Strains and culture conditions

P. aeruginosa strain PAO1 (obtained from the Manoil Lab at the University of Washington, Seattle, WA, USA) and its isogenic mutant ΔDS (diol synthase deletion mutant) were used throughout the entire study. Deletion of the diol synthase operon was performed by allelic exchange using the suicide vector pEX100Tlink and a previously described method34 Briefly, a fragment of P. aeruginosa chromosome containing the genes PA2078 and PA2077 plus ∼300 bp of chromosomal flanking regions were amplified by PCR using the primers JCG1 (5′-ggcgaaagcttcgccttcctgccg-3′) and JCG2 (5′-ggcgggaattctggtcaccaccttct-3′). These primers introduced sites EcoRI and HindIII at the ends of the amplified fragment that were used for cloning into the suicide vector pEX100Tlink to obtain the plasmid pEX-PA77-78. From this plasmid a SmaI-StuI fragment internal to the diol synthase operon was deleted to obtain the final construction pEXΔDS. This plasmid was used to make an in-frame deletion of PA2077-78, through allelic exchange in the chromosome of PAO1 (Supplementary Fig. 1A). The diol-synthase-activity-lacking strain obtained was named ΔDS. The mutant genotype was confirmed by PCR and sequencing, and its oxylipin-negative phenotype was confirmed by TLC (see below). Complementation of the mutant was performed by replacing the mutated allele with the original copy from the parental strain PAO1, also through allelic exchange. Green and red fluorescent P. aeruginosa were obtained by transformation with the plasmids pMF230 or pMF440, which constitutively express the GFP mut2 and mCherry fluorescent proteins, respectively. Plasmids pMF230 and pMF440 (Addgene plasmids # 62546 and 62550) were a gift from Michael Franklin (Montana State University). Other P. aeruginosa strains used were the transposon mutants of genes encoding exotoxin A (exoA, strain PW3079), phospholipase C (plcN, PW6586), exoenzyme S (exoS, PW7479), quorum sensing transcriptional regulator (lasR, PW3597), type 4 fimbrial precursor (pilA, PW8622) and the flagellar motor switch protein (fliM, PW3621), which were acquired from the transposon library collection of University of Washington. Escherichia coli DH5α (Invitrogen) was the host for plasmid constructions and E. coli S17-1 λpir (a gift from Jorge Benitez—Morehouse School of Medicine) was used as a donor strain for bacterial conjugation when required.

The strains were routinely grown in lysogeny broth (LB) medium at 30 °C, to which agar was added when solid medium was required. LB agar without NaCl plus 15% sucrose was used to segregate suicide plasmid from merodiploids during construction of ΔDS by allelic exchange. Biofilm formation was performed in M63 media supplemented with 2% glucose, 5% casaminoacid and MgSO 4 1 mM (M63 complete). Antibiotics were added, when necessary, at the following concentrations: Ampicillin (Amp), 100 μg ml−1; Carbenicillin (Cb), 300 μg ml−1. Oleic acid 90% (Sigma 364525) was added to cultures for oxylipin production and purification. M63 complete media was supplemented with oleic acid 99% (Sigma O1008) or purified oxylipins when required for the study of biofilm formation over polystyrene surface of microtiter plates or over biotic surfaces.

Thin layer chromatography

TLCs were carried out on silica gel (60 Å) plates of 20 × 10 cm, 200 μm thickness (Whatman). A mix of hexane, ether and acetic acid (80/20/5) was used as mobile phase. TLC plates were revealed with 10% phosphomolybdic acid in ethanol.

Purification of 10-HOME and 7,10-DiHOME oxylipins

The supernatant of a 500 ml PAO1 culture grown in M63 complete supplemented with 1% oleic acid was used to purify oxylipins derived from the diol synthase activity. The culture was centrifuged at 8000g for 15 min, then the supernatant was recovered and acidified (pH=2) with hydrochloric acid. Then a one vol/vol organic extraction was performed using ethyl acetate. The organic phase was evaporated and the dried mixture obtained was dissolved in 3 ml of ethyl acetate and used for purification of the oxylipins on an Isco Teledyne Combiflash Rf 200 with four channels with 340CF ELSD (evaporative light scattering detector). Universal RediSep solid sample loading pre-packed cartridges (5.0 g silica) were used to absorb crude product and purified on 24 g silica RediSep Rf Gold Silica (20–40 μm spherical silica) columns using an increasing gradient of ethyl acetate (solvent B) over hexane (solvent A) (Supplementary Fig. 3A). Fractions collected for each detected peak were combined and evaporated, then dissolved in methanol. The purity of the oxylipins was checked by HPLC/MS analysis.

HPLC/MS analysis

Purified 7,10 Di-HOME and 10-HOME were prepared at 1 mg ml−1 in methanol (stock solution), from which samples to be analysed were prepared by diluting in ddH 2 O with 0.1% formic acid. For each sample, a 20 μl injection was loaded onto a Synergi Hydro-RP 80A 250 × 2 mm C18 column (Phenomenex) using a Shimadzu Prominence System Binary Pump (Shimadzu Scientific Instruments, Inc., Columbia, MD, USA) at a flow rate of 350 μl min−1 using ddH 2 O with 0.1% formic acid and acetonitrile with 0.1% formic acid for mobile phase A/B respectively. The gradient proceeded from 10 to 80%B over 11 min, then to 100%B at 14 min, then re-equilibrated back at initial conditions for 6 min for a total of 20 min per evaluation using the SCIEX 4000 Triple Quadrupole Mass Spectrometer (Concord, Ontario, Canada) in the ESI negative ion mode. Nitrogen was used as a nebulizer and curtain gas (CUR=20). The collision gas, collision energy and temperature were set at 10 °C (−30 eV for 10-HOME, −34 eV for 7,10-DiHOME) and 600 °C, respectively. GS1 and GS2 gases were set at 40 °C and 60 °C respectively. Analyst 1.6.2 software controlled the LC-MS/MS system.

Motility assays

Swimming, swarming and twitching motilities were studied using the methods described by Rashid and Kornberg35. To capture videos of twitching motility, 100 μl aliquots of the twitching medium35 were deposited over microscope cover slips. Once the medium solidified, it was punctured in the centre up to the bottom with an extended inoculation loop embedded with a bacterial suspension (OD 600 =1) of the strain to be filmed. The cover slips were incubated for 6 h at 37 °C, then placed inverted on the microscope stage (Olympus BX53 system microscope) and the twitching motility filmed using an XM10 incorporated camera (Olympus) controlled by the software cellSens Standard 1.6 (Olympus).

Quantification of biofilm formation

Biofilm assays were performed following the O’Toole protocol36. Briefly, P. aeruginosa strains were cultured overnight in LB agar plates at 37 °C. Bacterial suspensions were prepared in M63 medium to an OD 600 =1. Ten microliters of bacterial suspension was inoculated into each well of a 96-well microtiter plate containing 200 μl of M63 complete media. Oleic acid or pure oxylipins were added to the medium at desired concentrations when required. Biofilms were allowed to form at 30 °C overnight. For biofilm quantification the wells were washed two times with 1 × PBS and 200 μl of 0.1% crystal violet was added to the wells and incubated for 10 min. Wells were washed three times with 1 × PBS, then crystal violet-stained biofilm was solubilized with 250 μl of 30% acetic acid and the absorbance was measured at 550 nm.

Bacterial attachment to microtiter plates

To study bacterial attachment and microcolony formation similar conditions to the previous section experiment were used, but using 105 bacteria per well constitutively expressing GFP from the plasmid pMF230. Subsequently, the plates were incubated for 3 h at 37 °C, observed under the fluorescence microscope and pictures were taken.

Detection of eDNA and EPS in the extracellular matrix

Microcolonies induced by the addition of 10-HOME or 7,10-DiHOME, obtained as described in the previous section, were stained with 4′,6-diamidino-2-phenylindole and concanavalin A-FITC conjugate at 0.5 μg ml−1 and 50 μg ml−1, respectively. In this case P. aeruginosa constitutively expressing mCherry RFP from the plasmid pMF440, was used to distinguish green fluorescence of the FITC conjugate. After 30 min of incubation with the fluorescent compounds the wells were washed with PBS 1 × and observed under the fluorescence microscope.

Biofilm assay over A549 monolayers

A549 human alveolar epithelial cells (ATCC CCL-185) were plated into black clear bottom 96-well-tissue culture plates at 20,000 cells per well in Dulbecco's modified Eagle medium supplemented with bovine fetal serum and glutamine and allowed to grow up to confluence. Cells were washed with PBS 1 × and 100 μl of M63 complete medium—with or without oxylipins as required—were added to each well. GFP-expressing P. aeruginosa was added at 20 bacteria per A549 cell and incubated for 3 h. Plates were washed two times with PBS 1 × and the cell monolayers with attached green fluorescent bacteria were observed and pictures captured with a fluorescence microscope (EVOS FL Cell Imaging System, Life Technologies) using the same settings for each picture. For presentation of figures all relevant pictures were assembled as panels into a single file using Adobe Photoshop. All comparable pictures were subjected to the same transformations during the process (resizing, resolution change), which is specified in each pertaining figure legend. This also applies to pictures of the next section (crop imaging).

P. aeruginosa imaging inside Drosophila crops

All experiments involving D. melanogaster were done using 3-day-old flies from both sexes of the WT Oregon R (acquired from Carolina Company). P. aeruginosa colonization of D. melanogaster crop was performed as previously described by Mulcahy et al.20 P. aeruginosa strains constitutively expressing GFP were cultured on LB agar plates. Bacteria were resuspended in LB to OD600=1. Then 100 μl of the suspension was spotted onto a sterile filter (Whatman) that was placed on the surface of 5 ml of LB agar containing 5% sucrose. The medium was supplemented with 10-HOME or 7,10-DiHOME when required. Flies were allowed to grow under this condition for 20 h and then killed. Crops were placed on a drop of PBS on a microscope slide, sealed with a coverslip and observed using an EVOS FL Cell Imaging System. Pictures were captured using the same settings for each picture.

Virulence assay in orally inoculated Drosophila

Drosophila flies were inoculated with the tested strains as described in the previous section, but instead of killing the flies they were incubated at room temperature (∼25 °C) and fly survival was followed and recorded for 14 days.

Virulence assay in Drosophila inoculated by pricking

Flies were anaesthetized with FlyNap (Carolina) and pricked in the thorax using a needle (BD Ultra-Fine Nano Pen Needles 32 g 5/32 inch) that was loaded with the tested strain of P. aeruginosa by dipping in a bacterial suspension at OD 600 =1.0 in M63 medium. After inoculation, the flies were placed into a vial (15 flies per vial) containing Formula 4-24 Instant Drosophila Medium (Carolina). Flies were maintained at room temperature and survival was monitored and recorded up to 36 h post inoculation.

Detection of oxylipins in P. aeruginosa infected flies

Groups of 20 infected flies were homogenized using an Omni THQ homogenizer with disposable Omni Tips plastic generator probes (OMNI international) in 2 ml of PBS 1 × . The homogenates were centrifuged to eliminate fly and bacterial debris and total fatty acids were extracted as described above (see section Purification of diol synthase-derived oxylipins). Extracted samples were analysed first by TLC and then by HPLC/MS (see above sections for TLC and HPLC/MS analyses) to identify the presence of 10-HOME and 7,10-DiHOME. To determine the presence of the oxylipins, samples were analysed by the MRM method using mass transitions m/z 297.3/155.1 for 10-HOME and 313.3/141.1 for 7,10-DiHOME.

Lettuce infection

Individual lettuce leaves taken from the external foliage of romaine hearts (Lactuca sativa L. var. longifolia) were inoculated at 2–3 cm apart intervals into the central nervure of each leaf with the P. aeruginosa strain to be tested (3 μl of a bacterial suspension at an OD 600 =3) or the inoculation vehicle alone (M63 medium) using a 25 5/8 G needle connected to a pipette. When required 10-HOME or 7,10-DiHOME, or both were added to the inocula or the control vehicle at a concentration of 0.5 mg ml−1). Inoculated leaves were placed in plastic beakers with the inferior part of the central nervure submerged in a solution of 10 mM MgSO 4 in water. The leaves were incubated for 5 days at room temperature. In the case of the leaves coinoculated with bacteria and oxylipins, 3 μl of each oxylipins at 0.5 mg ml−1 were deposited over the inoculation sites once a day up to day third. The vehicle alone or with oxylipins at the same concentrations used for bacterial inoculation were used as negative controls. At the end of the experiment the leaves were evaluated for necrosis around the inoculation points. A dark brownish necrotized area of more than 5 mm of diameter was recorded as a successful infective event.

Statistical analysis

Kaplan-Meier plots of Drosophila flies survival experiments were compared using the log-rank (Mantel-Cox) test. We used 15 flies per condition to be able to detect an effect size proportion of surviving subjects=0.5 with 99% power using a type I error of 0.01 (calculated using GraphPad StatMate 2). For randomization of Drosophila the flies were anesthetized and distributed to the final experimental container disregarding sex or size. In the case of the lettuce leaves, we were careful to take only external leaves of romaine hearts (up to 2–3 layer of leaves were taken per lettuce plant). Leaves coming from different plants were mixed and randomized, then the leaves were taken and distributed to the final containers. One lab member distributed and inoculated the flies or lettuce leaves and assigned letter codes to each container. Then other lab member counted dead flies and lettuce lesions. At the end of the experiments letter codes were reconciled with the respective experimental condition used. In the rest of the experiments one-way ANOVA or t-test (two-sided) were used as needed to calculate significant differences between means of different conditions after appreciating the variance was similar between groups being compared. All statistical analyses were performed using GraphPad Prism 7 software.

Data availability

The authors declare that the data supporting the findings of this study are available within the article and its supplementary information files, or from the corresponding author on request.