Pseudomonas fluorescens SBW25 produces furanomycin, a non-proteinogenic amino acid with selective antimicrobial properties

Abstract

Background: Pseudomonas fluorescens SBW25 has been extensively studied because of its plant growth promoting properties and potential as a biocontrol agent. The genome of SBW25 has been sequenced, and among sequenced strains of pseudomonads, SBW25 appears to be most closely related to P. fluorescens WH6. In the authors' laboratories, WH6 was previously shown to produce and secrete 4-formylaminooxyvinylglycine (FVG), a non-proteinogenic amino acid with selective herbicidal and antimicrobial activity. Although SBW25 does not have the genetic capacity to produce FVG, we were interested in determining whether this pseudomonad might produce some other type of non-proteinogenic amino acid.

Results: P. fluorescens SBW25 was found to produce and secrete a ninhydrin-reactive compound with selective antimicrobial properties. This compound was purified from SBW25 culture filtrate and identified as the non-proteinogenic amino acid L-furanomycin [2S,2'R,5'S)-2-amino-2-(5'methyl-2',5'-dihydrofuran-2'-yl)acetic acid].

Conclusions: The identification of furanomycin as a secondary metabolite of SBW25 is the first report of the production of furanomycin by a pseudomonad. This compound was known previously only as a natural product produced by a strain of Streptomyces. This report adds furanomycin to the small list of non-proteinogenic amino acids that have been identified as secondary products of pseudomonads. This study also extends the list of bacteria that are inhibited by furanomycin to include several plant pathogenic bacteria.

Figure 1

Thin-layer chromatograms of 90% ethanol extracts of dried culture filtrates from P. fluorescens SBW25 and WH6. A. Ninhydrin-stained cellulose TLC chromatograms. B. Ninhydrin-stained GHL-silica TLC chromatograms. Sample preparation and application and the solvent systems used to develop the chromatograms are described in the Methods section. The ninhydrin band corresponding to FVG is indicated on the WH6 chromatogram.

Figure 2

The distribution of antimicrobial activity and ninhydrin-banding after TLC fractionation of an 85% ethanol extract of dried culture filtrate from P. fluorescens SBW25. The 85% ethanol extract was prepared and applied to cellulose TLC plates as described in Methods. One of the developed plates was sprayed with ninhydrin (Figure 2A) and the replicate plate was divided into zones, as indicated, for removal and extraction of the cellulose. The aqueous extracts of the cellulose from each zone were assayed for antimicrobial activity according to the standard assay described in the Methods section. The resulting antimicrobial activity against Dickeya dadantii (Figure 2B) was measured after 48 h. Zones without bars did not result in a cleared zone when assayed with either D. dadantii or P. syringae pv. maculicola M4.

Figure 3

Initial Sephadex G-15 column fractionation of an 85% ethanol extract of dried culture filtrate from Pseudomonas fluorescens SBW25. The solids from 840 mL of dried SBW25 culture filtrate were extracted with 85% ethanol as described in the Methods section. A portion of the extract equivalent to 800 mL of original culture filtrate was taken to dryness in vacuo and dissolved in 6 mL of deionized water for application to a Sephadex G-15 column equilibrated in the same solvent. The column was eluted with deionized water. Fractions (6 mL each) were collected and analyzed for reaction with the Fe- and Cu-CAS reagents as described in the Methods section. The fractions corresponding to the largest Cu-binding peak were pooled (as indicated by the double arrow) for concentration and further purification by preparative TLC fractionation.

Figure 4

Final Sephadex G-15 column purification of the partially purified ninhydrin-reactive compound recovered from preparative TLC chromatograms. A sterile aqueous solution containing the partially purified SBW25 ninhydrin-reactive compound was prepared by extraction of the appropriate zone of preparative TLC chromatograms as described in the Methods section. This solution was taken to dryness in vacuo, and the recovered solids were dissolved in 5 mL of deionized water for application to the Sephadex G-15 column. The column was eluted with deionized water. Fractions (5 mL each) were collected and analyzed for reaction with the Fe- and Cu-ChromeAzurol S reagents. The fractions corresponding to the Cu-binding peak were pooled (as indicated by the double arrow) and concentrated for structural identification.

Figure 5

Molecular structure of L-furanomycin.

Figure 6

The effect of selected amino acids on the antimicrobial activity of furanomycin. The indicated amino acids were added to aliquots of P. fluorescens SBW25 culture filtrate to give a final amino acid concentration of 10 mM. The resulting solutions were filter sterilized and tested for antimicrobial activity against D. dadantii in our agar diffusion assay as described in the Methods section. The areas of the cleared zones in the bacterial lawns surrounding the central well containing the test solutions are the averages of three replicates. The error bars represent Standard Error of the Mean values.