Absence of 4-Formylaminooxyvinylglycine Production by Pseudomonas fluorescens WH6 Results in Resource Reallocation from Secondary Metabolite Production to Rhizocompetence

Abstract

Pseudomonas fluorescens WH6 produces the non-proteinogenic amino acid 4-formylaminooxyvinylglycine (FVG), a secondary metabolite with antibacterial and pre-emergent herbicidal activities. The gvg operon necessary for FVG production encodes eight required genes: one regulatory (gvgR), two of unknown functional potential (gvgA and C), three with putative biosynthetic function (gvgF, H, and I), and two small ORFs (gvgB and G). To gain insight into the role of GvgA and C in FVG production, we compared the transcriptome of knockout (KO) mutants of gvgR, A, and C to wild type (WT) to test two hypotheses: (1) GvgA and GvgC play a regulatory role in FVG production and (2) non-gvg cluster genes are regulated by GvgA and GvgC. Our analyses show that, collectively, 687 genes, including the gvg operon, are differentially expressed in all KO strains versus WT, representing >10% of the genome. Fifty-one percent of these genes were similarly regulated in all KO strains with GvgC having the greatest number of uniquely regulated genes. Additional transcriptome data suggest cluster regulation through feedback of a cluster product. We also discovered that FVG biosynthesis is regulated by L-glu, L-asp, L-gln, and L-asn and that resources are reallocated in KO strains to increase phenotypes involved in rhizocompetence including motility, biofilm formation, and denitrification. Altogether, differential transcriptome analyses of mutants suggest that regulation of the cluster is multifaceted and the absence of FVG production or its downregulation can dramatically shift the lifestyle of WH6.

Keywords: Pseudomonas fluorescens; natural herbicide; regulation of secondary metabolites; vinylglycine.

Figure 1

Structure of FVG and the gvg cluster. (A) Chemical structure of 4-formylaminooxyvinylglycine (FVG). (B) The organization of genes and promoters in the gvg cluster. The genes encode a putative regulator (light purple), proteins of unknown function (green, orange and dark purple), biosynthetic genes (blue), and transporters (brown). Arrows show approximate site of promoters.

Figure 2

Differentially expressed genes in ΔgvgR, A, and C strains. (A) Genes significantly differentially expressed (|Fold change| > 2, FDR p-value < 0.05) in Δgvg R, A, and C strains plotted across the WH6 genome. Red circles indicate the gvg cluster genes. The inset shows the numbers of up- and down-regulated genes in each knockout strain. (B) Overlap of differentially expressed genes for each strain. The top diagram represents the significantly different data set, while the bottom represents the manually curated data set for similar trends in the data. (C) Overlap of up- (top) and down-regulated (bottom) statistically significant differentially expressed genes in Δgvg R, A, and C strains.

Figure 3

Differential expression of gvg cluster genes and growth of gvg cluster mutants. (A) Downregulation of all genes in the gvg cluster in Δgvg R, A, and C strains. (B) Reads per kilobase of transcript, per million mapped reads (RPKM) for each gene plotted across the WH6 genome. Color coding indicates predicted gene function, including transcription and translation (blue); energy production (green), and gvg production (red). (C) Growth of gvg cluster mutants. Points represent a single experiment with 8 replicates. Error bars represent standard error. Two additional experiments were conducted with similar results.

Figure 4

Functional annotation of differentially regulated genes in Δgvg R, A, and C strains. (A) Radar plot of differentially regulated genes categorized into Clusters of Orthologous Groups. Labels reflect those groups which were significantly differentially regulated vs. WT and the axis indicates the numbers of genes regulated per category. Orange = ΔgvgC, green = ΔgvgA, and purple = ΔgvgR strains. (B) Venn diagrams of essential gene sets that are differentially down- (left side) or up-regulated (right side) in Δgvg R, A, and C strains as determined with a gene enrichment analysis of KEGG pathway categories. The numbers and descriptions of the gene sets are shown in similar colors.

Figure 5

Upregulation of biofilm and flagellar biosynthesis pathways lead to increased biofilm formation and motility. (A) Biofilm formation as a percent of WT WH6. Bars represent the average of two independent experiments with four replicates each. (B) Swimming motility of ΔgvgA, C, and R and WT WH6. Top panel shows a representative swim plate 48 h after inoculation and the bottom panel bars represents the average area of growth after 48 h from three independent experiments with three replicates each. Error bars = standard error. *** = p ≤ 0.001 by Student’s t-Test.

Figure 6

Downregulation of FVG production by acidic amino acids and their amides. (A) Schematic of uptake pathway of acidic amino acid and the regulation of pathway genes in gvg cluster mutants. The numbers in the brackets next to the gene product represent the fold difference from WT of ΔgvgR, A, and C, respectively. (B) Growth inhibition of E. amylovora by FVG in culture filtrates (CF) of WH6 grown in alternative N-sources. Bars represent the average of three independent experiments with six replicates each. (C) Swimming motility of WH6 grown in alternative N-sources after 6 days of growth. Bars represents the average colony area of three independent experiments with four to five replicates each. Error bars = standard error. *** = p ≤ 0.001 by Student’s t-Test.