Bacterial strains, plasmids, and genetic constructs

A complete list of strains, constructs, and oligonucleotides used in the experiments is listed in Suppl. Table. 2. All P. aeruginosa strains are based on the wild-type strain PAO1. Expression vectors and mutator plasmids were transformed into E. coli DH5α for cloning, and E. coli SM10 λ pir+ for conjugation, respectively. Endogenous fusion proteins used in this study were stably introduced by allelic exchange of the wild-type gene in the P. aeruginosa PAO1 genome by two-step homologous recombination for native expression of the proteins of interest95. Similarly, strains with gene deletions were generated by homologous recombination and verified by Sanger Sequencing. The ∆tse6tsi6 deletion strain was labeled fluorescently by expressing sYFP2 from a Tn7 transposon as described by Schlechter and colleagues96. For ectopic protein expression in P. aeruginosa, the corresponding genes were cloned into pJN105/pUCP20/pMMB67EH-based expression plasmids. These plasmid constructs were sequence-verified (Microsynth Seqlab) before being introduced into the respective bacterial strain via heat-shock transformation97. Primers used for constructing the plasmids of this study are listed in Suppl. Table 3.

Bacterial cultivation

P. aeruginosa strains used in this study were routinely grown in Luria-Bertani (LB) medium at 37 °C with shaking at 180 rpm. Overnight cultures were inoculated in triplicates if required. For strains carrying pJN105-based plasmids, Gentamicin (Gm, 40 µg/ml) was also added to ensure plasmid stability. For strains carrying pUCP20 or pMMB67EH-based plasmids, ampicillin (Amp, 300 µg/ml) was also added to ensure plasmid stability. To prepare day cultures, overnight cultures at the stationary phase were inoculated into fresh LB medium with antibiotics (if required) to an OD 600 of 0.1. To induce ectopic gene expression from the pJN105 plasmid, L-arabinose was also added at a final concentration of 0.2% (w/v). To provide non-secreting or secreting conditions for the T3SS, day cultures were cultivated in non-secreting or secreting medium, LB supplemented with 20 mM MgCl 2 , 200 mM NaCl, 0.4% (w/v) glycerol and either 5 mM CaCl 2 (to inhibit T3SS secretion) or 5 mM EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, to induce T3SS secretion), respectively.

Fluorescence microscopy

For fluorescence microscopy, day cultures were cultivated according to the specific experimental conditions and collected after 3 h of growth. Upon completion of the experimental incubation, 600 µl of culture was collected by centrifugation (3 min at 2400 g, room temperature (RT)) and washed once with minimal medium. Samples were then resuspended in 100 µl of minimal medium (100 mM 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethane-1-sulfonic acid (HEPES) (pH 7.2), 5 mM (NH 4 ) 2 SO 4 , 100 mM NaCl, 20 mM sodium glutamate, 10 mM MgCl 2 , 5 mM K 2 SO 4 , 50 mM glycine and 0.2% casamino acids (w/v)). To establish T3SS non-secreting/secreting conditions, 5 mM CaCl 2 or 5 mM EGTA was added, respectively. 0.2% (w/v) L-arabinose was added to maintain ectopic gene expression, where required. From this bacterial suspension, 2 µl were spotted onto an agarose pad (1.5% low-melting agarose (Sigma-Aldrich) in minimal medium with required supplements as discussed above) on glass depression slides (Marienfeld), topped with a coverslip (ThermoFisher Scientific). Imaging was conducted using a Deltavision Elite Optical Sectioning Microscope equipped with a UPlanSApo 100×/1.40 oil objective (Olympus) and an EDGE sCMOS_5.5 camera (Photometrics). The GFP/YFP signal was captured using a GFP filter set (excitation: 480/25 nm, emission: 535/28 nm) with an exposure time of 0.3 s, and the mCherry signal was visualized using a mCherry filter set (excitation: 575/25 nm, emission: 625/45 nm) with 0.6 s exposure time. Z-stacks were acquired with 8 slices (∆z = 0.15 µm) per fluorescence channel. The corresponding images with differential interference contrast channel (DIC) were processed using FIJI (ImageJ 2.1.0). Selected fields of view were identically adjusted for brightness and contrast across compared image sets, and the cell counter macro was used for cells and foci counting. Counting was repeated once for each selected microscopy field to ensure data reliability. For intensity measurements, the background of each selected microscopy field was subtracted individually. The statistical analysis and graph generation were done in GraphPad 10.0 (Dotmatics). For the statistical analysis of the co-existence of two virulence factors, a 2*2 contingency table was used, based on the statistical analysis built into a χ2 test.

Statistics and reproducibility

All numerical data values and p-values are provided in Suppl. Data 1. The number of biological replicates and details on statistical analyses are provided in the respective figure legends. Error bars represent standard deviations, unless specifically mentioned. Calculations of confidence intervals are based on the t-distribution for smaller sample sizes (n 75%) were used in the data analysis. To correct for the minor donor photobleaching during steps (b) and (d), we performed linear fitting (RStudio) of the donor fluorescence signal versus time for both pre- and post-acceptor photobleaching 25-frame curves. A final statistical analysis and graph generation were done via GraphPad 10.0.

c-di-GMP determination via LC-MS/MS

The respective overnight cultures were inoculated in LB medium supplemented with Gentamicin and L-arabinose for c-di-GMP enzyme expression, as described above. The cultures were grown at 37 °C for 3 h and then measured for 0D600. 1 ml of each culture was added into 2 ml Eppendorf tubes pre-filled with 1 ml cold quenching buffer (−80 °C, 70% (v/v) methanol), and mixed well gently. Quenched samples were centrifuged (15 min at 13,000 × g, −10 °C) and the supernatant was removed carefully. Extraction buffer (0.5 mM EDTA, 5 mM Trizma base, 50% (v/v) methanol, pH 7.0) and chloroform were added at equal volumes (300 µl/1 ODu cells) to each sample, and vortexed until no pellets were visible. Samples were then placed in the shaker (Eppendorf Thermomixer) for 2 h (0 °C, 600 rpm). Afterward, samples were centrifuged (15 min at 21,000 × g, −10 °C) for phase separation. 200 µl of the top-layer supernatant of each sample was filter-transferred (0.2 µm PTFE, Phenomenex) to a new Eppendorf tube. 20 µl of the filtered sample was loaded into 2 ml vial (VWR Avantor) and stored at −20 °C until further analysis. Semi-quantitative determination of cyclic di-GMP was performed using an LC-MS/MS. The chromatographic separation was performed on an Agilent Infinity II 1290 HPLC system using a SeQuant ZIC-pHILIC column (150 × 2.1 mm, 5 μm particle size, peek coated, Merck) connected to a guard column of similar specificity (20 × 2.1 mm, 5 μm particle size, Phenomenex) a constant flow rate of 0.1 ml/min with mobile phase A comprised of 10 mM ammonium acetate in water, pH 9, supplemented with medronic acid to a final concentration of 5 μM and mobile phase B comprised of 10 mM ammonium acetate in 90:10 acetonitrile to water, pH 9, supplemented with medronic acid to a final concentration of 5 μM at 40 °C. The injection volume was 5 µl. The mobile phase profile consisted of the following steps and linear gradients: 0–1 min constant at 75% B; 1–6 min from 75 to 40% B; 6 to 9 min constant at 40% B; 9–9.1 min from 40 to 75% B; 9.1 to 20 min constant at 75% B. An Agilent 6495 ion funnel mass spectrometer was used in negative ionization mode with an electrospray ionization source and the following conditions: ESI spray voltage 3000 V, nozzle voltage 1000 V, sheath gas 400 °C at 11 l/min, nebulizer pressure 20 psig and drying gas 100 °C at 11 l/min. The compound was identified based on its mass transition and retention time compared to a chemically pure standard. Chromatograms were integrated using MassHunter software (Agilent, Santa Clara, CA, USA). Metabolite abundance was determined based on peak areas. Mass transitions, collision energies, Cell accelerator voltages, and Dwell times have been optimized using a chemically pure standard. The parameter settings are listed in Suppl. Table 4.

Biofilm staining

Biofilm formation was examined during growth in a 96-well plate, using crystal-violet staining as described previously105. In brief, overnight cultures were diluted to OD 600 of 0.05 into fresh LB medium or secreting medium, supplemented with Gentamicin and L-arabinose if required. Diluted samples were added to the 96-well microplate (100 µl/strain/per well). The wells at the perimeter were filled with sterile water, and the lid was sealed to prevent evaporation. Incubation was carried out at 37 °C for 24-h with shaking at 180 rpm. After the incubation, the supernatant was carefully removed, and the microplate was rinsed twice in distilled water to further remove the debris. The biofilm biomass attached to each well surface was stained by 150 µl crystal violet (0.1% (w/v)) staining for 15 min. Excess crystal violet was carefully rinsed off with distilled water twice, and then the microplate was left at room temperature for drying overnight before taking digital photographs. After the image recording, 150 µl of 30% acetic acid was added per well with shaking for 15 min to dissolve the crystal violet. 100 µl of the dissolved solution per well was transferred to a new microplate and sent to the plate reader for quantification. Buffer-only wells were included for background subtraction. Crystal violet was read at absorbance 550 nm. Data processing and graph generation were done using GraphPad 10.0.

Flagellum swimming motility assay

Flagellum swimming motility was examined as previously published106. Overnight cultures were inoculated into day cultures (non-secreting or secreting medium) and incubated at 37 °C for 3 h. After that, respective cultures were toothpick-inoculated into swimming plates (secreting and non-secreting medium supplied with 0.3% (w/v) agar) and incubated at 37 °C for 24 h. Digital photographs were taken, and the diameters of the swim zones were measured in millimeters.

Western blotting

For protein expression and stability tests, bacteria were cultured according to the experimental conditions described above. 2 ml of culture was collected and centrifuged (3 min at 2400 × g, RT) to remove the supernatant. The cell pellets were resuspended in 1% (w/v) SDS buffer with the volume calculated to accommodate the SDS–PAGE loading concentration at 0.3 ODu/15 µl. Resuspended samples were boiled for 10 min at 99 °C, and then sonicated for 30 seconds. The processed samples were loaded identically into 2 SDS–PAGE gels with 15 µl/well. Proteins were separated on 15% (w/v) SDS–PAGE gels with Precision Plus All Blue Prestained (BioRad) as a size standard. One SDS–PAGE gel was stained with FastGene-Q-stain (NipponGenetics) for visualization, and the counterpart was transferred onto a nitrocellulose membrane with BioRad semi-dry transfer system (1.3 A; 25 V; 20 min) for immunoblotting. Membranes were blocked with non-fat milk (5% (w/v) in PBS) and rinsed with PBS-T (0.2% (v/v) Triton X-100 in PBS) for subsequent antibody attachment. Primary rabbit antibodies against mCherry (Biovision 5993, 1:2000) were used in combination with secondary anti-rabbit antibodies conjugated to horseradish peroxidase (HRP) (Sigma, A8275, 1:5000). Primary mouse antibodies against GFP (Proteintech 66002-1, 1:1000) were used in combination with secondary anti-mouse antibodies conjugated to horseradish peroxidase (HRP) (GE Healthcare NXA931, 1:5,000). For the detection of chemiluminescence signals, ECL chemiluminescence substrate (Millipore, WBLUF0500) was used in a LAS-4000 Luminescence imager (GE Healthcare).