Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/24/2016
Publication Date: 2/20/2017
Citation: Okrent, R.A., Trippe, K.M., Maselko, M.B., Manning, V. 2017. Functional analysis of a biosynthetic cluster essential for production of 4-formylaminooxyvinylglycine, a germination-arrest factor from Pseudomonas fluorescens WH6. Microbiology. 163(2):207-217.
Interpretive Summary: Pseudomonas fluorescens WH6 is a soil bacterium that produces a novel compound, 4-formylaminooxyvinyl glycine (FVG). FVG halts the germination of grasses and is toxic to certain bacteria that cause plant diseases. Thus, FVG could be an active ingredient in herbicides or in topical sprays to control weeds or plant diseases. One barrier that prevents the commercial use of FVG is that WH6 does not produce the molecule in abundant quantities. Understanding how WH6 produces FVG could therefore inform our efforts to produce the compound in large quantities. To better understand the pathway that WH6 uses to make FVG, we identified the genes that are required for FVG biosynthesis. We previously identified a gene cluster that is necessary for WH6 to produce FVG. This cluster contains 12 genes. In order to determine which of the 12 genes are necessary for FVG production, we constructed several strains of WH6 that have one or more of these genes deleted from the genome. We then tested these stains for the presence of FVG. This study determined that 10 of the 12 are necessary for FVG production. Two of these genes are unusually small; therefore the function of these genes are difficult to predict. The remaining 8 genes are likely involved in the regulation, synthesis, and the export of FVG from the bacterium. The basic identification of these genes will allow researchers to develop an approach where WH6, or an alternate bacterium, can produce agriculturally relevant quantities of FVG.
Technical Abstract: Rhizosphere-associated Pseudomonas fluorescens WH6 produces the germination-arrest factor, 4-formylaminooxyvinylglycine (FVG). FVG has previously been shown to both arrest the germination of weedy grasses and to inhibit the growth of the bacterial plant pathogen Erwinia amylovora. Very little is known about the mechanism by which FVG is produced. Although a previous study identified a region of the genome that may be involved in FVG biosynthesis, it has not yet been determined which genes within that region are sufficient and necessary for FVG production. In the current study, we explored the role of each of the putative genes encoded in that region by constructing deletion mutations. Mutant strains were assayed for their ability to produce FVG with a combination of biological assays and thin-layer chromatographic analyses. This work defined the core FVG biosynthetic gene cluster and revealed several interesting characteristics of FVG production. We determined that FVG biosynthesis requires two small open reading frames of less than 150 nucleotides and that multiple transporters have overlapping but distinct functionality. In addition, two genes in the center of the biosynthetic gene cluster are not required for FVG production, suggesting that additional products may be produced from the cluster. Transcriptional analysis indicated that at least three active promoters play a role in the expression of genes within this cluster. The results of this study enrich our knowledge regarding the diversity of mechanisms by which bacteria produce non-proteinogenic amino acids like vinylglycines.