Erwinia activity similar to that of GAF. The effects of GAF on Erwinia were reversed by particular amino acids. Conclusions:  The biological properties of GAF include a rather specific antimicrobial activity against Erw. amylovora. This may be a general property of oxyvinylglycines as AVG exhibited similar activity. The ability of particular amino acids to reverse GAF inhibition is consistent with a potential effect of this compound on the activity of aminotransferases. Significance and Impact of the Study:  The results presented here demonstrate a novel antimicrobial activity of oxyvinylglycines and suggest that GAF and/or GAF‐producing bacteria may have potential for the control of fire blight.

The genetic basis of the biosynthesis of the germination-arrest factor (GAF) produced by Pseudomonas fluorescens WH6, and previously identified as 4-formylaminooxyvinylglycine, has been investigated here. In addition to inhibiting the germination of a wide range of grassy weeds, GAF exhibits a selective antimicrobial activity against the bacterial plant pathogen Erwinia amylovora. We utilized the in vitro response of E. amylovora to GAF as a rapid screen for loss-offunction GAF phenotypes generated by transposon mutagenesis. A Tn5 mutant library consisting of 6364 WH6 transformants was screened in this Erwinia assay, resulting in the identification of 18 non-redundant transposon insertion sites that led to loss of GAF production in WH6, as confirmed by TLC analysis. These insertions mapped to five different genes and four intergenic regions. Three of these genes, including two putative regulatory genes (gntR and iopB homologues), were clustered in a 13 kb chromosomal region containing 13 putative ORFs. A GAF mutation identified previously as affecting an aminotransferase also maps to this region. We suggest that three of the genes in this region (a carbamoyltransferase, an aminotransferase and a formyltransferase) encode the enzymes necessary to synthesize dihydroGAF, the putative immediate precursor of GAF in a proposed GAF biosynthetic pathway. RT-qPCR analyses demonstrated that mutations in the gntR and iopB regulatory genes, as well as in a prtR homologue identified earlier as controlling GAF formation, suppressed transcription of at least two of the putative GAF biosynthetic genes (encoding the aminotransferase and formyltransferase) located in this 13 kb region.