16 research outputs found
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Genetics of germination-arrest factor (GAF) production by Pseudomonas fluorescens WH6: identification of a gene cluster essential for GAF biosynthesis
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-of-function 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.Keywords: Rhizosphere bacteria, Sequence, Antimetabolite, Aeruginosa, Rhizobitoxine, Pathway, Construction, Family, Amino acid
An improved, high-quality draft genome sequence of the Germination-Arrest Factor-producing Pseudomonas fluorescens WH6
<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas fluorescens </it>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. <it>P. fluorescens </it>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.</p> <p>Results</p> <p>We used a hybrid next-generation sequencing approach to develop a high-quality draft genome sequence for <it>P. fluorescens </it>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 <it>P. fluorescens </it>and 1567 were unique to WH6. Comparative genomic studies of WH6 revealed high similarity in synteny and orthology of genes with <it>P. fluorescens </it>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.</p> <p>Conclusions</p> <p>The improved high-quality draft genome sequence of WH6 contributes towards resolving the <it>P. fluorescens </it>species, providing additional impetus for establishing two separate lineages in <it>P. fluorescens</it>. 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 <it>P. fluorescens </it>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.</p
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Negative regulation of germination-arrest factor production in Pseudomonas fluorescens WH6 by a putative extracytoplasmic function sigma factor
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
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MillsDalliceBotanyPlantPathologyGeneticsGerminationArrest.pdf
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-of-function 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