Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum

Ralfuranones, aryl-furanone secondary metabolites, are involved in the virulence of Ralstonia solanacearum in solanaceous plants. Ralfuranone I (6) has been suggested as a biosynthetic precursor for other ralfuranones; however, this conversion has not been confirmed. We herein investigate the biosynthesis of ralfuranones using feeding experiments with ralfuranone I (6) and its putative metabolite, ralfuranone B (2). The results obtained demonstrated that the biosynthesis of ralfuranones proceeded in enzymatic and non-enzymatic manners. Graphical abstract Proposed biosynthetic pathway of ralfuranones in Ralstonia solanacearum.

Ralstonia solanacearum is a soil Gram-negative bacterium that causes "bacterial wilt" on a wide range of host plants, including economically important crops such as the tomato, tobacco, potato, peanut, and banana. 1) The bacterium invades the intercellular spaces of roots through openings such as wounds, accumulates around the stele, and then breaks into and fills xylem vessels through the action of cellulolytic enzymes on vessel walls. 2) The ability of this pathogen to cause host wilting has mainly been attributed to its production of extracellular polysaccharide (EPS). 3) The production of EPS was previously shown to be regulated by quorum sensing (QS) consisting of phc regulatory elements, in which the global virulence regulator PhcA plays a central role. [3][4][5] We recently discovered that R. solanacearum strains possess a phc QS system mediated by either (R)-methyl 3-hydroxymyristate or (R)-methyl 3-hydroxypalmitate. 6) In addition to EPS, phc QS also regulates the production of secondary metabolites, and ralfuranones A (1) and B (2) (Fig. 1) were identified as phc QS-dependent secondary metabolites from R. solanacearum strain GMI1000, a widely used model strain. 7) Thereafter, we reported the identification of three new derivatives, ralfuranones J (3), K (4), and L (5) (Fig. 1), from R. solanacearum strain OE1-1, a strain isolated from egg plants in Japan, and the involvement of the production of ralfuranone in its virulence in tomato plants. 8) Since ralfuranones did not exhibit apparent toxicity against host plants, these compounds may enhance bacterial activities related to virulence.
The key enzymes involved in ralfuranone biosynthesis have been studied in detail. The aminotransferase RalD converts L-phenylalanine to phenylpyruvic acid, which is then loaded to the furanone synthase RalA, composed of a tridomain nonribosomal peptide synthetase (NRPS)-like enzyme (Scheme 1). 9) Two phenylpyruvic acids are condensed by an aldol reaction on RalA, and the product is released from the enzyme by ester hydrolysis. The condensation product, named ralfuranone I (6) (Scheme 1), has been suggested as a common biosynthetic precursor for other ralfuranones. 10) However, these conversions have not yet been examined in R. solanacearum. We previously reported that a ralA-deletion mutant of OE1-1 (ΔralA) did not produce any ralfuranones, but started to produce them again when the ralA gene was complemented. 8) This finding indicated that enzymes involved in the biosynthesis of ralfuranones other than ralA remain active in ΔralA. Thus, using this mutant, feeding studies on ralfuranones may be conducted without preparing their isotopically labeled precursors. In order to elucidate the biosynthetic pathway of ralfuranones in R. solanacearum, we herein prepared ralfuranone I (6) by culturing OE1-1 and then conducted feeding experiments on ΔralA. The results obtained showed that the key steps in ralfuranone biosynthesis (ralfuranone I (6) → B (2) → A (1)) proceeded in a non-enzymatic manner.
Bacterial strains and growth conditions. Seed cultures of OE1-1 and ΔralA were grown in B medium 8) at 30°C overnight. These were centrifuged to remove medium, and the bacterial cells were then suspended in the same volume of MGRLS medium (MGRL medium 8) supplemented with 3% sucrose). The suspensions were diluted with MGRLS medium in Erlenmeyer flasks and incubated at 30°C with rotation (130 rpm).

Feeding of ralfuranones.
ΔralA was grown in MGRLS medium (100 mL) at 30°C with rotation (130 rpm) for 2 days. EtOH solutions of the isolated ralfuranone I (6) or synthetic ralfuranone B (2) were added to the culture, and the bacterial cultures were incubated for a further 2 days. Following growth, the bacterial cultures were extracted three times with an equal volume of EtOAc. The combined EtOAc extracts were dried over Na 2 SO 4 and evaporated to dryness. The residues were dissolved in MeOH (500 μL for experiments with 2.5 μM ralfuranone I (6) and 10 μM B (2), 5 mL for experiments with 200 μM I (6)) and subjected to an HPLC analysis: column, InertSustain C18 (150 × 4.6 mm, 3 μm, GL Sciences); column oven, 40°C; flow rate, 1 mL/min; eluent, MeCN in H 2 O (20-95% linear gradient in 24 min, then 95% MeCN for 6 min); injection volume, 10 μL. As control experiments, ralfuranones I (6) or B (2) were added to MGRLS media and incubated at 30°C with rotation (130 rpm) for 2 days. The analytical samples were prepared in accordance with the method for bacterial samples. Quantification data were shown as the mean ± SD of three independent experiments.
The results of the feeding and conversion experiments suggested a pathway for the biosynthesis of ralfuranones in R. solanacearum, as shown in Scheme 2. Ralfuranone B (2) was identified as the main product of ralfuranone I (6). This conversion may have occurred non-enzymatically through the attack of H 2 O on the exocyclic double bond and subsequent decarboxylation and was also supported by the absolute configuration of the oxymethine at C-10 in ralfuranone B (2) being racemic. 8) Furthermore, Pauly et al. reported that ralfuranone I (6) non-enzymatically reacted with thiols by the Michael reaction, and the products underwent decarboxylation to form various artificial ralfuranones. 10) This reaction manner may essentially be the same as the formation of ralfuranone B (2) from I (6). The conversion of ralfuranone B (2) to A (1) may have spontaneously occurred through a retro-aldol reaction. When ralfuranone B (2) was incubated in MGRLS medium, benzaldehyde accumulated in the medium in accordance with the formation of ralfuranone A (1) (Fig. S2). This result suggests the involvement of a retro-aldol reaction in the conversion of ralfuranone B (2) to A (1). To the best of our knowledge, this is the first time that such a unique reaction has led to the formation of natural furanones. As described previously, 8) ralfuranones J (3) and K (4) were the enzymatic oxidation products of ralfuranone B (2). We expected ralfuranone B (2) to be oxidized to J (3) and then further oxidized to K (4) in OE1-1; however, the reaction order and enzymes involved were not examined in this study. Ralfuranone L (5) was synthesized from ralfuranone I (6) presumably by the attack of hydride on the exocyclic double bond and subsequent decarboxylation. Since it may be hard to form hydride in medium, we expected this conversion to need R. solanacearum cells and an enzyme.
In conclusion, we herein demonstrated that ralfuranone I (6) was a precursor for other ralfuranones in R. solanacearum. By analyzing the conversion of ralfuranones I (6) and B (2), we suggested a biosynthetic pathway for ralfuranones, in which R. solanacearum uniquely synthesized the aryl-type furanones by combining non-enzymatic and enzymatic reactions. In a previous study, ΔralA exhibited reduced virulence in host plants as well as the ablation of ralfuranone production. 8) These findings strongly suggest that ralfuranones play an important role in this wilt disease. We are now investigating the mechanisms by which ralfuranones enhance bacterial activity in order to induce wilt disease in host plants. The present results may indicate that ralfuranone I (6) does not exhibit important biological activity due to its rapid conversion to other ralfuranones. Future studies on these unique metabolites may provide an insight into the ability of R. solanacearum to cause wilting in host plants.

Disclosure statement
No potential conflict of interest was reported by the authors. Scheme 2. Proposed conversion pathway from ralfuranone I (6) to other ralfuranones.

Supplemental materials
The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2015.1116931.