Bacterial strains
P. aeruginosa PAO1 strains were cultured in Luria-Bertani broth supplemented with 50 mM 3-(N-morpholino) propanesulfonic acid at 37°C with 250 rpm shaking overnight. PA1895-1897-knockout mutant was made by homologous recombination using reconstructed plasmid pEXG2 with insertion of PA1895-1897 genes. Plasmid pJN105 was reconstructed by inserting PA1895-1897 genes to overexpress the operon. These plasmids were created by overlapping PCR products, which are assembled in E. coli DH5 as previously described (Kostylev et al. 2015). pJN105.PA1895-1897 was transferred into PA1895-1897-knockout mutant by electroporation. For the selection of plasmid pJN105.PA1895-1897, media were supplemented with gentamicin (100 µg/ml).
Sample preparation for metabolomics
When reaching the OD600nm of 1.8, the cultures of P. aeruginosa wildtype (WT), PA1895-1897-knockout (KO) mutant, and KO mutant with 0.1% arabinose-induced overexpression plasmid pJN105.PA1895-1897 (OE) were centrifuged at 2,000g (5 min, 4°C). The pellets were collected and frozen at -80°C for later metabolomics analysis. The supernatants were also collected for 3OC12-HSL, C4-HSL and pyocyanin measurements.
Metabolomics analysis
Frozen pellets were ground and the powder was extracted with 70% aqueous methanol solution. The extracts were filtered for high-performance liquid chromatography (HPLC) analysis coupled with a mass spectrometer (Applied Biosystems, Foster City, CA, USA). The accurate precursor spectral ion and product spectral ion values were compared with the standard compounds, and the structure was analysed by referencing to existing databases such as MassBank and METLIN. We used pathway-associated metabolite sets to analyse pathway enrichment. KEGG pathways were further analysed with OmicsBean (GFK Inc., Shanghai, China).
3OC12-HSL, C4-HSL and pyocyanin measurements
Acyl-homoserine lactones were extracted using ethyl acetate, and 3OC12-HSL and C4-HSL concentrations were measured using reporter strains as previously described (Ding et al. 2018; Ding et al. 2022). Pyocyanin was extracted in hydrochloric acid-water according to reported methods (Kurachi 1958). At 520 nm, the absorbance was measured and multiplied by 17.072 to get the concentration of pyocyanin.
Exogenous fatty acids treatment
PA1895-1897-knockout mutant and wildtype PAO1 were grown, respectively, in 100 mL Luria-Bertani broth medium supplemented with palmitoleic acid (0.01 mg/ml, 0.05 mg/ml, and 0.5 mg/ml) or acetic acid (0.001%, 0.005%, and 0.025% v/v) at 37°C with 250 rpm shaking. At the OD600nm of 0.5, 1.0, 1.5, and 2.0, a 2 ml-aliquot of cultures were collected for 3OC12-HSL, C4-HSL, and pyocyanin measurements.
RNA isolation and qRT-PCR
When reaching the indicated OD600nm, cells were pelleted and preserved in RNA Protect Bacteria reagent before being stored at -80°C. The total RNA was extracted using RNeasy kit and then purified using the RNeasy MinElute cleanup kit (Qiagen, Hilden, Germany). cDNA was prepared using the iScript cDNA Synthesis kit, and the expression of target genes was analysed by following the protocol for the iQ SYBR Green SuperMix (Bio-Rad Laboratories, Hercules, CA, USA) on a ViiA 7 Real-Time PCR System (Applied Biosystems, CA, USA). The rplU was used as a reference gene, and primers are listed in Table S1. Three biological replicates were done.
Detection of palmitoleic acid and acetic acid
For palmitoleic acid detection, culture mixed with chloroform methanol (2:1 v/v) underwent ultrasonication of 30 min. Fatty-acid methyl esterification was achieved by 30 min of 80℃ water bath and later mixed with n-hexane. The supernatant was subjected to an Agilent Model 7890A/5975C gas chromatography-mass spectrometry (GC-MS) system. Palmitoleic acid quantification was performed by making a calibration curve using Supelco 37-component FAME mix (Sigma-Aldrich). For acetic acid detection, 50% H2SO4 was added to the culture for acidification, and an Agilent 7890B gas chromatograph system coupled with an Agilent 5977B mass spectrometer was used for GC-MS analysis. The standard curve of acetic acid concentration was calculated using the Agilent chemistry analysers.
Statistical analysis
Data were analysed by GraphPad Prism (version 9; GraphPad Software, San Diego, CA, USA), and graphs were drawn accordingly. For normally distributed variables, t-tests or variance was used for data analysis; while for non-normally distributed variables, Mann-Whitney U test was used. P < 0.05 was considered statistically significant.