The pvc Operon Regulates the Expression of the Pseudomonas aeruginosa Fimbrial Chaperone/Usher Pathway (Cup) Genes

The Pseudomonas aeruginosa fimbrial structures encoded by the cup gene clusters (cupB and cupC) contribute to its attachment to abiotic surfaces and biofilm formation. The P. aeruginosa pvcABCD gene cluster encodes enzymes that synthesize a novel isonitrile functionalized cumarin, paerucumarin. Paerucumarin has already been characterized chemically, but this is the first report elucidating its role in bacterial biology. We examined the relationship between the pvc operon and the cup gene clusters in the P. aeruginosa strain MPAO1. Mutations within the pvc genes compromised biofilm development and significantly reduced the expression of cupB1-6 and cupC1-3, as well as different genes of the cupB/cupC two-component regulatory systems, roc1/roc2. Adjacent to pvc is the transcriptional regulator ptxR. A ptxR mutation in MPAO1 significantly reduced the expression of the pvc genes, the cupB/cupC genes, and the roc1/roc2 genes. Overexpression of the intact chromosomally-encoded pvc operon by a ptxR plasmid significantly enhanced cupB2, cupC2, rocS1, and rocS2 expression and biofilm development. Exogenously added paerucumarin significantly increased the expression of cupB2, cupC2, rocS1 and rocS2 in the pvcA mutant. Our results suggest that pvc influences P. aeruginosa biofilm development through the cup gene clusters in a pathway that involves paerucumarin, PtxR, and different cup regulators.

Biofilm development by P. aeruginosa occurs in several stages [19][20][21].In the initial stages, bacteria attach reversibly to abiotic or biotic surfaces, followed by irreversible attachment and maturation of the biofilm [19][20][21].During the maturation stage, the bacteria multiply, form microcolonies, and produce the EPS matrix around the microcolonies [19][20][21].The final stage involves dispersion and focal dissolution of the biofilm [19][20][21].Attachment is accomplished through two cell-associated structures, the flagella and type IV pili [21][22][23].Type IV pili also allow the bacteria to climb a biofilm formed by other bacteria and colonize the top of that biofilm [22].Biofilm formation by P. aeruginosa also involves fimbrial structures that are components of and assembled on the outer surface of the bacteria by the conserved chaperone/usher pathways termed Cup [21,24,25].The Cup consist of an usher (outer membrane protein), one or two chaperone (periplasmic protein), and the fimbrial subunits [25,26].Previous studies identified four P. aeruginosa cup gene clusters, cupA1-5, cupB1-6, cupC1-3, and cupE1-6, that code for an usher, one or two chaperones, and at least one fimbrial subunit [26][27][28].Within these clusters, cupA1 and cupA4, cupB1 and cupB6, and cupC1 code for the major fimbrial subunits; cupA2 and cupA5, cupB2 and cupB4, and cupC2 encode the chaperones; and cupA3, cupB3, and cupC3 code for the usher proteins [26,27].While cupE4 and cupE5 encode a chaperon and usher, respectively; cupE1, cupE2, cupE3, and cupE6 encode proteins that have none of the characteristics of other archetypal systems of the chaperon usher pathway [28].An additional cluster, cupD1-5, exists in the P. aeruginosa strain PA14 [29].In this cluster, cupD1 codes for the major fimbrial subunit, cupD2 codes for the chaperon, and cupD3 codes for an outer membrane usher [29].cupD4 and cupD5 code for a predicted adhesin and a secondary chaperon, respectively [29].Further studies described the two roc regulatory systems that control the expression of cupB and cupC genes [30,31].
Pyoverdine is a high affinity iron-chelating peptide derived molecule that functions as the primary siderophore in P. aeruginosa [32][33][34].The pyoverdine molecule consists of a variable cyclic peptide moiety and a conserved bicyclic chromophore [32][33][34].Several P. aeruginosa genes encode enzymes required for the synthesis of the pyoverdine molecule [34].Besides pyoverdine, Stintzi et al. [35], identified pseudoverdine; a fluorescent bicyclic metabolite that structurally resembles the pyoverdine chromophore.Enzymes synthesizing the pseudoverdine are encoded by the pvcA-D gene cluster [36].However, further studies indicated that the pvcA-D gene cluster encode enzymes involved in the synthesis of a novel secondary metabolite termed paerucumarin [37], rather than pyoverdine.So far, no physiological role has been assigned for either the pvcA-D gene cluster or paerucumarin [36].The pvcA-D gene cluster is located downstream of ptxR, which encodes a transcriptional activator that regulates the expression of different P. aeruginosa genes including toxA and the quorum sensing (QS) genes [36,38,39].pvcA-D expression is negatively regulated by iron and positively regulated by PtxR [36].
In this study, we examined the effect of the pvcA-D gene cluster on biofilm development in the P. aeruginosa strain MPAO1.Our results confirmed that pvcA-D constitutes the pvc operon, and that pvc influences biofilm development by regulating the expression of cupB and cupC.Additionally, the pvc operon mediates its effect on the cupB and cupC genes through the secondary metabolite, paerucumarin in a pathway that includes ptxR.

Strains, Plasmids and General Growth Conditions
Pseudomonas aeruginosa strains and plasmids used in this study are listed in Table 1.Bacterial cultures were grown in Luria Bertani (LB) broth at 37uC with shaking at 250 r.p.m. for 14-16 h.These cultures were used to inoculate fresh LB broth or tryptone broth (1% tryptone, 5 mM MgCl 2 , 2.5 mM KCl 2 and 25 mM FeCl 3 ).Plasmids were isolated using FastPlasmid Mini Kit (5 Prime) and electroporated into P. aeruginosa strains using a Gene Pulser (Bio-Rad Laboratories) as previously described [40].Antibiotics were added to the media at the following concentrations: 300 mg carbenicillin ml 21 and 60 mg tetracycline ml 21 .

Biofilm Development and Analysis
We used the air-liquid interface method of biofilm development described by Kulasekara et al. [30] with some modification.Overnight cultures of P. aeruginosa strains were subcultured at a starting OD 600 of 0.02 into 3 ml of tryptone broth in polypropylene tubes (BD Biosciences) and incubated under static condition at 37uC for 16 h.Biofilms developed at the air-liquid interface were washed with sterile distilled water to remove planktonic cells.Four ml of 1X PBS was added to each tube and vigorously vortexed to release the cells from the biofilm.The bacterial suspensions were serially diluted tenfold in 1X PBS.A 10-ml aliquot of each dilution was spotted on LB agar plates in triplicates and the agar plates were incubated at 37uC for 16 h.The number of microorganisms (colony forming units, CFU) was calculated using the formula: CFU 6 dilution factor 6 100.Each experiment was repeated three times.
To visualize the biofilms, plasmid pMRP9-1 (Table 1), which expresses GFP was introduced into MPAO1, PW4830 and PW4832, the transposon mutants of pvcA and pvcB (Table 1), by electroporation.Prior to visualization, the biofilms within the polypropylene tubes were washed with distilled water.Biofilms were examined by CLSM using an Olympus 1X71 Fluoview 300 confocal laser scanning microscope (Olympus).Image stacks were analyzed with the COMSTAT program using MATLAB [41] and various aspects of biofilm structure were determined.

Construction of the pvcC-D Isogenic Mutant
MPAO1DpvcCD was constructed from MPAO1 by the gene replacement technique as previously described [39].A 600-bp BglII fragment that carries the entire pvcC gene and part of pvcD was deleted from pAH54 and replaced by a 1.4 kb fragment that carries the tetracycline resistance gene [39].The recombinant plasmid pNC80 was introduced into PAO1 by electroporation and the transformants were screened as previously described [39,40].Construction of the mutant was confirmed by PCR and Southern blot hybridization.

Reverse Transcription Quantitative PCR (RT-qPCR)
Overnight cultures of different P. aeruginosa strains were subcultured in fresh LB broth to an OD 600 of 0.02 and incubated at 37uC with shaking for 16 h.Cultures were then mixed with twice the volume of the culture of RNAprotect Bacteria Reagent (QIAGEN) for 5 min at room temperature.The cells were pelleted and stored at 280uC.Bacterial pellets were first lysed with lysozyme and proteinase K for 15 min at room temperature and the RNA was subsequently extracted using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's recommendations.The RNA solution was then digested with the RNase-free DNase Set (QIAGEN).RNA was purified from DNase by the RNA cleanup protocol (QIAGEN) with the exception that on-column DNase digestion was applied to eliminate any remaining traces of genomic DNA.RNA was quantified by NanoDropH spectrophotometer (NanoDrop Products) and the integrity of the RNA was assessed using RNA Nano Chip on an Agilent 2100 Bioanalyzer (Agilent).
Synthesis of cDNA from the extracted RNA was performed using the QuantiTect Reverse Transcription Kit (QIAGEN).A 200-ng aliquot of cDNA was mixed with SYBR Green PCR Master Mix (Life Technologies) and 250 nM of specific primer (Table S1).Amplification and detection of the product was conducted using StepOne Plus real-time PCR system (Life Technologies).For each experiment, we used three independent biological replicates for RNA extraction.Additionally, each PCR reaction was set up in triplicate.The quantity of cDNA in different samples was normalized using 30S ribosomal RNA (rplS) as an internal standard.Gene expression analysis was performed using StepOne Plus software version 2.2.2 (Life Technologies).Positive control samples containing genomic DNA as a template and negative control samples containing RNA as a template were included in the experiment (data not shown).

Thin Layer Chromatography (TLC)
Overnight cultures of P. aeruginosa strains were subcultured in fresh LB broth to an OD 600 of 0.02 and incubated at 37uC for 16 h with shaking.Cells were pelleted and supernatant fractions were separated.A 400 ml sample of each supernatant was added to 1200 ml of acidified ethyl acetate and vortexed for 2 min.Samples were centrifuged at 10,6006g for 3 min to separate phases.The upper phase was collected, vacuum dried and dissolved in 30 ml of 1:1 ethyl acetate:acetonitrile. Eight ml of each sample was loaded onto an activated high performance TLC gel 60F 254 (EMD).TLC plates were developed in a mixture of methylene chloride, acetonitrile and 1,4-dioxane (17:2:1) for 2 h.Plates were illuminated with long wave UV light and photographed with a FluorChem 8000 imaging system (Alpha Innotech).

Purification of Paerucumarin and Synthesis of Pseudoverdine
Paerucumarin was purified from the supernatant of PAO1 as previously described in detail by Clarke-Pearson & Brady [37].Pseudoverdine was synthesized through a commercial source (Eburon Organics, Lubbock, TX) based on the previously reported structure of the molecule [35,37].

Results
pvcA Enhances Biofilm Formation by MPAO1 P. aeruginosa forms biofilms at different infection sites including acute and chronic wounds and the lungs of cystic fibrosis patients [11][12][13][14][15]42]. Biofilm formation, which is a major virulence attribute in P. aeruginosa, is controlled by numerous characterized genes and possibly additional, as yet uncharacterized, genes.To determine if pvcA influences biofilm formation, we utilized the previously described static biofilm system in which P. aeruginosa forms a ring of biofilm at air-liquid interface [24,26].While MPAO1 and PW4830 (DpvcA; Table 1) formed biofilm rings, the amount of the biofilm, as measured by CFU ml 21 , formed by PW4830 was significantly less than that formed by MPAO1 (Figure 1A).We obtained similar results when we analyzed the biofilms using the staining assay (Text S1, Figure S1).To analyze the structure of the biofilm, we introduced pMRP9-1 (Table 1), which carries the gene for green fluorescent protein (GFP) [43], into both strains.Again, while both strains formed biofilm rings, the amount of biofilm formed by PW4830/pMRP9-1 was significantly less than that formed by MPAO1/pMRP9-1 (data not shown).Visualization of the biofilms by confocal laser scanning microscopy (CLSM) revealed that MPAO1/pMRP9-1 formed a dense, well-developed biofilm with clusters of pillar-like microcolonies that extended to a considerable height (Figure 1B).In contrast, PW4830/pMPR9-1 formed a flat, less developed biofilm with sparse microcolonies (Figure 1C).Quantitative analysis of the biofilms using the COMSTAT program [41] revealed significant differences between them in the mean thickness and total biovolume.The total biovolume of MPAO1/pMRP9-1 biofilm was 2.3 mm 3 /mm 2 while that of PW4830/pMRP9-1 was 0.079 mm 3 /mm 2 (Table 2).
To confirm these findings, we conducted complementation experiments.A 1730-bp DNA fragment containing intact pvcA was synthesized from the MPAO1 chromosome by PCR and cloned into the pCR2.1-1.8 vector at the TA cloning site downstream of the lac promoter to generate recombinant plasmid pLL1 (Table 1).The defect in biofilm formation by PW4830 was complemented by pLL1 (Figure 1D).These results suggest that pvcA is required for biofilm formation by MPAO1.

The pvc Operon Contributes to Biofilm Formation by MPAO1
The contribution of pvcA to biofilm development in MPAO1 may also involve the function of the adjacent genes pvcB, pvcC, and pvcD.It has been suggested that the four genes constitute one operon [36]; yet, computer analysis suggested that the four genes constitute two operons: pvcA-B, and pvcC-D (data not shown) as the intergenic region between pvcA-B, pvcB-C, and pvcC-D are 17, 51, and 28 bp, respectively (Figure 2A).To determine which scenario is correct, we analyzed the transcript(s) produced from the four genes using RT-PCR and specific primer sets overlapping the intergenic regions between pvcA and pvcB, pvcB and pvcC, and pvcC and pvcD (Figure 2A).We detected three specific products from pvcA-B (463 bp), pvcB-C (556 bp), and pvcC-D (324 bp) (Figure 2B), indicating the presence of one transcript.Thus, pvcABCD constitute the pvc operon.Therefore, a mutation in any of the other genes besides pvcA should produce a phenotype similar to that observed in PW4830.Indeed, compared with MPAO1, PW4832, which carries a transposon insertion that inactivates pvcB, produced a significantly reduced biofilm (Table 1, Figure 1E, Figure S1).Additionally, this defect in biofilm formation was complemented by plasmid pLL2, which carries an intact copy of pvcB (Table 1, Figure 1F).Furthermore, the defects in biofilm formation in both PW4830 and PW4832 were complemented by plasmid pLL5, which carries the 3-kb region containing intact pvcA and pvcB (Table 1; data not shown).To analyze the role of the pvcC-D genes, we constructed an MPAO1 isogenic mutant (MPAO1DpvcCD) in which we deleted a 600-bp BglII fragment that encompasses portions of pvcC and pvcD (Table 1, Materials and Methods).Compared with MPAO1, MPAO1DpvcCD produced a significantly reduced biofilm (Figure 1G).These results suggest that the entire pvc operon contributes to the development of biofilm formation by P. aeruginosa.Further studies were conducted using strain PW4830.
pvc does not Enhance Biofilm Formation by MPAO1 Through either Flagellum or Type IV Pili Biofilm formation is a complex process involving three distinct stages: attachment, maturation, and dispersal [21].Bacteria initiate biofilm by first reversibly and then irreversibly attaching to host tissues or abiotic surfaces, processes that depend on the flagellum and type IV pili [20][21][22].Therefore, we next examined PW4830 for possible defects in the flagellummediated swimming motility or pili-mediated twitching motility.PW4830 showed comparable motility to MPAO1 on both swimming and twitching agar plates (data not shown).To confirm these results, we determined if the mutation in pvcA affected the expression of the flagellar and/or pilin genes.Using RT-qPCR, we analyzed the expression of the flagellar biosynthesis genes fliC and flgK [44] in MPAO1 and PW4830.Similarly, we examined the expression of the pilin biosynthesis gene pilA in both strains.The levels of expression of flgK and pilA in MPAO1 and PW4830 were comparable, while the level of fliC expression was higher, rather than lower (Figure 3A).These results suggest that pvcA does not enhance biofilm formation by MPAO1 through either the flagellum or the pili.
Other components that contribute to biofilm development in P. aeruginosa are the polysaccharides including Psl and Pel, which are required to maintain the biofilm structure in P. aeruginosa non-mucoid strains [45,46].Pel is required for pellicle development at the air-medium interface [47].We examined the possibility that the pvc operon influences biofilm formation in MPAO1 through either Pel or Psl by analyzing the level of expression of pelA, pslA, and pslD in MPAO1 and PW4830.We detected no significant difference in the level of expression of these genes (Figure S2).

pvc Influences Chaperone/Usher Pathway Systems in MPAO1
As the pvc operon does not affect biofilm formation in MPAO1 through either the flagellum or the type IV pili, pvc may affect it through the fimbriae.Fimbrial assembly in P. aeruginosa occurs through the proteins encoded by cup gene clusters, cupA1-5, cupB1-6, cupC1-3, and cupE1-6 [26].To examine the effect of pvc on the cup genes, we first compared the level of expression of the fimbrial subunit genes cupA1, cupB1and cupC1, between MPAO1 and PW4830 using RT-qPCR.The expression of cupB1 and cupC1, in PW4830 was significantly reduced by 17-and five-fold, respectively, while the expression of cupA1 was only reduced by 2.2fold (Figure 3B).At this time, we decided to focus our efforts on analyzing the effect of pvc operon on the expression of cupB and cupC genes.
Since pvcA affects the expression of the cupB1 and cupC1 fimbrial genes, we examined the effect of pvcA mutation on the expression of other genes within each cluster.In addition to the chaperones, fimbrial subunits, and the usher protein, the cupB cluster includes cupB5, which codes for a protein that is homologous to the Bordetella pertussis hemagglutinin and other nonfimbrial adhesion molecules [26,48].The level of expression of cupB1-6 in PW4830 was significantly reduced by 17-, 64-, four-, 111-, 29-and 18-fold, respectively, compared with MPAO1 (Figure 4A).Similarly, the level of expression of cupC1-3 in PW4830 was lower than MPAO1 by five-, 14-, and four-fold, respectively (Figure 4B).As we analyzed the effect of pvc on biofilm development using the same air-liquid assay that was previously used to analyze the role of the Cup pathways and the fimbriae in biofilm formation [24,26,28], these results suggest that the pvc operon influences biofilm formation in MPAO1 primarily through the fimbrial cupB and cupC gene systems.Results of preliminary experiments suggest that the pvc operon also regulates the expression of the cupE1-6 genes as expression of cupE4 was reduced in PW4380 (data not shown).
pvc Enhances the Expression of cupB and cupC Through the roc Systems The expression of the cupB and cupC gene clusters is regulated by two different two-component systems, roc1 and roc2 [28].The roc1 system consists of RocS1, a membrane bound sensor kinase homologous to the B. pertussis multi-domain sensor kinase BvgS [30,49]; RocA1, a conventional response regulator; and RocR, which contains an EAL domain and antagonizes RocA1 function [30].The roc2 system consists of an unorthodox sensor kinase, RocS2 and a conventional response regulator, RocA2 [30].We examined the possibility that the pvc operon regulates the expression of one or more of these genes by comparing the level of their expression between MPAO1 and PW4830 (DpvcA).Compared with MPAO1, the levels of expression of rocS1, rocR, rocS2, and rocA2 in PW4830 were reduced by 43-, 17-, 29-, and 39-fold, respectively; however, the level of rocA1 expression was decreased by only three-fold (Figure 5A).Compared with other genes of the roc1/roc2 systems, rocA1 expression in MPAO1 is considerably lower (Figure S3).It is possible that in MPAO1, rocA1 is not responsive to rocS1 transcriptional enhancement.Yet, Kulasekara et al. [30] previously showed that rocS1 overexpression from the tac promoter significantly increased rocA1    expression in P. aeruginosa PAK.To confirm that rocA1 responds to rocS1, we examined the effect of rocS1 overexpression on the level of rocA1 expression in MPAO1.Using PCR, we synthesized a 4060-bp fragment containing intact rocS1 from MPAO1 chromosome and cloned it in pCR2.1-1.8 generating pLL4, in which rocS1 is expressed from the lac promoter (Table 1), an E. coli promoter that is constitutively expressed in P. aeruginosa [50].Compared with PW4830/pCR2.1-1.8, rocS1 and rocA1 expression was significantly enhanced in PW4830/ pLL4 indicating the responsiveness of rocA1 to rocS1 in this strain (Figure 5B).As the roc systems regulate expression of the cup genes, we also examined whether pLL4 affects expression of cupB2 and cupC2 in PW4830.Overexpression of rocS1 resulted in significant enhancement in expression of both genes (Figure 5C) suggesting that pvc regulates cup gene expression through the roc systems.
ptxR Affects Expression of cup Genes Through Intact pvc Stintzi et al. [36] previously provided evidence suggesting that ptxR positively regulates pvc expression in PAO1.Additionally, to analyze the different components encoded by the pvc operon in the P. aeruginosa strain PAK, Clarke-Pearson & Brady [37] overexpressed pvc using a ptxR plasmid.Therefore, we first examined the effect of ptxR on the expression of individual genes of the pvc operon in MPAO1.Either a vector control (p18.230) or pJAC7-1, which carries an intact copy of ptxR (Table 1), was introduced in MPAO1.The transformants were grown in LB broth for 16 h and the level of expression of different genes was determined.Compared with MPAO1/p18.230,the level of expression of pvcA, pvcB, pvcC, and pvcD in MPAO1/pJAC7-1 was increased by 26-, 14-, 25-, and 55-fold, respectively (Figure 6A).This confirms that a ptxR plasmid also drives overexpression of the individual genes of the pvc operon in MPAO1.
As ptxR regulates the expression of pvc in MPAO1, it would likely increase the expression of the cup genes, which are regulated by pvc.To test this possibility, we examined the expression of cupB2 in MPAO1/p18.230and MPAO1/pJAC7-1.The presence of pJAC7-1 increased cupB2 expression in MPAO1 by 3.7-fold (Figure 6B).To confirm that ptxR enhances cupB2 expression through pvc, we examined the level of cupB2 expression in PW4830 and PW4832 carrying pJAC7-1 or p18.230.In contrast to MPAO1/pJAC7-1, the level of cupB2 expression in PW4830/pJAC7-1 and PW4832/pJAC7-1 was not significantly different from that in the strains carrying p18.230 (Figure 6B).Similar results were obtained upon examination of the level of cupC2 expression in MPAO1, PW4830, and PW4832 in the presence and absence of pJAC7-1 (data not shown).
Results described in Figure 6A and 6B were obtained using strains carrying multiple copies of ptxR (pJAC7-1 plasmid).To confirm that the phenomenon is truly ptxR-related and not artificially produced by multiple copies of ptxR, we compared the level of pvcA, pvcB, cupB2, and cupC2 expression between MPAO1 and its ptxR mutant PW4833.We also compared the level of rocS1 and rocS2 in the two strains to further prove that ptxR regulates the expression of cupB and cupC through the rocS genes.The level of pvcA, pvcB, rocS1, rocS2, cupB2 and cupC2 expression in PW4833 was reduced by 5-, 4.7-, 1.6-, 2.5-, 2.2-and 4.6-fold respectively, compared to their expression in MPAO1 (Figure 6C).

Paerucumarin Enhances the Expression of cupB2, cupC2, rocS1 and rocS2 in PW4830
Clarke-Pearson & Brady [37] previously showed that the major product of the pvc operon is a novel isonitrilefunctionalized cumarin, paerucumarin.Paerucumarin was detected in the extract of the supernatant fraction of PAO1 in which ptxR was overexpressed [37].Thus, we investigated the possibility that paerucumarin enhances the expression of cupB2, cupC2, rocS1 and rocS2 in MPAO1.We first confirmed that paerucumarin is produced by MPAO1/pJAC7-1 but not PW4830 or PW4832.Strains carrying either pJAC7-1 or p18.230 were grown in LB broth for 16 h at 37uC.The supernatant fraction was extracted with ethyl acetate and analyzed using thin layer chromatography.MPAO1/pJAC7-1 but not MPAO1/p18.230produced a considerable amount of paerucumarin (Figure 7A).Despite the presence of the ptxR plasmid, neither PW4830 nor PW4832 produced detectable levels of paerucumarin (Figure 7A).Thus, production of paerucumarin requires functional pvc.
Next, we examined if exogenous paerucumarin affects the expression of cupB2, cupC2, and rocS1 and rocS2 in PW4830.Either ethyl acetate (mock) or purified paerucumarin was added to PW4830 and the expression of these genes was examined by RT-qPCR after 16 h of growth at 37uC.Compared with ethyl acetate, exogenously added paerucumarin significantly enhanced the expression of all four genes (Figure 7B).These results strongly suggest that exogenous paerucumarin bypasses the defect of PW4830 in the expression of the cup and roc genes.However, exogenously added paerucumarin had no effect on the expression of pilA and flgK genes (data not shown), which confirms the specificity of the paerucumarin effect on the expression of the cup genes.Besides the cup genes, the roc1/roc2 regulon includes the mexAB-oprM operon, which is negatively regulated by the roc2 system.Exogenously added paerucumarin significantly reduced the expression of mexA in PW4830 (data not shown), suggesting that paerucumarin may also regulate the expression of other genes of the roc1/roc2 regulon.To confirm that the observed effect on the cup genes is unique to paerucumarin, we examined the effect of pseudoverdine on cupB2 and cupC2 expression.Pseudoverdine, the N-formyl adduct of paerucumarin, is also synthesized by the pvc operon; however, its function is unknown [37].Therefore, we synthesized pseudoverdine using a commercial facility (Eburon Organics, Lubbock, TX).However, exogenously added pseudoverdine had no effect on either cupB2 or cupC2 expression in PW4830 (Figure S4, data not shown).
To confirm that paerucumarin enhances the expression of cup genes through rocS1/rocS2, we determined the effect of exogenously added paerucumarin on cupB2 expression in the rocS1 mutant strain PW7672 and the rocS2 mutant strain PW6105.At a concentration of 300 mM, paerucumarin had no effect on cupB2 expression in either mutant (data not shown).However, at a concentration of 600 mM, paerucumarin significantly enhanced cupB2 expression in the rocS1 mutant (Figure 8).The same level of paerucumarin also slightly increased cupB2 expression in the rocS2 mutant, although this increase was not significant (Figure 8).

Discussion
The results of this study indicate that the pvcA-D gene cluster constitutes an operon (pvc) (Figure 2).The entire pvc operon, rather than the effect of its individual genes, affects biofilm development in P. aeruginosa by regulating cupB and cupC fimbrial synthesis systems (Figures 1, 3 and 4).The four synthesis enzymes encoded by pvc are unlikely to be individually involved in fimbrial synthesis or biofilm development.Rather, our results strongly suggest that the effect is due to paerucumarin, the isonitrile-functionalized cumarin that is the product synthesized by the four PVC enzymes (Figure 7).
Initial studies indicated that pvc codes for pseudoverdine, a fluorescent bicyclic metabolite (cumarate derivative) that is structurally related to the chromophore of the pyoverdine molecule [35].However, further studies identified the main product of the pvc operon as 2-isocyano-6,7-dihydroxycoumarin, or paerucumarin [37].Paerucumarin, a novel molecule, is basically pseudoverdine in which the N-formyl group is replaced with an isonitrile moiety [37,51].PvcA belongs to the family of isonitrile synthases [51][52][53] and PvcB belongs to a family of alphaketo glutarate dependent oxygenases [51,54] that function in the production of isocyano derivatives of amino acids.Homologues for PvcA and PvcB exist in other bacteria including Vibrio cholerae, Erwinia carotovora, and Legionella pneumophila [37].Similarly to pvcA-D, gene clusters that contain the PvcA/PvcB homologues in other bacteria synthesize metabolites with no known function [37].Based on the results of this study, paerucumarin may function as an effector molecule that activates a P. aeruginosa transactivator.Upon its activation, the protein enhances the expression of several target genes including the cupB and cupC genes.In this capacity, paerucumarin would be similar to the P. aeruginosa QS-molecules including 3OC 12 -HSL, C 4 -HSL, and PQS that activate LasR, RhlR, and PqsR, respectively [55,56].Unlike QS-molecules, paerucumarin is not detected in the supernatant of P. aeruginosa under laboratory conditions; that is, in supernatants of cultures grown in LB broth at 37uC (Figure 7A) [37].Paerucumarin was detected only in the supernatant of P. aeruginosa strains in which pvc is overexpressed either from a strong exogenous promoter or in the presence of a ptxR plasmid (Figure 7A) [37].Despite this, wild-type strains of P. aeruginosa such as MPAO1 contain a sufficient amount of intracellular paerucumarin to influence bacterial functions including biofilm formation.Mutations within the pvc operon significantly reduced the expression of different cup gene clusters (Figures 3 and 4) as well as compromising biofilm development (Figure 1).Increasing paerucumarin production by overexpressing pvc through ptxR enhanced cupB2 expression in MPAO1 but not in PW4830 (DpvcA) or PW4832 (DpvcB) (Figure 6B).Whether the environmental conditions within the host at certain infection sites induce ptxR expression leading to increased paerucumarin production is not known at this time.
Based on these results, we propose the following model to explain our findings: PtxR enhances the expression of the pvc operon to produce more paerucumarin (Figure 9).Paerucumarin activates a potential transcriptional activator which enhances the expression of different cup genes (Figures 6 and 7) and facilitates biofilm development (Figures 1 and S1).At this time, we have no direct experimental evidence for such a transcriptional activator.However, extrapolating from the results of previous analyses, we propose PtxR may be this potential activator.In support of this possibility, we recently observed that paerucumarin exogenously added to the ptxR mutant PW4833 did not enhance the expression of either cupB2 or cupC2 (data not shown).However, further experiments will be conducted to fully explore the role of PtxR in regulating the roc1/roc2 systems and the cup genes.PtxR belongs to the LysR family of transcriptional activators [39].Most members of this family are activated by specific effector metabolites that bind to an effector binding or co-inducer binding domain within the carboxy terminus regions of these proteins [57].Co-inducer binding induces a conformational change within the tertiary structure of the LysR protein which enhances its binding to the DNA target sequence [57].For example, the co-inducers for CysB, NodD, and OxyR are N-acetylserine, flavonoids, and hydrogen peroxide, respectively [58][59][60].Computer analysis indicated that the region spanning amino acid residues 99-297 within the carboxy terminus region of PtxR represents a potential co-inducer or effector binding domain (data not shown).However, that specific co-inducer has not yet been defined.
PtxR may resemble the P. aeruginosa PqsR/MvfR, another LysR DNA-binding protein that activates expression of the pqsA-E operon [61,62].PQS, the product of the pqsA-E operon, enhances PqsR binding to the pqsA-E upstream region thereby increasing pqsA-E expression and producing more PQS, which functions as a co-inducer [56].Despite numerous attempts, including using multiple expression systems and commercial facilities, we have been unable to produce a sufficient amount of purified PtxR to examine its binding to the pvc upstream region, whether in the presence or absence of paerucumarin (data not shown).Currently, we are trying to determine the PtxR target sequence within the PAO1 chromosome using the ChIP-on-chip technique which does not require a purified protein [63].

Figure 1 .
Figure 1.pvc genes affect biofilm development in MPAO1. A. Mutation in pvcA reduces biofilm development in the MPAO1 isogenic mutant PW4830.Strains were transformed with pMRP9-1, which expresses GFP.Overnight cultures were subcultured into tryptone broth as described in Materials and Methods.Bacterial biofilms formed in a ring at the air-liquid interface were washed to remove planktonic cells and the biofilm cells were removed by vortexing in PBS, diluted tenfold, and plated to quantify the viable microorganisms within the biomass (CFU ml 21 ).B and C. Representative photomicrographs of the biofilms formed by (B) MPAO1 and (C) PW4830 (DpvcA) visualized with CLSM at 40X magnification.Z slices of 0.5 mm were generated; the zy and zx planes of the Z images are shown to the left and below the flat field, respectively.Bars equal 50 nm.D-G.Biofilms were developed and CFU assayed as described in A. D. Plasmid pLL1 carrying intact pvcA constitutively expressed from the lac promoter complements the defect of PW4830 in biofilm formation.Strains were transformed with pLL1 or pCR2.1-1.8 (vector control).E. Mutation in pvcB also reduces biofilm development in the MPAO1 isogenic mutant PW4832.F. Plasmid pLL2 carrying intact pvcB constitutively expressed from the lac promoter complements the defect of PW4832 (DpvcB) in biofilm formation.Strains were transformed with pLL1 or pCR2.1-1.8 (vector control).G. Mutation in pvcC-D reduces biofilm development in the MPAO1 isogenic mutant MPAO1DpvcC-D.Values in A and D-G represent the average of three independent experiments 6 standard error of the mean (SEM).Statistical significance in viable biomass between the strains was calculated by Student's unpaired t-test.P,0.05 (*); P,0.01 (**).doi:10.1371/journal.pone.0062735.g001

e
Variation in the thickness of the biofilm, or heterogeneity.f Total of the area occupied in each image stack.g Portion of the biofilm exposed to nutrients; biovolume divided by the surface area of the substratum.doi:10.1371/journal.pone.0062735.t002

Figure 2 .
Figure 2. pvcA-D gene cluster constitutes an operon in MPAO1.Confirmation that the pvcA-D gene cluster constitutes an operon was accomplished through a series of RT-PCR experiments using primers that correspond to regions within adjacent genes.A. Order and direction of transcription of pvcABCD on the MPAO1 chromosome is indicated by block arrows.The intergenic region between each pair of genes is shown above and the location of the primers for each pair of genes is marked by the inward-facing arrows below: (1) pvcC-pvcD, solid; (2) pvcB-pvcC, dashed; (3) pvcA-pvcB, dotted.Sizes of the expected products are indicated below the primer locations.B. RNA from MPAO1 grown in LB broth for 16 h was obtained, processed, and reverse transcribed to produce cDNA as described in Materials and Methods.PCR reactions using each pair of primers were run and the products separated on a 1% (w/v) agarose gel and stained with ethidium bromide.(1) 324-bp product from pvcC-pvcD; (2) 556-bp product from pvcB-pvcC; (3) 463-bp product from pvcA-pvcB; (NT) no cDNA template control (Std) molecular size standard.doi:10.1371/journal.pone.0062735.g002

Figure 3 .
Figure 3. pvcA mutation does not interfere with the expression of pilA or flgK.pvcA mutation does not interfere with the expression of pilA or flgK but reduces cup gene expression.The relative level of expression for each gene in PW4830 (DpvcA) compared to MPAO1 was determined by RT-qPCR as described in Materials and Methods.The quantity of cDNA in different samples was normalized using 30S ribosomal RNA (rplS) as an internal standard.A. Mutation in pvcA does not interfere with the expression of either pilA or flgK but enhanced expressions of fliC in MPAO1.B. Mutation in pvcA affects expression of cupA1, cupB1 and cupC1 in MPAO1.Values in A and B represent the average of triplicate PCR experiments conducted on three independently obtained RNA preparations 6 SEM (n = 3); P,0.05 (*); P,0.01 (**); P,0.001 (***).doi:10.1371/journal.pone.0062735.g003

Figure 4 .
Figure 4. pvcA mutation reduces the expression of cupB and cupC gene clusters.The relative level of expression for each gene in PW4830 (DpvcA) compared to MPAO1 was determined by RT-qPCR.The quantity of cDNA in different samples was normalized using 30S ribosomal RNA (rplS) as an internal standard.A. pvcA mutation in MPAO1 significantly reduces the expression of all genes of the cupB gene cluster.Order and direction of transcription of cupB1-6 on the MPAO1 chromosome is indicated by block arrows above the graph.B. pvcA mutation in MPAO1 significantly reduces the expression of all cupC genes.Order and direction of transcription of cupC1-3 on the MPAO1 chromosome is indicated by block arrows above the graph.Values in A and B represent the average of triplicate PCR experiments conducted on three independently obtained RNA preparations 6 SEM (n = 3); P,0.01 (**); P,0.001 (***).doi:10.1371/journal.pone.0062735.g004

Figure 7 .Figure 8 .
Figure 7. Exogenous paerucumarin enhances the expression of different genes within the roc and cup systems.The relative level of expression for each gene in each pair of strains was determined by RT-qPCR.The quantity of cDNA in different samples was normalized using 30S ribosomal RNA (rplS) as an internal standard.A. Overexpression of pvc by pJAC7-1 leads to accumulation of paerucumarin within the supernatant of MPAO1 but not those of PW4830 (DpvcA) and PW4832 (DpvcB).Strains carrying p18.230 (V) or pJAC7-1 (PtxR) were grown for 16 h at 37uC.Cells were pelleted and the supernatants isolated by centrifugation.Paerucumarin was extracted from the supernatant fractions by ethyl acetate and detected using TLC as described in Materials and Methods.Asterisk indicates position of paerucumarin.Purified paerucumarin served as a positive control (C).B. Exogenously added paerucumarin enhances the expression of rocS1, rocS2, cupB2, and cupC2 in PW4830 (DpvcA).An overnight culture of PW4830 was subcultured into fresh LB broth to an OD 600 of 0.02.A 15ml aliquot of either ethyl acetate (mock) or purified paerucumarin 300 mM) was added at the time of subculturing.Cells were grown at 37uC for 16 h.The levels of gene expression in PW4830 plus paerucumarin were compared to the levels in mock-treated PW4830.Values in B represent the average of triplicate PCR experiments conducted on three independently obtained RNA preparations 6 SEM (n = 3); P,0.05 (*); P,0.01 (**); P,0.001 (***).doi:10.1371/journal.pone.0062735.g007

Figure 9 .
Figure 9. Diagram of the proposed regulatory circuits through which the pvc operon enhances the expression of cup genes.(1) PtxR enhances pvc expression and the proteins encoded by the pvc operon synthesize paerucumarin.(2) As a molecular signal, paerucumarin may activate the roc system through histidine kinases RocS1 and RocS2.Alternatively, paerucumarin may activate a potential transcriptional activator which enhances the expression of rocS1-rocA1 operon, rocS2, and rocA2.(3) RocS2 and RocS1 then activate RocA1, which in turn enhances cupC1-3 expression.(4) Both RocS2 and RocS1 enhance cupB1-6 expression by activating an unknown regulator (X)[31].Y indicates potential paerucumarin activated transcriptional regulator.Direction of expression is indicated by placement of genes above or below the black lines.doi:10.1371/journal.pone.0062735.g009

Table 1 .
Bacterial strains and plasmids used in this study.

Table 2 .
Quantification of the effect of pvcA mutation on biofilm formation.
b Image stacks were acquired in triplicate at 10X magnification and analyzed using the COMSTAT program; values represent the mean 6 SEM.c Biomass of the biofilm.d Spatial size of the biofilm.