The ppuI-rsaL-ppuR Quorum-Sensing System Regulates Biofilm Formation of Pseudomonas putida PCL1445 by Controlling Biosynthesis of the Cyclic
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《细菌学杂志》
Leiden University, Institute of Biology, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands
ABSTRACT
Pseudomonas putida strain PCL1445 produces two cyclic lipopeptides, putisolvin I and putisolvin II, which possess surface tension-reducing abilities and are able to inhibit biofilm formation and to break down existing biofilms of several Pseudomonas spp., including P. aeruginosa. Putisolvins are secreted in the culture medium during growth at late exponential phase, indicating that production is possibly regulated by quorum sensing. In the present study, we identified a quorum-sensing system in PCL1445 that is composed of ppuI, rsaL, and ppuR and shows very high similarity with gene clusters of P. putida strains IsoF and WCS358. Strains with mutations in ppuI and ppuR showed a severe reduction of putisolvin production. Expression analysis of the putisolvin biosynthetic gene in a ppuI background showed decreased expression, which could be complemented by the addition of synthetic 3-oxo-C10-N-acyl homoserine lactone (3-oxo-C10-AHL) or 3-oxo-C12-AHL to the medium. An rsaL mutant overproduces AHLs, and production of putisolvins is induced early during growth. Analysis of biofilm formation on polyvinylchloride showed that ppuI and ppuR mutants produce a denser biofilm than PCL1445, which correlates with decreased production of putisolvins, whereas an rsaL mutant shows a delay in biofilm production, which correlates with early production of putisolvins. The results demonstrate that quorum-sensing signals induce the production of cyclic lipopeptides putisolvin I and II and consequently control biofilm formation by Pseudomonas putida.
INTRODUCTION
Bacteria can form multicellular aggregates generally referred to as biofilms on biotic and abiotic surfaces. Such communities are ubiquitous in natural environments but can also be found in industrial and clinical settings, for example, on artificial surfaces of medical devices, thereby greatly contributing to infections (34).
Pseudomonas putida strain PCL1445 is capable of forming biofilms on roots and on polyvinylchloride (PVC) in a commonly used biofilm assay (21). We have shown that P. putida PCL1445 produces two novel lipodepsipeptides, putisolvin I and II, consisting of a C6 lipid moiety and a 12-amino-acid peptide, which are produced by a putisolvin synthetase gene designated as psoA (21). A mutant with impaired putisolvin biosynthesis was shown to form a thicker biofilm than the wild-type strain. In addition, purified putisolvins I and II inhibit biofilm formation and break down existing biofilms of various Pseudomonas spp., including the opportunistic human pathogen P. aeruginosa (21). The production of putisolvins occurs at the end of the exponential growth phase (21), which may indicate that the production is mediated through a quorum-sensing mechanism. The term quorum sensing describes an environmental sensing system which allows bacteria to monitor their own population density. Quorum sensing in gram-negative bacteria relies on the interaction of small diffusible signal molecules belonging to the class of N-acyl homoserine lactones (AHLs). They are synthetized via the LuxI protein, whereas the transcriptional activator protein LuxR couples cell population density to gene expression (12, 35). These signal molecules can traffic in and out of the bacterial cell. Once a certain intracellular threshold concentration has been reached, the signals induce transcription of a set of target genes (11). AHLs play a role in regulating different bacterial functions such as antibiotic biosynthesis, production of virulence factors, bacterial swarming, and transition to the stationary growth phase. In this paper we describe (i) the identification and characterization of the regulatory quorum-sensing genes affecting cyclic lipopeptides putisolvins I and II in PCL1445, (ii) the involvement of the quorum-sensing system in the regulation of biofilm formation of PCL1445, and (iii) the direct relationship between production of quorum-sensing signals, production of cyclic lipopeptides, and reduction of the size of the biofilm formed by P. putida PCL1445.
MATERIALS AND METHODS
Bacterial strains and growth conditions. Bacterial strains used in this study are listed in Table 1. Pseudomonas strains were grown in King's medium B (17) or in a defined BM medium (25) supplemented with 2% of glycerol (BDH Laboratory Supplies Pool, England) at 28°C. Escherichia coli strains were grown in Luria-Bertani medium (30) at 37°C. Media were solidified with 1.8% agar (Select Agar; Invitrogen Life Technologies, Paisley, United Kingdom). The antibiotics kanamycin, tetracycline, gentamicin, and carbenicillin were added, when necessary, to final concentrations of 50, 40, 2, and 100 μg ml–1, respectively.
Extraction and detection of AHLs autoinducers from spent culture medium. To isolate autoinducer activity, 3 volumes of dichloromethane were added to 7 volumes of supernatant of a 50-ml BM bacterial culture and shaken for 1 h at 120 rpm. The organic phase was removed and dried by evaporation under a vacuum (27). Supernatant extracts were redissolved in 100 μl of ethyl acetate, and 10 μl was fractionated on a C18 reverse-phase thin-layer chromatography (TLC) plate (Merck, Darmstadt, Germany) developed in methanol-water (60:40, vol/vol).
To detect autoinducer activity, overnight cultures of E. coli DH5 containing pAK211 (22) or pSB1075 (37) were grown in LB medium supplemented with 20 μg of chloramphenicol per ml for 10 h. TLC plates were overlaid with 0.8% LB top agar containing 50 μl of the pAK211 or pSB1075 strains per ml and then incubated at 28°C for 16 h. Autoinducer activity was then detected by the emission of light after Fuji medical X-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan) was applied to the TLC plates.
Isolation and identification of quorum-sensing gene homologues. A plasmid library of chromosomal fragments of strain PCL1445 was constructed by cloning 1.5- to 3.0-kb fragments of chromosomal DNA digested with EcoRI into pBluescript (Stratagene, La Jolla, CA). The resulting fragment library was introduced into an E. coli strain harboring the lux reporter plasmid pAK211 (22). After overnight growth on LB agar plates, clones that induced the luciferase reporter were identified using photographic film. To remove pAK211 (22) from the E. coli reporter strain, the total plasmid was isolated and reintroduced into DH5 cells by standard transformation protocols (31) followed by carbenicillin selection, whereas chloramphenicol selection was omitted. The DNA sequence of the chromosomal fragment inserted in the selected plasmid pMP5548 was determined using universal primers, including M13-20 and M13 reverse primers flanking the multiple cloning sites of pBluescript.
Construction of ppuI, ppuR, and rsaL mutant strains. ppuI mutant derivatives of strains PCL1445 and PCL1633 were constructed by homologous recombination. A 0.5-kb internal fragment of ppuI of strain PCL1445 was obtained by PCR using primers oMP902 (5'-ATGCATAAACTTCGGGCA-3') and oMP903 (5'-CATTTTCTCGACCCCCAC-3'), cloned into the pGEM-T Easy Vector System I (Promega Corporation, Madison, WI), and ligated as an EcoRI-EcoRI insert in the pMP5285 suicide plasmid (20) derived from pME3049 (8), resulting in pMP7568. pMP7568 was transferred to Pseudomonas PCL1445 by triparental mating using pRK2013 as a helper plasmid (31) and using selection on KB agar medium (17) supplemented with kanamycin (50 μg ml–1). Strain PCL1636 was obtained as a kanamycin-resistant colony resulting from single homologous recombination. The insertion of the suicide construct was confirmed by sequence analysis. To construct a PCL1633 ppuI mutant, the pGEM-T vector containing the 0.5-kb fragment of ppuI and a gentamicin resistance cassette cloned as a SalI-SalI fragment resulting in pMP7583 was used as a suicide plasmid. Single homologous recombination in PCL1633 carried out using pMP7583 resulted in PCL1639. A P. putida PCL1445 ppuR mutant was constructed by using a similar mutagenesis strategy. The ppuR fragment for the construction of the pMP5285-based suicide plasmid pMP7571 resulted from a PCR using primers oMP905 (5'-AATTCTTCGAAGAAGCCGCCG-3') and oMP906 (5'-TTGCTGGATGGCTTTGAGCACC-3') and chromosomal DNA of strain PCL1445 as a template. Single homologous recombination in ppuR of PCL1445 resulted in strain PCL1637. The P. putida PCL1445 rsaL mutant was constructed using the pMP7575 suicide plasmid based on pMP5285 obtained after cloning a 0.21-kb KpnI-SalI PCR fragment of the central part of the rsaL gene of PCL1445 obtained using primers oMP897 (3'-TACCTCAGCTGTGCGCGAGGT-5') and oMP898 (3'-GGTGGGCCAGGTCGCTTTCCT-5'). Single homologous recombination in rsaL of PCL1445 resulted in strain PCL1638.
Complementation of ppuI, ppuR, and rsaL mutants of PCL1445. Complementation of strain PCL1636 (ppuI) was carried out using pMP7565, a shuttle vector derived from pME6010 (14) in which a 1.4-kb fragment containing ppuI and rsaL of strain PCL1445 was inserted. This insert was obtained by EcoRI digestion from pMP5548. pMP7565 was transferred to strain PCL1636 by triparental mating as described above, and transformants were selected on KB agar medium supplemented with tetracycline (40 μg ml–1). To complement the ppuR insertion in PCL1637, a 1.1-kb PCR fragment containing the ppuR gene of strain PCL1445 was obtained using primers oMP883 (3'-TGTATATCCTGCTGCGCCTTTA-5') and oMP884 (3'-CATGTGCATCGTGGTGCTGCCT-5') and cloned into pME6010, resulting in pMP7566. To complement the rsaL insertion in PCL1638, a 1.6-kb PCR fragment containing the rsaL gene of strain PCL1445 was obtained using primers oMP1011 (3'-TTGTCAAGCAGTGCCACTGGTTCTAGAAAA-5') and oMP1012 (3'-ATCAGCGACATCTAGTCGTGGGAGCTCAAA-5') and cloned into pME6010, resulting in pMP7587.
Biosurfactant production. The production of biosurfactant activity was detected using the drop-collapsing assay, in which the reduction of the water surface tension can be observed in the collapse of a round droplet placed on a hydrophobic surface, as described previously (16).
To quantify the biosurfactant production in culture medium, the decrease of surface tension between culture medium and air was determined using a Du Nouy ring (K6 Kruss, GmbH, Hamburg, Germany).
Extraction and HPLC analysis of putisolvins. To quantify the production of putisolvins in BM culture medium, 10 ml of a BM culture supernatant was extracted with 1 volume of ethyl acetate (Fluka Chemie, Zwijndrecht, The Netherlands) as described previously (21). Ethyl acetate extracts were evaporated under a vacuum to dryness and dissolved in 55% acetonitrile (Labscan Ltd., Dublin, Ireland). The dried pellet obtained from the 10-ml culture was resuspended in 500 μl of 50/50 acetonitrile/water (vol/vol) and filtered using a 0.45-μm-pore-size SpinX centrifuge tube filter (Corning Costar Corporation, Cambridge, MA). A 500-μl volume of the samples was separated by use of a high-performance liquid chromatography (HPLC) system (Jasco International Co. Ltd., Japan) with a reverse-phase C8 5-μm Econosphere column (Alltech, Deerfield, IL), a PU-980 pump system (B&L Systems, Boechout, Belgium), an LG-980-02 gradient unit (Jasco), and an MD 910 detector (Jasco). Separation was performed using a linear gradient, starting at 35/65 acetonitrile/water (vol/vol) and ending at 20/80 after 50 min at a flow rate of 1 ml min–1. Chromatograms were analyzed in the wavelength range between 195 nm and 420 nm. Fractions that corresponded to the retention times of 20 min for putisolvin I and 21 min for putisolvin II were collected and tested for activity in the drop-collapsing assay. The amount of putisolvin produced was quantified as the peak area in microabsorbance units at 206 nm.
Quantification of bioluminescent Tn5luxAB reporter strains. Expression of Tn5luxAB genes was determined by quantification of bioluminescence during culturing. Cells from overnight cultures were washed with fresh medium and diluted to an optical density at 620 nm (OD620) of 0.1. Cultures were grown in BM medium in a 10-ml volume with vigorous shaking. During growth, 100-μl samples were taken in triplicate to quantify luminescence. A 100-μl volume of a 0.2% n-decyl-aldehyde substrate solution (Sigma, St. Louis, MO) in a 2.0% bovine serum albumin solution was added, and luminescence was determined with a MicroBeta 1450 TriLux luminescence counter (Wallac, Turku, Finland), which was normalized to the luminescence per OD620 unit. The synthetic AHL molecules N-hexanoyl-L-homoserine lactone (C6-AHL) (Fluka, Zwijndrecht, The Netherlands), N-octanoyl-L-homoserine lactone (C8-AHL) (Fluka), N-decanoyl-L-homoserine lactone (C10-AHL) (Fluka), N-dodecanoyl-L-homoserine lactone (C12-AHL), N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-AHL), N-(3-oxo-octanoyl)-L-homoserine lactone (3-oxo-C8-AHL), N-(3-oxo-decanoyl)-L-homoserine lactone (3-oxo-C10-AHL), N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-AHL), and N-(3-oxo-tetradecanoyl)-L-homoserine lactone (3-oxo-C14-AHL) were tested for the ability to induce the Tn5luxAB reporter strains. Briefly, cells were grown in BM-2% glycerol medium for 48 h, washed, and resuspended to an OD620 of 0.1 in fresh medium supplemented with either 5 μM of synthetic AHL or 25 μl of a 1,000-fold-concentrated crude extract of spent culture supernatant dissolved in 100% acetonitrile.
Biofilm assay. Biofilm formation on PVC was conducted as previously described by O'Toole and Kolter (28) and adapted for strain PCL1445 as previously described by Kuiper et al. (21). When the effect of AHLs on biofilm formation was tested, the culture medium and planktonic cells were removed after 4 h. Subsequently, 100 μl of M63 medium containing 1 μl of 5 μM synthetic 3-oxo-C12-AHL dissolved in 100% acetonitrile was added to the wells. An equal volume of acetonitrile was added to control wells. All conditions were tested in triplicate.
Nucleotide sequence accession numbers. The nucleotide sequences of the P. putida PCL1445 ppuI-rsaL-ppuR DNA region and putisolvin synthetase promoter region reported in this paper have been deposited in the GenBank database under accession numbers DQ151886 and DQ151887, respectively.
RESULTS
Production of AHLs by P. putida PCL1445. To test the possible production and secretion of AHLs, a crude dichloromethane extract of the spent BM-glycerol medium of a culture with an OD620 of 1 was tested for induction of an E. coli reporter strain based on the lux quorum-sensing system of Vibrio fischeri (Fig. 1A) and the las system of P. aeruginosa (Fig. 1B). After separation on C18 reverse-phase TLC, four compounds with Rf values similar to those of 3-oxo-C12-, 3-oxo-C10-, 3-oxo-C8-, and 3-oxo-C6-AHLs were detected (Fig. 1A and B). Furthermore, when the standard molecules were mixed with PCL1445 dichloromethane extracts, the four detected compounds comigrated with the standard AHLs (Fig. 1A and B, lanes 3). Dichloromethane extracts of the putisolvin biosynthetic mutant PCL1633 showed the same profile as the wild-type strain (data not shown).
Identification of quorum-sensing genes of P. putida PCL1445. To isolate a chromosomal fragment of strain PCL1445 containing luxI and luxR homologues, an EcoRI chromosomal library of PCL1445 was introduced into E. coli DH5 containing pAK211, a reporter strain for AHLs based on the lux system of Vibrio fischeri (22). The plasmid of one luminescent transformant, pMP5548, was isolated for analysis. Nucleotide sequence analysis of the 2.2-kb genomic fragment present in pMP5548 revealed the presence of several open reading frames which show homologies to suhB, ppuI, rsaL, and ppuR of P. putida. The identified sequences of the genes showed 99% identity with the ppu locus characterized in P. putida strains IsoF (33) and WCS358 (1), 57% identity with lasI of P. aeruginosa (29), and 51% identity with mupI of P. fluorescens (10) (Fig. 2A). The sequence of the gene located upstream of ppuI showed 100% identity with rsaL gene in P. putida IsoF (33) and WCS358 (1), and 60% identity with rsaL of P. aeruginosa (6). The rsaL gene was described first as a repressor of virulence genes in P. aeruginosa and later as a repressor of the ppuI gene in P. putida strains IsoF and WCS358. The open reading frame located downstream of ppuI showed 91% identity with the suhB of P. putida IsoF (33) and 78% with suhB of P. aeruginosa. The latter gene was suggested to possess inositol monophosphatase activity in E. coli (26).
To test whether the ppuI gene present in pMP5548 was responsible for the production of C10-, 3-oxo-C10-, C12-, and 3-oxo-C12-AHLs, dichloromethane extracts of the DH5 reporter containing pAK211 with or without pMP5548 were subjected to TLC analysis. The results showed the presence of the four AHLs detected in PCL1445 crude extracts with Rf values similar to those of C10-, 3-oxo-C10-, C12-, and 3-oxo-C12-AHLs (data not shown).
In the region upstream of ppuI and ppuA, nucleotide sequences identical to ppuI and ppuA lux box elements found in P. putida strains IsoF (33) and WCS358 (1) were found. A 16-bp palindromic sequence with high similarity to the lux box elements, which are located in the promoter region of quorum-sensing regulated genes of P. putida (33), P. aeruginosa (36), P. chlororaphis (2), and V. fischeri (7), is present 92 bp upstream of the psoA gene start codon (Fig. 2B) (9). These palindromes might constitute a binding site for the LuxR response regulator.
Expression of the putisolvin biosynthetic gene psoA is stimulated by AHLs. To analyze the effect of a mutation in the AHL biosynthetic gene ppuI on the expression of psoA, ppuI was mutated in strain PCL1633 (psoA::Tn5luxAB), resulting in strain PCL1639 (psoA/ppuI), in which psoA expression was quantified by measuring luminescence. The psoA expression appeared to be 10-fold lower in strain PCL1639 (ppuI/psoA) than the transcriptional activity detected in PCL1633 (psoA) (Table 2). The transcriptional activity of the psoA promoter in strain PCL1639 (ppuI/psoA) was analyzed in liquid culture at an OD620 of 1.5 after addition of crude 1,000-fold-concentrated dichloromethane extracts of the wild-type strain culture supernatant, or of the ppuI mutant, to an early-log-phase culture (OD620 of 0.2) of PCL1639. PCL1445 dichloromethane extract, but not ppuI mutant extract, was able to complement part of the psoA promoter activity in PCL1639 (Table 2).
The effect of C4-, C6-, 3-oxo-C6-, C8-, C10-, 3-oxo-C10-, C12-, and 3-oxo-C12-AHLs, added at a concentration of 5 μM to an early-log-phase culture (OD620, 0.2) of PCL1639, on psoA transcriptional activity was quantified at an OD620 of 1.5. The addition of AHLs without a 3-oxo group or with short acyl chains (C4, C6, and C8) did not significantly affect psoA::luxAB expression (Table 2). However, the psoA promoter activity was stimulated by addition of 3-oxo-C10-AHLs and even more strongly stimulated by addition of 3-oxo-C12-AHLs (Table 2).
Construction and characterization of ppuI, ppuR, and rsaL mutants. To investigate whether ppuI, ppuR, and rsaL are involved in putisolvin production, insertion mutants were constructed by single homologous recombination using suicide plasmids pMP7568, pMP7571, and pMP7575, respectively (see Materials and Methods), resulting in strains PCL1636, PCL1637, and PCL1638, respectively. The proper integration of plasmids pMP7568, pMP7571, and pMP7575 by homologous recombination into the chromosome was confirmed by sequencing the region flanking the suicide plasmids after isolation of the chromosomal DNA recombinants.
Putisolvin production by mutant strains PCL1636 (ppuI), PCL1637 (ppuR), and PCL1638 (rsaL) was investigated by two different approaches. Firstly, biosurfactant production by strains PCL1626 (ppuI), PCL1637 (ppuR), and PCL1638 (rsaL) was quantified by the Du Nouy ring method during growth until the stationary phase was reached (Fig. 3A). Secondly, the production of putisolvins I and II by strains PCL1445, PCL1636 (ppuI), PCL1637 (ppuR), and PCL1638 (rsaL) was tested by HPLC analysis (Fig. 3B and 3C).
Culture supernatants of PCL1636 (ppuI) and PCL1637 (ppuR) were not able to decrease the surface tension between culture medium and air compared to the wild type, indicating a lack of biosurfactant production (Fig. 3A). The culture supernatant of strain PCL1638 (rsaL) caused a dramatic decrease of surface tension during the early exponential phase (to 32 mN m–1 at an OD of 1), indicating an earlier production of biosurfactant than that of the wild-type strain (48 mN m–1 at OD 1) (Fig. 3A).
Mutants PCL1636 (ppuI) and PCL1637 (ppuR) showed a significant reduction (85%) of putisolvin production (Fig. 3B, bars c and e, respectively). Introduction of pMP7565 harboring the genomic fragment from pMP5548 with ppuI and pMP7566 harboring ppuR restored putisolvin production to wild-type levels in both strains PCL1636 (ppuI) and PCL1637 (ppuR) (Fig. 3B, bars d and f, respectively). The production of putisolvins by PCL1445 and mutant PCL1638 (rsaL) was compared by HPLC analysis at different stages of bacterial growth (Fig. 3C). Mutant PCL1638 (rsaL) shows a significantly increased putisolvin production during the early exponential phase compared to the wild type (fourfold at an OD620 of 0.6). This difference in production tends to decrease when the cells reach the stationary phase (twofold at an OD620 of 1.1 and hardly any difference at an OD620 of 2). Introduction of pMP7587 harboring rsaL into PCL1638 (rsaL) significantly decreased putisolvin production during exponential phase more than for the wild-type strain (twofold lower at an OD620 of 0.6). This is possibly due to the multiple-copy effect of the plasmid used for complementation of rsaL mutation (Fig. 3C).
To investigate the involvement of ppuI in AHL biosynthesis in PCL1445 and of rsaL in regulation of AHL biosynthesis in PCL1445, AHL production by strains PCL1445, PCL1636 (ppuI), PCL1636 harboring pMP7565 (ppuI), PCL1638 (rsaL), and PCL1638 harboring pMP7587 (rsaL) was examined by TLC analysis. Control vector pME6010 did not influence the AHL production, which remained the same as that of the wild type without pME6010 (Fig. 1C, lane 2). Mutant PCL1636 (ppuI) showed a total absence of AHL production (Fig. 1C, lane 3). AHL production by PCL1636 (ppuI) was restored by introduction of pMP7565 harboring a functional ppuI gene (Fig. 1C, lane 4). Finally, introduction of a mutation into rsaL (PCL1638) had a strongly positive effect on the production of AHLs by PCL1445 (Fig. 1C, lane 5). The AHL production by PCL1628 (rsaL) decreased dramatically compared to that of the wild type following introduction of pMP7587 harboring a functional rsaL gene (Fig. 1C, lane 6).
Effect of ppu quorum-sensing system on biofilm formation by PCL1445. Biofilm formation on PVC titer wells by PCL1445 and its mutants PCL1633 (psoA), PCL1636 (ppuI), PCL1637 (ppuR), and PCL1638 (rsaL) was measured at various times after inoculation (Fig. 4A). The size of the biofilms formed by mutants PCL1636 (ppuI) and PCL1637 (ppuR) was comparable to that of the putisolvin-deficient mutant (PCL1633) and considerably thicker than that of the wild type (Fig. 4A). To monitor the surfactant activity of the bacterial cells in the titer wells, culture samples were analyzed by the drop-collapsing assay. An index ranging from 0 to 4 was used to quantify biosurfactant production by bacterial cells in the biofilm assay (Fig. 4E). PCL1636 (ppuI) and PCL1637 (ppuR) did not have any detectable biosurfactant activity (Fig. 4B). Analysis of PCL1638 (rsaL) showed that biofilm formation decreased 1.5-fold compared to the wild-type strain (Fig. 4A), which correlates with an earlier appearance of biosurfactant activity (visible after 6 h) than that observed for PCL1445 (visible after 10 h) (Fig. 4B).
The effect of AHLs produced by PCL1445 on its biofilm-forming ability and consequently on the production of biosurfactants was analyzed in two different ways: (i) mutant PCL1636 (ppuI) was transformed with pMP5548 harboring ppuI, and (ii) exogenous 3-oxo-AHL (5 μM) was added to the medium. Biofilms were assayed after 24 h of incubation (Fig. 4C). Mutants PCL1636 (ppuI) (Fig. 4C, bar e) and PCL1633 (psoA) (Fig. 4C, bar d) form thicker biofilms than the wild type (Fig. 4C, bar b). Introduction of pMP5548 into PCL1636 (ppuI) restored biosurfactant production (Fig. 4D, lane f) and decreased biofilm formation to the same level as that reached by PCL1445 (Fig. 4C, bar f). Exogenous 3-oxo-C12-AHL signaling molecules also appeared to be able to stimulate production of biosurfactant by ppuI mutants (Fig. 4D, lane j) and reduce the thickness of the biofilm (Fig. 4C, bar j).
DISCUSSION
P. putida PCL1445 produces two cyclic lipopeptide biosurfactants (putisolvin I and II), which inhibit biofilm formation and degrade existing Pseudomonas biofilms (21). Initiation of putisolvin production starts at the onset of stationary phase (21), suggesting that putisolvin biosynthesis might be regulated by population density, which would imply that putisolvins are regulating the formation and thickness of the biofilm after the initial formation steps of the biofilm at high bacterial cell density. The aim of this work was to determine whether quorum sensing is regulating production of the cyclic lipopeptides putisolvin I and II by P. putida PCL1445 and, consequently, biofilm formation.
Using several bacterial reporter strains for the detection of AHLs we showed that PCL1445 produces at least four different inducing compounds, which are migrating at the same positions as 3-oxo-C6-, 3-oxo-C8-, 3-oxo-C10-, and 3-oxo-C12-AHL on TLC (Fig. 1). Two of these compounds, 3-oxo-C10- and 3-oxo-C12-AHLs, were shown to restore psoA promoter activity in double-mutant PCL1639 (ppuI/psoA) (Table 2). The AHLs lacking the 3-oxo group did not stimulate the psoA promoter (Table 2). Furthermore, we detected a palindromic sequence in the promoter region of psoA similar to the regulatory lux box (Fig. 2B), the presence of which is typical for genes under control of quorum sensing.
Regulation via quorum sensing involves a LuxI (homologous) protein, which directs the synthesis of signaling molecules, and the cognate transcriptional regulator LuxR, which binds to the operator of the target regulated gene. In strain PCL1445, a luxI homologous gene was identified as ppuI, and a luxR homologous gene was identified as ppuR (Fig. 2A). The ppuI and ppuR genes are transcribed in the same direction and separated by rsaL, which is transcribed in the opposite direction (Fig. 2A). RsaL was reported to play a role in the repression of lasI of P. aeruginosa (6) and of ppuI of P. putida WCS358 (1). A highly conserved palindromic sequence (lux box) was identified in the promoter regions of the ppuI and ppuR genes (Fig. 2A). Such a regulatory element is thought to represent the binding site for the LuxR homolog after activation by binding the appropriate AHL. The genetic organization of the ppu-rsaL-ppuR locus of PCL1445 is identical to that of the loci identified in P. putida IsoF (33) and P. putida WCS358 (1). Although the ppuI-rsaL-ppuR locus was reported to be involved in biofilm formation by P. putida IsoF, the molecular mechanism could not be explained (33). Members of the luxI and luxR families usually show weak homologies. The ppuI/ppuR quorum-sensing system is not widespread among P. putida members but seems to be evolutionarily well conserved and might regulate similar genes (33).
More detailed studies showed that a mutation in ppuI of PCL1445 abolishes the production of all four detected AHL compounds (Fig. 1C), indicating that ppuI is responsible for the production of AHLs. Mutation of ppuI and ppuR abolishes putisolvin production almost completely (Fig. 3B). Transcriptional analysis of the psoA promoter in a ppuI mutant background showed clearly that at least one of the quorum-sensing signals present in the medium (3-oxo-C12-AHL), which can be synthesized via ppuI, is able to induce putisolvin biosynthesis (Table 2). Our results show that ppuI and ppuR are responsible for production of AHLs and regulate putisolvin expression in PCL1445. Mutation of rsaL resulted in increased AHL production (Fig. 1C), suggesting that rsaL is involved in repressing ppuI and/or ppuR. Introducing a mutation into rsaL had a positive effect on putisolvin production during the lag phase (Fig. 3C), which can be explained by its repressive effect on AHL synthesis.
Biofilm formation in PVC titer wells indicated that ppuI and ppuR mutants, in which putisolvin production is strongly reduced (Fig. 4B), exhibit the same phenotype as a putisolvin biosynthetic mutant by forming a thicker biofilm (Fig. 4A), while a rsaL mutant forms even less biofilm than the wild type (Fig. 4A) and produces putisolvins at an earlier stage of biofilm formation (Fig. 4B). Most interestingly, when AHL signal molecules were added to the medium, the ppuI mutant started to produce biosurfactant and lost the ability to form a dense biofilm with a thickness comparable to that of a putisolvin biosynthetic mutant (Fig. 4C and 4D). These results show that biofilm formation in PCL1445 is regulated by the production of putisolvins in a cell population-dependent manner.
P. aeruginosa possesses two quorum-sensing systems, lasI/lasR and rhlI/rhlR, both of which are involved in the regulation of rhamnolipid surfactant production (5). In a recent study by Davey et al. (4) it was indicated that rhlI influences biofilm development. Rhamnolipids were shown to be involved in the maintenance of the P. aeruginosa biofilm architecture by keeping the water-filled channels of the biofilm opened (4). The observation that chemically unrelated molecules such as rhamnolipids and the cyclic lipopeptides putisolvin I and II, all of which have biosurfactant activity, are regulated by quorum sensing and are all involved in the regulation of biofilm formation and structure suggests that biosurfactants play an important role in biofilm structure and development.
The synthesis of the biosurfactant viscosin in P. fluorescens 5064 (3), as well as biosurfactants serrawettin W2 in Serratia liquefaciens (24) and a lipopeptide of unknown structure in Burkholderia cepacia (15), was also reported to be regulated by AHLs. The production of biosurfactants was shown to be essential for swarming motility of S. liquefaciens (24), P. aeruginosa (18), and B. cepacia (15). We have shown previously that putisolvins stimulate swarming motility (21), which could provide an explanation for the reducing effect of putisolvins on biofilm size or for the resultant breakdown of biofilm when they are added to a formed biofilm (21).
A role for AHL-mediated quorum sensing in biofilm formation was shown for B. cepacia (15), S. liquefaciens MG1 (23), and P. putida IsoF (33). For B. cepacia (15) and S. liquefaciens MG1 (23) it was demonstrated that expression of quorum-sensing system-controlled genes is crucial at a specific stage for the development and maturation of the biofilm. In contrast, wild-type P. putida IsoF produces a very homogenous biofilm while a quorum-sensing mutant appears to form a dense and structured biofilm with characteristic microcolonies and water-filled channels (33).
The present study clearly links quorum sensing in P. putida PCL1445 with the synthesis of the cyclic lipopeptides putisolvins I and II and thereby with biofilm formation. Putisolvins seem to function when the bacterial population reaches a high cell density. The high cell density could form a signal for the release of Pseudomonas putida cells. Such a release from the biofilm could be favorable when the nutrient level in the biofilm environment becomes limiting. Moreover, starvation-mediated stress could play an important role in cell detachment from biofilms since it has been shown for several Pseudomonas spp. that the stationary-phase sigma factor RpoS influences AHL production (1, 32). The production of biosurfactants could stimulate some of the bacteria to colonize other, more favorable, niches, therefore enhancing competitiveness (fitness), pollutant degradation capabilities, or even rhizosphere colonization.
ACKNOWLEDGMENTS
We thank P. Williams of the University of Nottingham, United Kingdom, for kindly providing the synthetic autoinducers C12-AHL, 3-oxo-C6-AHL, 3-oxo-C8-AHL, 3-oxo-C10-AHL, 3-oxo-C12-AHL, and 3-oxo-C14-AHL. We thank E. Lagendijk for technical assistance and P. Hock for processing the images and the graphs.
This research was supported by grant number 700.50.015 from the Council for Chemical Sciences of The Netherlands Foundation for Scientific Research (NWO/CW).
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ABSTRACT
Pseudomonas putida strain PCL1445 produces two cyclic lipopeptides, putisolvin I and putisolvin II, which possess surface tension-reducing abilities and are able to inhibit biofilm formation and to break down existing biofilms of several Pseudomonas spp., including P. aeruginosa. Putisolvins are secreted in the culture medium during growth at late exponential phase, indicating that production is possibly regulated by quorum sensing. In the present study, we identified a quorum-sensing system in PCL1445 that is composed of ppuI, rsaL, and ppuR and shows very high similarity with gene clusters of P. putida strains IsoF and WCS358. Strains with mutations in ppuI and ppuR showed a severe reduction of putisolvin production. Expression analysis of the putisolvin biosynthetic gene in a ppuI background showed decreased expression, which could be complemented by the addition of synthetic 3-oxo-C10-N-acyl homoserine lactone (3-oxo-C10-AHL) or 3-oxo-C12-AHL to the medium. An rsaL mutant overproduces AHLs, and production of putisolvins is induced early during growth. Analysis of biofilm formation on polyvinylchloride showed that ppuI and ppuR mutants produce a denser biofilm than PCL1445, which correlates with decreased production of putisolvins, whereas an rsaL mutant shows a delay in biofilm production, which correlates with early production of putisolvins. The results demonstrate that quorum-sensing signals induce the production of cyclic lipopeptides putisolvin I and II and consequently control biofilm formation by Pseudomonas putida.
INTRODUCTION
Bacteria can form multicellular aggregates generally referred to as biofilms on biotic and abiotic surfaces. Such communities are ubiquitous in natural environments but can also be found in industrial and clinical settings, for example, on artificial surfaces of medical devices, thereby greatly contributing to infections (34).
Pseudomonas putida strain PCL1445 is capable of forming biofilms on roots and on polyvinylchloride (PVC) in a commonly used biofilm assay (21). We have shown that P. putida PCL1445 produces two novel lipodepsipeptides, putisolvin I and II, consisting of a C6 lipid moiety and a 12-amino-acid peptide, which are produced by a putisolvin synthetase gene designated as psoA (21). A mutant with impaired putisolvin biosynthesis was shown to form a thicker biofilm than the wild-type strain. In addition, purified putisolvins I and II inhibit biofilm formation and break down existing biofilms of various Pseudomonas spp., including the opportunistic human pathogen P. aeruginosa (21). The production of putisolvins occurs at the end of the exponential growth phase (21), which may indicate that the production is mediated through a quorum-sensing mechanism. The term quorum sensing describes an environmental sensing system which allows bacteria to monitor their own population density. Quorum sensing in gram-negative bacteria relies on the interaction of small diffusible signal molecules belonging to the class of N-acyl homoserine lactones (AHLs). They are synthetized via the LuxI protein, whereas the transcriptional activator protein LuxR couples cell population density to gene expression (12, 35). These signal molecules can traffic in and out of the bacterial cell. Once a certain intracellular threshold concentration has been reached, the signals induce transcription of a set of target genes (11). AHLs play a role in regulating different bacterial functions such as antibiotic biosynthesis, production of virulence factors, bacterial swarming, and transition to the stationary growth phase. In this paper we describe (i) the identification and characterization of the regulatory quorum-sensing genes affecting cyclic lipopeptides putisolvins I and II in PCL1445, (ii) the involvement of the quorum-sensing system in the regulation of biofilm formation of PCL1445, and (iii) the direct relationship between production of quorum-sensing signals, production of cyclic lipopeptides, and reduction of the size of the biofilm formed by P. putida PCL1445.
MATERIALS AND METHODS
Bacterial strains and growth conditions. Bacterial strains used in this study are listed in Table 1. Pseudomonas strains were grown in King's medium B (17) or in a defined BM medium (25) supplemented with 2% of glycerol (BDH Laboratory Supplies Pool, England) at 28°C. Escherichia coli strains were grown in Luria-Bertani medium (30) at 37°C. Media were solidified with 1.8% agar (Select Agar; Invitrogen Life Technologies, Paisley, United Kingdom). The antibiotics kanamycin, tetracycline, gentamicin, and carbenicillin were added, when necessary, to final concentrations of 50, 40, 2, and 100 μg ml–1, respectively.
Extraction and detection of AHLs autoinducers from spent culture medium. To isolate autoinducer activity, 3 volumes of dichloromethane were added to 7 volumes of supernatant of a 50-ml BM bacterial culture and shaken for 1 h at 120 rpm. The organic phase was removed and dried by evaporation under a vacuum (27). Supernatant extracts were redissolved in 100 μl of ethyl acetate, and 10 μl was fractionated on a C18 reverse-phase thin-layer chromatography (TLC) plate (Merck, Darmstadt, Germany) developed in methanol-water (60:40, vol/vol).
To detect autoinducer activity, overnight cultures of E. coli DH5 containing pAK211 (22) or pSB1075 (37) were grown in LB medium supplemented with 20 μg of chloramphenicol per ml for 10 h. TLC plates were overlaid with 0.8% LB top agar containing 50 μl of the pAK211 or pSB1075 strains per ml and then incubated at 28°C for 16 h. Autoinducer activity was then detected by the emission of light after Fuji medical X-ray film (Fuji Photo Film Co., Ltd., Tokyo, Japan) was applied to the TLC plates.
Isolation and identification of quorum-sensing gene homologues. A plasmid library of chromosomal fragments of strain PCL1445 was constructed by cloning 1.5- to 3.0-kb fragments of chromosomal DNA digested with EcoRI into pBluescript (Stratagene, La Jolla, CA). The resulting fragment library was introduced into an E. coli strain harboring the lux reporter plasmid pAK211 (22). After overnight growth on LB agar plates, clones that induced the luciferase reporter were identified using photographic film. To remove pAK211 (22) from the E. coli reporter strain, the total plasmid was isolated and reintroduced into DH5 cells by standard transformation protocols (31) followed by carbenicillin selection, whereas chloramphenicol selection was omitted. The DNA sequence of the chromosomal fragment inserted in the selected plasmid pMP5548 was determined using universal primers, including M13-20 and M13 reverse primers flanking the multiple cloning sites of pBluescript.
Construction of ppuI, ppuR, and rsaL mutant strains. ppuI mutant derivatives of strains PCL1445 and PCL1633 were constructed by homologous recombination. A 0.5-kb internal fragment of ppuI of strain PCL1445 was obtained by PCR using primers oMP902 (5'-ATGCATAAACTTCGGGCA-3') and oMP903 (5'-CATTTTCTCGACCCCCAC-3'), cloned into the pGEM-T Easy Vector System I (Promega Corporation, Madison, WI), and ligated as an EcoRI-EcoRI insert in the pMP5285 suicide plasmid (20) derived from pME3049 (8), resulting in pMP7568. pMP7568 was transferred to Pseudomonas PCL1445 by triparental mating using pRK2013 as a helper plasmid (31) and using selection on KB agar medium (17) supplemented with kanamycin (50 μg ml–1). Strain PCL1636 was obtained as a kanamycin-resistant colony resulting from single homologous recombination. The insertion of the suicide construct was confirmed by sequence analysis. To construct a PCL1633 ppuI mutant, the pGEM-T vector containing the 0.5-kb fragment of ppuI and a gentamicin resistance cassette cloned as a SalI-SalI fragment resulting in pMP7583 was used as a suicide plasmid. Single homologous recombination in PCL1633 carried out using pMP7583 resulted in PCL1639. A P. putida PCL1445 ppuR mutant was constructed by using a similar mutagenesis strategy. The ppuR fragment for the construction of the pMP5285-based suicide plasmid pMP7571 resulted from a PCR using primers oMP905 (5'-AATTCTTCGAAGAAGCCGCCG-3') and oMP906 (5'-TTGCTGGATGGCTTTGAGCACC-3') and chromosomal DNA of strain PCL1445 as a template. Single homologous recombination in ppuR of PCL1445 resulted in strain PCL1637. The P. putida PCL1445 rsaL mutant was constructed using the pMP7575 suicide plasmid based on pMP5285 obtained after cloning a 0.21-kb KpnI-SalI PCR fragment of the central part of the rsaL gene of PCL1445 obtained using primers oMP897 (3'-TACCTCAGCTGTGCGCGAGGT-5') and oMP898 (3'-GGTGGGCCAGGTCGCTTTCCT-5'). Single homologous recombination in rsaL of PCL1445 resulted in strain PCL1638.
Complementation of ppuI, ppuR, and rsaL mutants of PCL1445. Complementation of strain PCL1636 (ppuI) was carried out using pMP7565, a shuttle vector derived from pME6010 (14) in which a 1.4-kb fragment containing ppuI and rsaL of strain PCL1445 was inserted. This insert was obtained by EcoRI digestion from pMP5548. pMP7565 was transferred to strain PCL1636 by triparental mating as described above, and transformants were selected on KB agar medium supplemented with tetracycline (40 μg ml–1). To complement the ppuR insertion in PCL1637, a 1.1-kb PCR fragment containing the ppuR gene of strain PCL1445 was obtained using primers oMP883 (3'-TGTATATCCTGCTGCGCCTTTA-5') and oMP884 (3'-CATGTGCATCGTGGTGCTGCCT-5') and cloned into pME6010, resulting in pMP7566. To complement the rsaL insertion in PCL1638, a 1.6-kb PCR fragment containing the rsaL gene of strain PCL1445 was obtained using primers oMP1011 (3'-TTGTCAAGCAGTGCCACTGGTTCTAGAAAA-5') and oMP1012 (3'-ATCAGCGACATCTAGTCGTGGGAGCTCAAA-5') and cloned into pME6010, resulting in pMP7587.
Biosurfactant production. The production of biosurfactant activity was detected using the drop-collapsing assay, in which the reduction of the water surface tension can be observed in the collapse of a round droplet placed on a hydrophobic surface, as described previously (16).
To quantify the biosurfactant production in culture medium, the decrease of surface tension between culture medium and air was determined using a Du Nouy ring (K6 Kruss, GmbH, Hamburg, Germany).
Extraction and HPLC analysis of putisolvins. To quantify the production of putisolvins in BM culture medium, 10 ml of a BM culture supernatant was extracted with 1 volume of ethyl acetate (Fluka Chemie, Zwijndrecht, The Netherlands) as described previously (21). Ethyl acetate extracts were evaporated under a vacuum to dryness and dissolved in 55% acetonitrile (Labscan Ltd., Dublin, Ireland). The dried pellet obtained from the 10-ml culture was resuspended in 500 μl of 50/50 acetonitrile/water (vol/vol) and filtered using a 0.45-μm-pore-size SpinX centrifuge tube filter (Corning Costar Corporation, Cambridge, MA). A 500-μl volume of the samples was separated by use of a high-performance liquid chromatography (HPLC) system (Jasco International Co. Ltd., Japan) with a reverse-phase C8 5-μm Econosphere column (Alltech, Deerfield, IL), a PU-980 pump system (B&L Systems, Boechout, Belgium), an LG-980-02 gradient unit (Jasco), and an MD 910 detector (Jasco). Separation was performed using a linear gradient, starting at 35/65 acetonitrile/water (vol/vol) and ending at 20/80 after 50 min at a flow rate of 1 ml min–1. Chromatograms were analyzed in the wavelength range between 195 nm and 420 nm. Fractions that corresponded to the retention times of 20 min for putisolvin I and 21 min for putisolvin II were collected and tested for activity in the drop-collapsing assay. The amount of putisolvin produced was quantified as the peak area in microabsorbance units at 206 nm.
Quantification of bioluminescent Tn5luxAB reporter strains. Expression of Tn5luxAB genes was determined by quantification of bioluminescence during culturing. Cells from overnight cultures were washed with fresh medium and diluted to an optical density at 620 nm (OD620) of 0.1. Cultures were grown in BM medium in a 10-ml volume with vigorous shaking. During growth, 100-μl samples were taken in triplicate to quantify luminescence. A 100-μl volume of a 0.2% n-decyl-aldehyde substrate solution (Sigma, St. Louis, MO) in a 2.0% bovine serum albumin solution was added, and luminescence was determined with a MicroBeta 1450 TriLux luminescence counter (Wallac, Turku, Finland), which was normalized to the luminescence per OD620 unit. The synthetic AHL molecules N-hexanoyl-L-homoserine lactone (C6-AHL) (Fluka, Zwijndrecht, The Netherlands), N-octanoyl-L-homoserine lactone (C8-AHL) (Fluka), N-decanoyl-L-homoserine lactone (C10-AHL) (Fluka), N-dodecanoyl-L-homoserine lactone (C12-AHL), N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-AHL), N-(3-oxo-octanoyl)-L-homoserine lactone (3-oxo-C8-AHL), N-(3-oxo-decanoyl)-L-homoserine lactone (3-oxo-C10-AHL), N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-AHL), and N-(3-oxo-tetradecanoyl)-L-homoserine lactone (3-oxo-C14-AHL) were tested for the ability to induce the Tn5luxAB reporter strains. Briefly, cells were grown in BM-2% glycerol medium for 48 h, washed, and resuspended to an OD620 of 0.1 in fresh medium supplemented with either 5 μM of synthetic AHL or 25 μl of a 1,000-fold-concentrated crude extract of spent culture supernatant dissolved in 100% acetonitrile.
Biofilm assay. Biofilm formation on PVC was conducted as previously described by O'Toole and Kolter (28) and adapted for strain PCL1445 as previously described by Kuiper et al. (21). When the effect of AHLs on biofilm formation was tested, the culture medium and planktonic cells were removed after 4 h. Subsequently, 100 μl of M63 medium containing 1 μl of 5 μM synthetic 3-oxo-C12-AHL dissolved in 100% acetonitrile was added to the wells. An equal volume of acetonitrile was added to control wells. All conditions were tested in triplicate.
Nucleotide sequence accession numbers. The nucleotide sequences of the P. putida PCL1445 ppuI-rsaL-ppuR DNA region and putisolvin synthetase promoter region reported in this paper have been deposited in the GenBank database under accession numbers DQ151886 and DQ151887, respectively.
RESULTS
Production of AHLs by P. putida PCL1445. To test the possible production and secretion of AHLs, a crude dichloromethane extract of the spent BM-glycerol medium of a culture with an OD620 of 1 was tested for induction of an E. coli reporter strain based on the lux quorum-sensing system of Vibrio fischeri (Fig. 1A) and the las system of P. aeruginosa (Fig. 1B). After separation on C18 reverse-phase TLC, four compounds with Rf values similar to those of 3-oxo-C12-, 3-oxo-C10-, 3-oxo-C8-, and 3-oxo-C6-AHLs were detected (Fig. 1A and B). Furthermore, when the standard molecules were mixed with PCL1445 dichloromethane extracts, the four detected compounds comigrated with the standard AHLs (Fig. 1A and B, lanes 3). Dichloromethane extracts of the putisolvin biosynthetic mutant PCL1633 showed the same profile as the wild-type strain (data not shown).
Identification of quorum-sensing genes of P. putida PCL1445. To isolate a chromosomal fragment of strain PCL1445 containing luxI and luxR homologues, an EcoRI chromosomal library of PCL1445 was introduced into E. coli DH5 containing pAK211, a reporter strain for AHLs based on the lux system of Vibrio fischeri (22). The plasmid of one luminescent transformant, pMP5548, was isolated for analysis. Nucleotide sequence analysis of the 2.2-kb genomic fragment present in pMP5548 revealed the presence of several open reading frames which show homologies to suhB, ppuI, rsaL, and ppuR of P. putida. The identified sequences of the genes showed 99% identity with the ppu locus characterized in P. putida strains IsoF (33) and WCS358 (1), 57% identity with lasI of P. aeruginosa (29), and 51% identity with mupI of P. fluorescens (10) (Fig. 2A). The sequence of the gene located upstream of ppuI showed 100% identity with rsaL gene in P. putida IsoF (33) and WCS358 (1), and 60% identity with rsaL of P. aeruginosa (6). The rsaL gene was described first as a repressor of virulence genes in P. aeruginosa and later as a repressor of the ppuI gene in P. putida strains IsoF and WCS358. The open reading frame located downstream of ppuI showed 91% identity with the suhB of P. putida IsoF (33) and 78% with suhB of P. aeruginosa. The latter gene was suggested to possess inositol monophosphatase activity in E. coli (26).
To test whether the ppuI gene present in pMP5548 was responsible for the production of C10-, 3-oxo-C10-, C12-, and 3-oxo-C12-AHLs, dichloromethane extracts of the DH5 reporter containing pAK211 with or without pMP5548 were subjected to TLC analysis. The results showed the presence of the four AHLs detected in PCL1445 crude extracts with Rf values similar to those of C10-, 3-oxo-C10-, C12-, and 3-oxo-C12-AHLs (data not shown).
In the region upstream of ppuI and ppuA, nucleotide sequences identical to ppuI and ppuA lux box elements found in P. putida strains IsoF (33) and WCS358 (1) were found. A 16-bp palindromic sequence with high similarity to the lux box elements, which are located in the promoter region of quorum-sensing regulated genes of P. putida (33), P. aeruginosa (36), P. chlororaphis (2), and V. fischeri (7), is present 92 bp upstream of the psoA gene start codon (Fig. 2B) (9). These palindromes might constitute a binding site for the LuxR response regulator.
Expression of the putisolvin biosynthetic gene psoA is stimulated by AHLs. To analyze the effect of a mutation in the AHL biosynthetic gene ppuI on the expression of psoA, ppuI was mutated in strain PCL1633 (psoA::Tn5luxAB), resulting in strain PCL1639 (psoA/ppuI), in which psoA expression was quantified by measuring luminescence. The psoA expression appeared to be 10-fold lower in strain PCL1639 (ppuI/psoA) than the transcriptional activity detected in PCL1633 (psoA) (Table 2). The transcriptional activity of the psoA promoter in strain PCL1639 (ppuI/psoA) was analyzed in liquid culture at an OD620 of 1.5 after addition of crude 1,000-fold-concentrated dichloromethane extracts of the wild-type strain culture supernatant, or of the ppuI mutant, to an early-log-phase culture (OD620 of 0.2) of PCL1639. PCL1445 dichloromethane extract, but not ppuI mutant extract, was able to complement part of the psoA promoter activity in PCL1639 (Table 2).
The effect of C4-, C6-, 3-oxo-C6-, C8-, C10-, 3-oxo-C10-, C12-, and 3-oxo-C12-AHLs, added at a concentration of 5 μM to an early-log-phase culture (OD620, 0.2) of PCL1639, on psoA transcriptional activity was quantified at an OD620 of 1.5. The addition of AHLs without a 3-oxo group or with short acyl chains (C4, C6, and C8) did not significantly affect psoA::luxAB expression (Table 2). However, the psoA promoter activity was stimulated by addition of 3-oxo-C10-AHLs and even more strongly stimulated by addition of 3-oxo-C12-AHLs (Table 2).
Construction and characterization of ppuI, ppuR, and rsaL mutants. To investigate whether ppuI, ppuR, and rsaL are involved in putisolvin production, insertion mutants were constructed by single homologous recombination using suicide plasmids pMP7568, pMP7571, and pMP7575, respectively (see Materials and Methods), resulting in strains PCL1636, PCL1637, and PCL1638, respectively. The proper integration of plasmids pMP7568, pMP7571, and pMP7575 by homologous recombination into the chromosome was confirmed by sequencing the region flanking the suicide plasmids after isolation of the chromosomal DNA recombinants.
Putisolvin production by mutant strains PCL1636 (ppuI), PCL1637 (ppuR), and PCL1638 (rsaL) was investigated by two different approaches. Firstly, biosurfactant production by strains PCL1626 (ppuI), PCL1637 (ppuR), and PCL1638 (rsaL) was quantified by the Du Nouy ring method during growth until the stationary phase was reached (Fig. 3A). Secondly, the production of putisolvins I and II by strains PCL1445, PCL1636 (ppuI), PCL1637 (ppuR), and PCL1638 (rsaL) was tested by HPLC analysis (Fig. 3B and 3C).
Culture supernatants of PCL1636 (ppuI) and PCL1637 (ppuR) were not able to decrease the surface tension between culture medium and air compared to the wild type, indicating a lack of biosurfactant production (Fig. 3A). The culture supernatant of strain PCL1638 (rsaL) caused a dramatic decrease of surface tension during the early exponential phase (to 32 mN m–1 at an OD of 1), indicating an earlier production of biosurfactant than that of the wild-type strain (48 mN m–1 at OD 1) (Fig. 3A).
Mutants PCL1636 (ppuI) and PCL1637 (ppuR) showed a significant reduction (85%) of putisolvin production (Fig. 3B, bars c and e, respectively). Introduction of pMP7565 harboring the genomic fragment from pMP5548 with ppuI and pMP7566 harboring ppuR restored putisolvin production to wild-type levels in both strains PCL1636 (ppuI) and PCL1637 (ppuR) (Fig. 3B, bars d and f, respectively). The production of putisolvins by PCL1445 and mutant PCL1638 (rsaL) was compared by HPLC analysis at different stages of bacterial growth (Fig. 3C). Mutant PCL1638 (rsaL) shows a significantly increased putisolvin production during the early exponential phase compared to the wild type (fourfold at an OD620 of 0.6). This difference in production tends to decrease when the cells reach the stationary phase (twofold at an OD620 of 1.1 and hardly any difference at an OD620 of 2). Introduction of pMP7587 harboring rsaL into PCL1638 (rsaL) significantly decreased putisolvin production during exponential phase more than for the wild-type strain (twofold lower at an OD620 of 0.6). This is possibly due to the multiple-copy effect of the plasmid used for complementation of rsaL mutation (Fig. 3C).
To investigate the involvement of ppuI in AHL biosynthesis in PCL1445 and of rsaL in regulation of AHL biosynthesis in PCL1445, AHL production by strains PCL1445, PCL1636 (ppuI), PCL1636 harboring pMP7565 (ppuI), PCL1638 (rsaL), and PCL1638 harboring pMP7587 (rsaL) was examined by TLC analysis. Control vector pME6010 did not influence the AHL production, which remained the same as that of the wild type without pME6010 (Fig. 1C, lane 2). Mutant PCL1636 (ppuI) showed a total absence of AHL production (Fig. 1C, lane 3). AHL production by PCL1636 (ppuI) was restored by introduction of pMP7565 harboring a functional ppuI gene (Fig. 1C, lane 4). Finally, introduction of a mutation into rsaL (PCL1638) had a strongly positive effect on the production of AHLs by PCL1445 (Fig. 1C, lane 5). The AHL production by PCL1628 (rsaL) decreased dramatically compared to that of the wild type following introduction of pMP7587 harboring a functional rsaL gene (Fig. 1C, lane 6).
Effect of ppu quorum-sensing system on biofilm formation by PCL1445. Biofilm formation on PVC titer wells by PCL1445 and its mutants PCL1633 (psoA), PCL1636 (ppuI), PCL1637 (ppuR), and PCL1638 (rsaL) was measured at various times after inoculation (Fig. 4A). The size of the biofilms formed by mutants PCL1636 (ppuI) and PCL1637 (ppuR) was comparable to that of the putisolvin-deficient mutant (PCL1633) and considerably thicker than that of the wild type (Fig. 4A). To monitor the surfactant activity of the bacterial cells in the titer wells, culture samples were analyzed by the drop-collapsing assay. An index ranging from 0 to 4 was used to quantify biosurfactant production by bacterial cells in the biofilm assay (Fig. 4E). PCL1636 (ppuI) and PCL1637 (ppuR) did not have any detectable biosurfactant activity (Fig. 4B). Analysis of PCL1638 (rsaL) showed that biofilm formation decreased 1.5-fold compared to the wild-type strain (Fig. 4A), which correlates with an earlier appearance of biosurfactant activity (visible after 6 h) than that observed for PCL1445 (visible after 10 h) (Fig. 4B).
The effect of AHLs produced by PCL1445 on its biofilm-forming ability and consequently on the production of biosurfactants was analyzed in two different ways: (i) mutant PCL1636 (ppuI) was transformed with pMP5548 harboring ppuI, and (ii) exogenous 3-oxo-AHL (5 μM) was added to the medium. Biofilms were assayed after 24 h of incubation (Fig. 4C). Mutants PCL1636 (ppuI) (Fig. 4C, bar e) and PCL1633 (psoA) (Fig. 4C, bar d) form thicker biofilms than the wild type (Fig. 4C, bar b). Introduction of pMP5548 into PCL1636 (ppuI) restored biosurfactant production (Fig. 4D, lane f) and decreased biofilm formation to the same level as that reached by PCL1445 (Fig. 4C, bar f). Exogenous 3-oxo-C12-AHL signaling molecules also appeared to be able to stimulate production of biosurfactant by ppuI mutants (Fig. 4D, lane j) and reduce the thickness of the biofilm (Fig. 4C, bar j).
DISCUSSION
P. putida PCL1445 produces two cyclic lipopeptide biosurfactants (putisolvin I and II), which inhibit biofilm formation and degrade existing Pseudomonas biofilms (21). Initiation of putisolvin production starts at the onset of stationary phase (21), suggesting that putisolvin biosynthesis might be regulated by population density, which would imply that putisolvins are regulating the formation and thickness of the biofilm after the initial formation steps of the biofilm at high bacterial cell density. The aim of this work was to determine whether quorum sensing is regulating production of the cyclic lipopeptides putisolvin I and II by P. putida PCL1445 and, consequently, biofilm formation.
Using several bacterial reporter strains for the detection of AHLs we showed that PCL1445 produces at least four different inducing compounds, which are migrating at the same positions as 3-oxo-C6-, 3-oxo-C8-, 3-oxo-C10-, and 3-oxo-C12-AHL on TLC (Fig. 1). Two of these compounds, 3-oxo-C10- and 3-oxo-C12-AHLs, were shown to restore psoA promoter activity in double-mutant PCL1639 (ppuI/psoA) (Table 2). The AHLs lacking the 3-oxo group did not stimulate the psoA promoter (Table 2). Furthermore, we detected a palindromic sequence in the promoter region of psoA similar to the regulatory lux box (Fig. 2B), the presence of which is typical for genes under control of quorum sensing.
Regulation via quorum sensing involves a LuxI (homologous) protein, which directs the synthesis of signaling molecules, and the cognate transcriptional regulator LuxR, which binds to the operator of the target regulated gene. In strain PCL1445, a luxI homologous gene was identified as ppuI, and a luxR homologous gene was identified as ppuR (Fig. 2A). The ppuI and ppuR genes are transcribed in the same direction and separated by rsaL, which is transcribed in the opposite direction (Fig. 2A). RsaL was reported to play a role in the repression of lasI of P. aeruginosa (6) and of ppuI of P. putida WCS358 (1). A highly conserved palindromic sequence (lux box) was identified in the promoter regions of the ppuI and ppuR genes (Fig. 2A). Such a regulatory element is thought to represent the binding site for the LuxR homolog after activation by binding the appropriate AHL. The genetic organization of the ppu-rsaL-ppuR locus of PCL1445 is identical to that of the loci identified in P. putida IsoF (33) and P. putida WCS358 (1). Although the ppuI-rsaL-ppuR locus was reported to be involved in biofilm formation by P. putida IsoF, the molecular mechanism could not be explained (33). Members of the luxI and luxR families usually show weak homologies. The ppuI/ppuR quorum-sensing system is not widespread among P. putida members but seems to be evolutionarily well conserved and might regulate similar genes (33).
More detailed studies showed that a mutation in ppuI of PCL1445 abolishes the production of all four detected AHL compounds (Fig. 1C), indicating that ppuI is responsible for the production of AHLs. Mutation of ppuI and ppuR abolishes putisolvin production almost completely (Fig. 3B). Transcriptional analysis of the psoA promoter in a ppuI mutant background showed clearly that at least one of the quorum-sensing signals present in the medium (3-oxo-C12-AHL), which can be synthesized via ppuI, is able to induce putisolvin biosynthesis (Table 2). Our results show that ppuI and ppuR are responsible for production of AHLs and regulate putisolvin expression in PCL1445. Mutation of rsaL resulted in increased AHL production (Fig. 1C), suggesting that rsaL is involved in repressing ppuI and/or ppuR. Introducing a mutation into rsaL had a positive effect on putisolvin production during the lag phase (Fig. 3C), which can be explained by its repressive effect on AHL synthesis.
Biofilm formation in PVC titer wells indicated that ppuI and ppuR mutants, in which putisolvin production is strongly reduced (Fig. 4B), exhibit the same phenotype as a putisolvin biosynthetic mutant by forming a thicker biofilm (Fig. 4A), while a rsaL mutant forms even less biofilm than the wild type (Fig. 4A) and produces putisolvins at an earlier stage of biofilm formation (Fig. 4B). Most interestingly, when AHL signal molecules were added to the medium, the ppuI mutant started to produce biosurfactant and lost the ability to form a dense biofilm with a thickness comparable to that of a putisolvin biosynthetic mutant (Fig. 4C and 4D). These results show that biofilm formation in PCL1445 is regulated by the production of putisolvins in a cell population-dependent manner.
P. aeruginosa possesses two quorum-sensing systems, lasI/lasR and rhlI/rhlR, both of which are involved in the regulation of rhamnolipid surfactant production (5). In a recent study by Davey et al. (4) it was indicated that rhlI influences biofilm development. Rhamnolipids were shown to be involved in the maintenance of the P. aeruginosa biofilm architecture by keeping the water-filled channels of the biofilm opened (4). The observation that chemically unrelated molecules such as rhamnolipids and the cyclic lipopeptides putisolvin I and II, all of which have biosurfactant activity, are regulated by quorum sensing and are all involved in the regulation of biofilm formation and structure suggests that biosurfactants play an important role in biofilm structure and development.
The synthesis of the biosurfactant viscosin in P. fluorescens 5064 (3), as well as biosurfactants serrawettin W2 in Serratia liquefaciens (24) and a lipopeptide of unknown structure in Burkholderia cepacia (15), was also reported to be regulated by AHLs. The production of biosurfactants was shown to be essential for swarming motility of S. liquefaciens (24), P. aeruginosa (18), and B. cepacia (15). We have shown previously that putisolvins stimulate swarming motility (21), which could provide an explanation for the reducing effect of putisolvins on biofilm size or for the resultant breakdown of biofilm when they are added to a formed biofilm (21).
A role for AHL-mediated quorum sensing in biofilm formation was shown for B. cepacia (15), S. liquefaciens MG1 (23), and P. putida IsoF (33). For B. cepacia (15) and S. liquefaciens MG1 (23) it was demonstrated that expression of quorum-sensing system-controlled genes is crucial at a specific stage for the development and maturation of the biofilm. In contrast, wild-type P. putida IsoF produces a very homogenous biofilm while a quorum-sensing mutant appears to form a dense and structured biofilm with characteristic microcolonies and water-filled channels (33).
The present study clearly links quorum sensing in P. putida PCL1445 with the synthesis of the cyclic lipopeptides putisolvins I and II and thereby with biofilm formation. Putisolvins seem to function when the bacterial population reaches a high cell density. The high cell density could form a signal for the release of Pseudomonas putida cells. Such a release from the biofilm could be favorable when the nutrient level in the biofilm environment becomes limiting. Moreover, starvation-mediated stress could play an important role in cell detachment from biofilms since it has been shown for several Pseudomonas spp. that the stationary-phase sigma factor RpoS influences AHL production (1, 32). The production of biosurfactants could stimulate some of the bacteria to colonize other, more favorable, niches, therefore enhancing competitiveness (fitness), pollutant degradation capabilities, or even rhizosphere colonization.
ACKNOWLEDGMENTS
We thank P. Williams of the University of Nottingham, United Kingdom, for kindly providing the synthetic autoinducers C12-AHL, 3-oxo-C6-AHL, 3-oxo-C8-AHL, 3-oxo-C10-AHL, 3-oxo-C12-AHL, and 3-oxo-C14-AHL. We thank E. Lagendijk for technical assistance and P. Hock for processing the images and the graphs.
This research was supported by grant number 700.50.015 from the Council for Chemical Sciences of The Netherlands Foundation for Scientific Research (NWO/CW).
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