The bacterial strains used in this study are listed in Table 1. All mutant strains were confirmed by PCR (data not shown). Growth was routinely performed in lysogeny broth (LB) or BM2 minimal medium (Overhage et al., 2008) at 37°C with shaking at 170 rpm unless otherwise indicated. When required, gentamicin was used at a final concentration of 30 μg/ml for P. aeruginosa transposon mutants and ampicillin and kanamycin at final concentrations of 100 and 30 μg/ml, respectively, for recombinant Escherichia coli.

For recording growth curves, bacterial cultures were grown overnight in LB and diluted in fresh LB to obtain starting optical densities of OD_600nm = 0.1. Aliquots of these dilutions were apportioned in 96-well microtiter plates (100 μl per well). Growth was recorded using a TECAN Infinite® 200 PRO plate reader (Tecan, Maennedorf, Switzerland) under shaking conditions. Two independent experiments were performed with three replicates for each strain or condition.

Since wild-type Hod protein is prone to oxidative dimerization by the formation of an intermolecular disulfide bridge, Hod protein carrying a substitution of Cys69 by serine, termed HodC, was used. HodC shows the same catalytic activity as Hod (Frerichs-Deeken et al., 2004). HodC-H251A protein, termed iHodC, which is virtually inactive due to its inability to initiate catalysis by deprotonating the organic substrate (Frerichs-Deeken et al., 2004), was used in control experiments. Purification of the recombinant His₆-tagged HodC proteins from recombinant E. coli strains (Table 1) was performed as described by Beermann et al. (2007). For storage at −80°C, 10% glycerol (vol/vol) was added to the protein stock solutions. To control the activity of HodC protein before and after an experiment, its catalytic activity was determined spectrophotometrically by measuring 3-hydroxy-2-methyl-4(1H)-quinolone consumption as described previously (Frerichs-Deeken et al., 2004). 1 Unit of enzyme activity is defined as the amount of HodC catalyzing the conversion of 1 μmol substrate per minute under the conditions of the assay. Concentrations of HodC were determined by absorption measurements using an extinction coefficient (ε_280nm) of 1.937 ml mg⁻¹ cm⁻¹ (Beermann et al., 2007).

Stability of HodC against different proteases produced by P. aeruginosa was monitored by measuring the enzyme activity in the presence of stationary phase culture supernatants of P. aeruginosa PA14, and of the PA14 mutants lasB::Tn, lasA::Tn, prpL::Tn, and aprA::Tn, respectively. Bacterial strains were grown in LB medium overnight at 37°C and 160 rpm. These cultures were used to inoculate 15 ml LB medium to an OD_{600 nm} of 0.05, and cells were further grown for 10 h at 37°C and vigorous shaking. Afterwards, cells were pelleted by centrifugation (9000 × g, 10 min, 4°C) and the culture supernatants were collected and filter sterilized. For analysis, 100 μl/ml culture supernatant was incubated in sodium phosphate buffer pH 8.0 with 0.75 μg/ml HodC at 37°C, and enzyme activity was measured at different time points.

The PQS cleavage product, N-octanoylanthranilic acid, was synthesized according to the method described by Wells et al. (1952) with some modifications. Briefly, octanoyl chloride (Sigma Aldrich, Taufkirchen, Germany) was added dropwise to a solution of methyl anthranilate (Sigma Aldrich, Taufkirchen, Germany) dissolved in ethyl acetate at 0°C while stirring. Following heating to 50°C for 5 min the solution was stirred over night at room temperature resulting in a clear solution. Subsequently, this solution was successively washed with water, 1 M sodium hydroxide, 1 M hydrochloric acid and finally with brine. After drying with Na₂SO₄ the organic solvent was removed by evaporation leading to a crude oil of methyl N-octanoylanthranilate which was used without further purification. Hydrolysis of the acylated anthranilate was carried out with 0.5 M sodium hydroxide solved in ethanol. The reaction mixture was refluxed for 3 h. After cooling, the solution was acidified with 4 M hydrochloric acid and extracted two times with n-hexane. Evaporation yielded crude N-octanoylanthranilic acid which was recrystallized in n-hexane. ESI-TOF/MS for C₁₅H₂₂NO₃⁺ ([M + H]⁺): calculated m/z = 264.159; found m/z = 264.114.

CORM-2, a carbon monoxide releasing molecule, and PQS were purchased from Sigma Aldrich (Taufkirchen, Germany).

Rapid attachment of bacterial cells to a surface was analyzed as described previously (Yeung et al., 2011). Briefly, overnight cultures grown in LB-medium were washed and diluted in LB medium to an OD_{600 nm} of 1.0. Aliquots (100 μl) of this suspension were used to inoculate each well of a microtiter plate. Cells were allowed to adhere for 60 min at 37°C prior to staining with crystal violet. All experiments were done in triplicates with 6 individual repeats per measurement (n = 18).

The abiotic solid surface assay was used to measure biofilm formation according to the method described by Friedman and Kolter (2004) with the following modifications. Overnight cultures were diluted 1:100 in fresh LB medium, inoculated into 96-well microtiter plates and incubated for 24 h at 37°C without shaking to allow bacterial adherence and biofilm formation. After incubation the biofilm cells were stained using 0.1% (w/v) crystal violet and the absorbance was measured at 595 nm using a TECAN Infinite® 200 PRO microtiter plate reader.

For the determination of colony forming units (CFU), biofilms were grown in glass bottom petri dishes as described above in the presence or absence of 70 U/ml HodC. After 24 h of incubation, planktonic cells were removed by gentle washing. The remaining, adherent cells were scraped off the petri dish surface using cell scrapers, transferred into a new test tube and vortexed vigorously for 10–30 s. Several dilutions were prepared and the CFU were determined by the drop plate method (Herigstad et al., 2001).

Extracellular DNA in biofilm cultures was determined according to the method described previously (Yang et al., 2007). Briefly, overnight cultures were diluted 1:100 in fresh LB medium supplemented with 0.05 mM propidium iodide and biofilms were grown in microtiter plates at 37°C under static conditions. After 24 h of incubation, the absorbance of propidium iodide was measured at 490 nm using a MultisKan MS photometer (Labsystems, Bradenton, USA) and cell density at 595 nm using a TECAN Infinite® 200 PRO.

For microscopic analyses, overnight cultures of P. aeruginosa PA14 lasB::Tn were diluted 1:100 in LB, HodC was added at a final concentration of 70 U/ml, and the suspensions were used to inoculate glass bottom petri dishes (MatTek, Ashland, USA). After 24 h of incubation at 37°C under static conditions, planktonic cells were removed from the medium by gently washing with LB medium and the attached viable biofilm cells were stained using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) for 3 h in the dark as described previously (Li et al., 2013). Fluorescence microscopy was carried out using an Axioplan 2 imaging system (Carl Zeiss, Oberkochem, Germany) with appropriate filter sets.

RNA for transcriptome analysis was isolated from biofilm cells of the P. aeruginosa PA14 lasB::Tn strain, grown for 24 h at 37°C under static conditions in microtiter plates in LB medium in the presence or absence of 70 U/ml HodC or iHodC, respectively. The biofilm cells of 40 wells for each condition were pooled, total RNA was extracted using RNeasy Midi columns (QIAGEN), and DNase treatment of isolated RNA samples were performed as described previously (Breidenstein et al., 2012). Depletion of rRNA was accomplished with the MICROBExpress™ bacterial mRNA enrichment kit (life technologies) according to the manufacturer's protocol. RNA-Seq was performed as described previously (Tettmann et al., 2014). Briefly, sequencing libraries were generated from 50 ng of rRNA depleted RNA samples following the Truseq RNA protocol (Illumina). Paired end reads (2 × 50 nucleotides) were obtained with a Hiseq1000 using SBS v3 kits (Illumina). Cluster detection and base calling were performed using RTAv1.13, and quality of reads was assessed with CASAVA v1.8.1 (Illumina). The reads were mapped against the genome of P. aeruginosa PA14 (accession number: NC_008463) using bowtie2 (Langmead and Salzberg, 2012). The genomic annotation of P. aeruginosa PA14 was downloaded from the Pseudomonas Genome database (www.pseudomonas.com) (Winsor et al., 2009). Gene expression was determined by counting for each gene the number of reads that overlapped with the annotation location using HTSeq (Anders et al., 2015). Differential expression was calculated using the R package DESeq2 (Love et al., 2014), and genes were assumed to be differentially expressed, if the fold-change was at least two-fold (±) and the P-value less than 0.05. Complete expression data is deposited at the Sequence Read Archive NCBI under the accession number SRP046054.

The 2-alkyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase HodC was previously shown to be able to quench PQS signaling in P. aeruginosa, leading to decreased production of several virulence factors, however, HodC activity in P. aeruginosa cultures was significantly reduced over time by proteolytic cleavage (Pustelny et al., 2009). In order to identify the extracellular protease(s) of P. aeruginosa PA14 involved in inactivation of HodC, its activity was determined in the presence of cell-free culture supernatants of P. aeruginosa PA14 and supernatants of the four protease deficient mutants lasB::Tn (inactivation of elastase LasB), lasA::Tn (LasA), prpL::Tn (protease IV), and aprA::Tn (alkaline metalloproteinase). While supernatants of P. aeruginosa PA14 as well as of the lasA, prpL, and aprA mutant strains strongly decreased HodC activity already after 10 min of incubation, no change in activity was observed for supernatants of P. aeruginosa PA14 lasB::Tn in comparison to the LB control (Figure 1). Therefore, the lasB mutant was chosen for all following experiments to ensure high HodC activity even during prolonged incubation periods.

Since PQS is important for biofilm formation (Diggle et al., 2003), we analyzed the effect of HodC on P. aeruginosa biofilm development. Addition of HodC to static biofilm cultures of P. aeruginosa PA14 resulted in a moderate increase in biofilm biomass after 24 h of incubation, whereas the lasB::Tn mutant exhibited a much stronger, five-fold increase in biofilm biomass in the presence of HodC (Figure 2A). This enhancement in biofilm formation increased with increasing concentrations of the enzyme in the culture media (Figure 2B). In contrast, addition of inactive HodC (iHodC) to biofilm cultures of both P. aeruginosa PA14 wild-type and the lasB mutant did not have an impact on biofilm development (Figure 2A). Remarkably, HodC addition even influenced established biofilms, as we observed an almost two-fold increase of biofilm biomass of pre-grown biofilms in the presence of the enzyme (Figure 3).

To analyze whether the observed enhancement in biofilm mass was indeed due to an increase in viable cells, we evaluated biofilms of P. aeruginosa PA14 lasB::Tn on glass bottom petri dishes using fluorescence microscopy in combination with CTC staining, which allows the visualization of metabolically active cells. The biofilms exhibited a much higher proportion of viable cells when grown in the presence of HodC (Figure 4). Moreover, determination of CFUs revealed 9.2 × 10⁸ (± 3 × 10⁸) CFU/ml and 22.5 × 10⁸ (± 2.7 × 10⁸) CFU/ml for untreated and HodC-treated biofilms, respectively, corresponding to a 2.4-fold increase in viable biofilm cells in the presence of HodC. However, HodC (70 U/ml) did not affect planktonic growth of P. aeruginosa PA14 in LB (data not shown). Since initial adherence of bacterial cells strongly impacts biofilm development, we investigated whether HodC affects biofilm growth already at the very initial phase of cell attachment. To this end, attachment of P. aeruginosa PA14 lasB::Tn was determined after 1 h of incubation with or without HodC. Quantification of surface-attached cells by crystal violet staining revealed that HodC addition did not alter initial cell adhesion of P. aeruginosa (Supplementary Figure 1A). In addition, quantification of extracellular DNA, which is an important factor in biofilm formation, revealed no differences in eDNA of biofilms grown in the presence of HodC compared to the iHodC control biofilms (Supplementary Figure 1B). Taken together, the results indicate that the enhanced biofilm in presence of HodC is due to the increase in viable biomass.

In order to identify the mechanisms underlying the stimulation in biofilm formation by HodC, we first analyzed whether N-octanoylanthranilic acid and carbon monoxide, the products of HodC-catalyzed PQS cleavage, have an effect on biofilm development of the P. aeruginosa PA14 lasB::Tn strain. In agreement with an earlier study by Murray et al. (2012), no significant increase in biofilm formation was observed in the presence of 10–20 μM of the CO-releasing molecule CORM-2, but biofilm formation was decreased strongly at higher concentrations of CORM-2 (Figure 5A). Given that supernatants of overnight cultures of P. aeruginosa PA14 contain between 5 and 25 μM PQS (Diggle et al., 2003; Lépine et al., 2003), we analyzed the impact of N-octanoylanthranilic acid on biofilm biomass at concentrations of up to 30 μM. The data (Figure 5B) indicated that this PQS cleavage product has also no influence on biofilm development. Overall, these results indicated that the two metabolites formed by HodC-mediated PQS cleavage are not responsible for the observed biofilm phenotype.

To get an insight into the molecular basis for the observed biofilm phenotype, transcriptome analyses were performed using Illumina sequencing, and the expression profile of P. aeruginosa PA14 lasB::Tn biofilms treated with HodC was compared to that of untreated control biofilm cells. This analysis identified a total set of 38 genes that were differentially regulated in the presence of HodC (Table 2). Most of the 34 genes that were down-regulated are directly or indirectly under control of the ferric uptake regulator Fur (Ochsner et al., 2002). Among them are the operon comprising the fumarate hydratase (fumC) and superoxide dismutase (sodM) genes (Hassett et al., 1997), and genes coding for the regulation and biosynthesis of the siderophore pyoverdine (pvdS, pvdL, pvdA, pvdQ) and for proteins involved in heme uptake and degradation (phuS, phuR, hemO). Additional transcriptome analyses for cells incubated with iHodC did not reveal any significant differences in gene expression except a 3.1-fold upregulation of the glpD gene, which is coding for glycerol-3-phosphate dehydrogenase, in comparison to the untreated control biofilms (data not shown). This glpD upregulation was comparable to the expression observed in response to HodC (Table 2) and is most likely due to the presence of glycerol in the enzyme stock solutions. Thus, the observed transcriptome data demonstrate that the changes in gene expression are entirely depending on the catalytic activity of HodC.

Because PQS exhibits iron-chelating properties and therefore sequesters iron from the culture medium (Bredenbruch et al., 2006; Diggle et al., 2007), we assumed that the observed downregulation of genes involved in iron metabolism by HodC is due to an increase in available iron in the culture medium as a result of PQS cleavage. This hypothesis is supported by the finding that in chemically defined medium, biofilm formation of the P. aeruginosa lasB mutant was significantly enhanced by the addition of 50 μM FeSO₄ or FeCl₃ (Figure 6).

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.