The bacterial strains and plasmids used in this study are listed in Table S1. Escherichia coli DH5α strain was used for standard DNA manipulations (Bertani, 1951). LB medium was used to cultivate E. coli strains. Batch cultivation of Pseudomonas aeruginosa strains was carried out at 37°C in ABT minimal medium (Clark, 1968) with 5 g L⁻¹ glucose (ABTG) or 2 g L⁻¹ glucose and 2 g L⁻¹ casamino acids (ABTGC). To maintain plasmids in E. coli, 2 ml LB was supplemented with 100 μg ampicillin (Ap) mL⁻¹, 15 μg mL⁻¹ gentamicin (Gm), 15 μg mL⁻¹ tetracycline (Tc), or 8 μg mL⁻¹ chloramphenicol (Cm). In P. aeruginosa, 30 μg mL⁻¹ Gm, 50 μg mL⁻¹ Tc, and 200 μg carbenicillin mL⁻¹ (Cb) were used for marker selection.

Bacterial cells in 5 ml ABTGC were harvested and pelleted by centrifugation at 13,000 g for 3 min. The supernatant was removed and the cell pellet was immediately snap-frozen in liquid nitrogen. The cell pellet was re-suspended in 1 ml of acetonitrile/methanol/water (40:40:20) mixture. An aliquot of cells (10 μl) was used for protein quantification. The cells were then lysed with a probe tip ultrasonicator (Amplitude 30%; 5 s ON, 5 s OFF) for 1 min in ice slurry. The cell debris was removed by centrifugation at 13000 g for 3 min. The liquid phase was then evaporated by using the vacuum concentrator, leaving behind the nucleotide precipitate. The samples were then re-suspended in 100 μl ddH₂O and centrifuged at 10,000 g, 4°C for 10 min. The solutions were transferred to glass vials and injected through liquid chromatography- mass spectrometry (LCMS).

For the LC portion, the samples in the glass vials were run through the BEH C18 (1.7 μm; 2.1 × 50 mm) column with injection volume of 5 μl at 0.3 ml min⁻¹ for a total runtime of 6 min, with the mobile phase A as 10 mM ammonium formate in water + 0.1% formic acid and mobile phase B as methanol + 0.1% formic acid. For the MS portion, the samples were then analyzed by Xevo TQ-S, Waters mass spectrometer, under the ESI positive ion mode (capillary voltage: 3.8 kV, desolvation temperature: 400°C). The cyclic di-GMP compound was detected by monitoring ion transition of 691.2 m/z to 152.0 m/z at collision energy 36 eV.

For protein quantification, the cell aliquot was treated in 1 ml of 5 M NaOH at 95°C for 5 min. After cooling the samples for 15 mins, the proteins were processed with the Qubit® protein assay kit (NanoOrange dye) and quantified by the Qubit® 2.0 Fluorometer (Invitrogen). The concentration of c-di-GMP was then normalized with protein quantity. Experiments were performed in triplicate, and results were shown as the mean ± s.d.

Bacterial cells in 5 ml ABTGC were harvested and pelleted by centrifugation at 13,000 g for 3 min. The supernatant was removed and the cell pellet was resuspended in 5 ml 0.9% NaCl. The cells were lysed with a probe tip ultrasonicator (Amplitude 30%; 5 s ON, 5 s OFF) for 3 min in ice slurry to obtain a crude extract. As previously described (Kuchma et al., 2007), the crude extracts were incubated with 5 ml of 5 mM bis(p-nitrophenyl) phosphate (bis-pNPP) in buffer (5 mM MgCl₂, 50 mM Tris-HCl [pH 9.3], 50 mM NaCl). The release of p-nitrophenol was quantified by using a microplate reader (Tecan Infinite Series 2000) at OD410 every 15 min for 16 h.

As described in the previous section, protein concentration was determined by the Qubit® 2.0 Fluorometer (Invitrogen).The PDE activity was then normalized with protein quantity. Experiments were performed in triplicate, and results were shown as the mean ± s.d.

PAO1, PAO1ΔwspF and PAO1/p_lac-yhjH were grown in 1 ml ABTGC in each well (triplicates) within a 24-well microplate (Nunc) for 7 h till late logarithmic phase in 37°C, 200 rpm shaking. Bacterial cells were first treated with RNA Protect (Qiagen, Netherlands) and then treated with lysozyme. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Netherlands). On-column DNase digestion with the RNase-free DNase Set (Qiagen) was used to remove DNA. The DNA contamination levels were assessed by using the Qubit®dsDNA High Sensitivity (HS) assay (PicoGreen dye) and the Qubit® 2.0 Fluorometer (Invitrogen). The integrity of total RNA was assessed by using the Bioanalyser RNA analysis kit (Agilent Technologies) and the Agilent 2100 Bioanalyzer (Agilent Technologies). The Ribo-Zero™ Magnetic Kit (Bacteria) (Epicentre) was used to deplete 16S, 23S, and 5S rRNAs from the samples.

Gene expression analysis of 2 biological replicates was conducted by RNA-Seq technology (Illumina). The RNA was fragmented to 200–300 bp fragments using divalent cations under elevated temperature.

First and second strand cDNA were then synthesized and treated by end repair and adapter ligation. After the 12-cycle PCR enrichment, the quality of the libraries was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies). The libraries were sequenced using the Illumina HiSeq2000 platform with paired-end protocol and read lengths of 100 nt.

The sequence reads were assembled and analyzed by “RNA-Seq and expression analysis” application of CLC genomics Workbench 6.0 (CLC Bio, Aarhus, Denmark). The PAO1 genome (http://www.ncbi.nlm.nih.gov/nuccore/110645304) was utilized as the reference genome. The following criteria were used to filter the unique sequence reads: maximum number of hits for a read of 1, minimum length fraction of 0.9, minimum similarity fraction of 0.8, and maximum number of two mismatches. Genes were annotated with Pseudomonas Genome Database (Winsor et al., 2011). The mapping results of RNA-Seq raw data from CLC genomics Workbench 6.0 were subjected to DESeq2 package for statistical analysis (Anders and Huber, 2010) by reading them into R/Bioconductor (Gentleman et al., 2004). The transcript counts were normalized to the effective library size. Hierarchical clustering analysis was performed and a heatmap was drawn for the 1000 most highly expressed genes of PAO1, PAO1ΔwspF, and PAO1/p_lac-yhjH using heatmap.2 package of R/Bioconductor (Gentleman et al., 2004). Furthermore, the normalized counts were stabilized according their variance as outlined in the DESeq2 package tutorial and a principle component analysis (PCA) plot was generated. The differentially expressed genes among PAO1, PAO1ΔwspF, and PAO1/ p_lac-yhjH were identified by performing a negative binomial test using the DESeq2 package of R/Bioconductor. Transcripts were stringently determined as differentially expressed when having a fold change larger than 5 and an adjusted p-value smaller than 0.05.

Accession number for the RNA-seq is PRJNA381683.

Total RNA from cells grown in 2 ml ABTGC was extracted using RNeasy Mini Kit (Qiagen) with on- column DNase digestion. The concentration and purity of the extracted RNA were measured by NanoDrop 2000 spectrophotometer (Thermo Scientific), while the integrity of RNA was analyzed by Agilent 2200 TapeStation System (Agilent Technologies). The elimination of contaminating DNA was confirmed via real time PCR amplification of the rpoD gene with total RNA as template.

First-strand cDNA was first synthesized from total RNA with the SuperScript® III First-Strand Synthesis SuperMix kit (Invitrogen). The cDNA was used as template for qRT-PCR with a kit of SYBR® Select Master Mix (Applied Biosystems, Life Technologies) on the StepOnePlus Real-Time PCR System (Applied Biosystems, Life Technologies). The gene rpoD was used as endogenous control. To verify specific single-product amplification, melting curves were analyzed.

Supernatants (2 ml) from P. aeruginosa strains grown in ABTGC in 37°C overnight were filtered through 0.2-μm filters, and the filtrates were collected. Overnight culture of the reporter strain ΔlasIΔrhlI/p_rhlA-gfp was adjusted to OD₆₀₀ = 0.2 using ABTGC medium. 100 μl of filtrate was added to 100 μl of ΔlasIΔrhlI/p_rhlA-gfp in a 96-well plate (Nunc, Denmark). Because ΔlasIΔrhlI does not produce BHL, p_rhlA-gfp was induced by the addition of serial diluted filtrates containing BHL. GFP fluorescence from p_rhlA-gfp expression (expressed in relative fluorescence units, RFU) was measured for each well using a microplate reader (Tecan Infinite 2000) and was normalized to the OD₆₀₀ of each well. Experiments were performed in triplicate, and results are shown as the mean ± s.d.

Supernatants (2 ml) from P. aeruginosa strains grown in ABTGC in 37°C overnight filtered through 0.2-μm filters and the filtrates were collected. Overnight culture of the reporter strain ΔpqsA/p_pqsA-gfp was adjusted to OD₆₀₀ = 0.2 using ABTGC medium. 100 μl of filtrate was added to 100 μl of ΔpqsA/ p_pqsA-gfp in a 96-well plate (Nunc, Denmark). Because ΔpqsA does not produce PQS, p_pqsA-gfp was induced by the addition of serial diluted filtrates containing PQS. GFP fluorescence from p_pqsA-gfp expression (expressed in relative fluorescence units, RFU) was measured for each well using a microplate reader (Tecan Infinite 2000) and was normalized to the OD₆₀₀ of each well. Experiments were performed in triplicate, and results are shown as the mean ± s.d.

Relative amounts of rhamnolipids produced by P. aeruginosa strains were quantified as previously described (Wittgens et al., 2011; Fong et al., 2016). The supernatant of strain grown in 2 ml ABTGC in 37°C overnight was filtered with 0.2-μm filter and treated with equal volumes of ethyl acetate. Samples were then mixed with vortexing for 30 s, with a phase separation by putting samples briefly in centrifuge for 30 s at 5,000 g. The upper organic phase containing rhamnolipids were transferred to new tube. The organic solvent was then removed by evaporation with a vacuum concentrator. The residue containing rhamnolipids was then re-suspended in 100 μl ddH₂O. 100 μl of sample was mixed with 100 μl of 1.6% orcinol in ddH₂O and 800 μl sulphuric acid (60% v/v). The samples were then incubated at 80°C for 30 mins and mixed periodically. After cooling the samples to room temperature for 15 mins, OD₄₂₀ was measured by the microplate reader (Tecan Infinite 2000). Experiments were performed in triplicates and results were shown as mean ± standard deviation.

Relative levels of pyocyanin produced by P. aeruginosa strains were quantified as previously described (Frank and Demoss, 1959; Fong et al., 2016). The supernatant of strain grown in 2 ml ABTGC in 37°C overnight was filtered with 0.2-μm filter and treated with equal volumes of chloroform. Samples were then mixed with vortexing for 30 s, with a phase separation by putting samples in centrifuge for 30 s at 5,000 g. The lower organic phase containing pyocyanin was transferred to new tube. 200 μl of 0.1 M hydrochloric acid (HCl) was then added to the chloroform phase and mixed with vortexing for 30 s, with a phase separation by putting samples briefly in centrifuge. The pink coloration which subsequently formed by acidification of pyocyanin, in the HCl phase at the top layer was then transferred to a 96-well microplate (Nunc, Denmark) and OD₅₀₀ was measured by the microplate reader (Tecan Infinite 2000). Experiments were performed in triplicates and results were shown as mean ± standard deviation.

The murine macrophage cell line RAW264.7 (ATCC No. TIB-71) was grown in 15 ml Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies), supplemented with 10% fetal bovine serum (FBS) (Gibco). Cells were incubated in 75 cm² cell culture flasks (Nunc, Denmark) at a density of 5.0 × 10⁶ cells ml⁻¹ for 72 h, at 37°C, 5% CO₂ and 90% humidity.

The cells were checked against Mycoplasma contamination by using the PCR Mycoplasma detection kit (Abmgood, USA) before experiments.

To test the ability of cells to kill macrophages, cytotoxicity of macrophages was determined by monitoring cell integrity in 1 ml DMEM + 10% FBS + 20 μM propidium iodide (PI), as previously described (Chua et al., 2016b). Cells that stained by PI under the epifluorecent microscopy (Zeiss) with 20 × objective were observed as dead. The ratio of dead cells to live cells, enumerated from five images (each image contained approximately 200 macrophages), was then calculated. Experiments were performed in triplicates and results were shown as mean ± standard deviation.

We used RNA-sequencing to compare the transcriptomes of P. aeruginosa PAO1 cells “locked” in conditions of either high or low intracellular c-di-GMP contents. The late log phase P. aeruginosa ΔwspF mutant cells possessed a high intracellular c-di-GMP content due to the constitutively expressed WspR DGC protein (D'argenio et al., 2002; Rybtke et al., 2012; Chua et al., 2016a; Figure 1A), contributing to the overproduction of exopolysaccharides and low motility in ΔwspF cells. Hence, we used ΔwspF cells to mimic the biofilm stage. On the other hand, the late log phase P. aeruginosa p_lac-yhjH mutant cells contained reduced c-di-GMP content due to the constitutively expressed YhjH PDE protein (Gjermansen et al., 2010; Chua et al., 2013; Figure 1A), thus mimicking cells freshly dispersed from the biofilms. We also corroborated our findings by detecting increased enzymatic PDE activity in the p_lac-yhjH harboring cells, as compared to wild type and ΔwspF mutant cells (Figure 1B).

The ΔwspF and PAO1/p_lac-yhjH mutants demonstrated distinct gene expression profiles according to the heat map diagram and PCA analysis (Figures 2A,B). 431 genes were up-regulated and 595 genes were down-regulated in the p_lac-yhjH mutant as compared to the ΔwspF mutant (Data Sheet 1), including genes regulated by QS (in particular regulated by the rhl and pqs encoded systems). Specifically, rhlR, rhlA and rhlB were highly induced in the p_lac-yhjH strain compared to the ΔwspF mutant (Data Sheet 1). This finding was validated by qRT-PCR analysis (Figure 2C) and the rhl QS reporter fusion p_rhlA-gfp (Yang et al., 2009; Figure 3A). Accordingly, the p_lac-yhjH strain was found to produce more rhamnolipids (Figure 3B), PQS (Figure 3C) and pyocyanin (Figure 3D) than the ΔwspF mutant, and was thus more cytotoxic to murine macrophages than the ΔwspF mutant (Figure 3E). This showed that the reduction of c-di-GMP levels lead to induction of the QS systems, which in turn stimulate production of a subset of QS-controlled virulence factors. Conversely, the ΔwspF mutant with the “mimicked” biofilm phenotype had higher expression of biofilm-related genes important for exopolysaccharide production (pelB, pelC, pelD and PelF) and siderophore production (pvdD, pvdJ, pvdL, pvdO), as compared to the PAO1/p_lac-yhjH mutant.

We also noticed that genes from the pqs operon, such as pqsA, pqsB, pqsC, and pqsH were highly induced in cells with low intracellular c-di-GMP content (Data Sheet 1). The qRT-PCR analysis confirmed these findings (Figure 2C). Since the pqs operon is controlled by PqsR, we next hypothesized that PqsR could be crucial to the induction of pqs QS system under conditions of low c-di-GMP levels. Since the rhl encoded QS system can be induced by the pqs QS system (McKnight et al., 2000), it was also possible that activation of the pqs QS system boosted rhamnolipid production. Furthermore, a ΔpqsR/p_lac-yhjH mutant produced lesser rhamnolipids than the p_lac-yhjH mutant (Figure 4A). Accordingly, we showed that a PqsR deficient ΔpqsR/p_lac-yhjH mutant expressed only low levels of pyocyanin, BHL and PQS compared with the p_lac-yhjH mutant (Figures 4B–D). This correlated well with lower cytotoxicity to macrophages by ΔpqsR (Figure 4E). Hence, PqsR appeared to be a key regulator for induction of QS systems by low intracellular c-di-GMP content.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.