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Key Laboratory of Microbial Metabolism, Ministry of Education, College of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China
Correspondence
Yuquan Xu
xuyq{at}sjtu.edu.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence of the rhlI gene and the partial sequence of the rhlR gene of Pseudomonas sp. M18 is DQ345445.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Thomashow & Weller, 1996). Various pseudomonad strains used in bio-control synthesize a battery of antibiotics, including polyketides and phenazines, as well as others (Bender et al., 1999; Turner & Messenger, 1986). The biosynthesis and secretion of these antibiotics occur mostly after the cell has entered into the stationary phase of growth, when bacterial densities have reached a certain threshold; this threshold is detected by a process called quorum sensing (QS) (Fuqua et al., 2001; Withers et al., 2001). QS regulates gene expression via signalling molecules, such as N-acyl-homoserine lactones (AHLs), in Gram-negative bacteria (Taga & Bassler, 2003; Waters & Bassler, 2005). The concentration of AHL increases along with the increasing bacterial cell density. When the concentration of AHL reaches a threshold level, it binds to specific transcriptional regulators belonging to the LuxR family, and forms various activated protein–AHL complexes. These complexes regulate the transcription of specific target genes, including various antibiotic biosynthesis gene clusters in Pseudomonas spp. (Haas & Keel, 2003).
The QS system, and its function in the regulation of antibiotic production, have been thoroughly investigated in some phenazine-producing strains, particularly Pseudomonas aeruginosa PAO1. There are at least two QS systems in P. aeruginosa PAO1, and these are called the las (LasI–LasR) and rhl (RhlI–RhlR) systems (Gambello & Iglewski, 1991; Ochsner et al., 1994). The major AHLs synthesized by LasI and RhlI are N-(3-oxododecanoyl)-homoserine lactone and N-butyrylhomoserine lactone (BHL), respectively (Pearson et al., 1994, 1995). Furthermore, these two QS systems do not function independently, but rather arrange in a cascade to positively control the expression of biosynthetic genes for phenazine production (Brint & Ohman, 1995; Pesci et al., 1997; Schuster et al., 2003). In three other phenazine-producing Pseudomonas strains, i.e. P. aureofaciens 30-84, P. fluorescens 2-79 and P. chlororaphis PCL1391, a conserved system called the PhzI–PhzR QS system has been found to positively regulate phenazine production (Chin-A-Woeng et al., 2001; Khan et al., 2005; Wood et al., 1997). In addition, biosynthesis of certain polyketide antibiotics depends on the QS system; for example, the biosynthesis of mupirocin (pseudomonic acid) requires the MupI–MupR QS system in P. fluorescens NCIMB 10586 (El-Sayed et al., 2001).
Pyoluteorin (Plt) is a polyketide compound that can effectively suppress several oomycete fungi including the seed- and root-rotting pathogen Pythium ultimum (Bailey et al., 1973; Howell & Stipanovic, 1980; Maurhofer et al., 1992). Plt is produced by several strains of Pseudomonas spp., including the soil-borne bacteria P. fluorescens Pf-5 and CHA0, and the regulation of Plt biosynthesis has been investigated in both of these strains. Studies have revealed that the regulatory circuit controlling Plt production includes positive autoregulation, with Plt acting as a signalling molecule (Brodhagen et al., 2004). The extracellular concentration of Plt increases in parallel with cell density, and accumulates to detectable levels until the cells begin to enter the stationary phase (Brodhagen et al., 2004). In addition, PltR, the LysR-type transcriptional activator, has been presumed to be a candidate receptor (Brodhagen et al., 2004). These properties are very similar to those of AHLs; however, AHLs have not yet been found in pseudomonad strains such as P. fluorescens Pf-5 and CHA0, which are known to produce Plt and PltR. Except for these preliminary results that suggest the presence of AHLs, little is known about the relationship between the QS system and Plt production.
The Pseudomonas sp. strain M18 adopted in this study is an effective bio-control agent against soil-borne phytopathogens (Hu et al., 2005). This capability is primarily due to its ability to produce two antibiotics: phenazine-1-carboxylic acid (PCA) and Plt. To the best of the authors' knowledge, this pseudomonad is the first strain that has been reported to produce these two different types of antibiotics together in a single cell (Ge et al., 2004; Hu et al., 2005). In previous work, we described how Plt is positively regulated, and PCA is negatively regulated, by a global regulator called GacA (Ge et al., 2004); however, the reverse relationship occurs in Pseudomonas sp. M18 through another global regulator, RsmA (Zhang et al., 2005). We have also identified a novel pathway-specific regulator of PltZ that could specifically repress Plt biosynthesis (Huang et al., 2004). In a more recent study, we characterized a putative Plt-induced ABC transporter cassette required for Plt production in Pseudomonas sp. M18 (Huang et al., 2006); however, no research has yet been done on the regulation of Plt production by potential AHL signalling molecules secreted by this strain.
This study was initiated using a red-pigment-negative transposon mutant M18-T510, which was derived from Pseudomonas sp. M18. We then identified an rhl QS system, and, to the best of our knowledge, this is the first report indicating that this system can regulate Plt production in Pseudomonas sp. M18. We further demonstrate that Plt production is negatively controlled by this rhl QS system at the transcriptional level, and that this regulation is partially mediated by PltR. In addition, we present evidence that expression of a Plt-specific ABC transporter is also negatively regulated by the rhl QS system in a Plt-dependent manner. Finally, we demonstrate that cell growth and red pigment production are also under the control of the rhl QS system.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The other bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli was routinely grown at 37 °C in Luria–Bertani (LB) medium (Sambrook et al., 1989). Pseudomonas sp. M18 and its derivatives were incubated at 28 °C in King's medium B (KMB; King et al., 1954). Normal growth conditions for strain M18 and its derivatives were as follows: one colony was inoculated into a 250 ml flask containing 25 ml KMB broth, and grown at 28 °C, with shaking at 220 r.p.m., in a C25KC incubator shaker (New Brunswick Scientific). After 10 h growth, 7.5 ml of each strain was transferred to a 500 ml flask containing 150 ml KMB broth, and shaken at 28 °C, as described above. Antibiotics were used at the following concentrations (µg ml–1): for pseudomonads, gentamicin (Gm) 40, kanamycin (Km) 50, spectinomycin (Sp) 100, and tetracycline (Tc) 125; for E. coli, Km 50, Gm 15, ampicillin (Ap) 100.
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). Restriction fragments were purified from agarose gels using the DNA gel extraction kit (V-gene Biotech). Primers used are listed in Table 1
. PCR products were recovered, and directly sequenced at the Sangon Biological Engineering Technology and Service (Shanghai, China).
Plasmid and mutant construction.
Genomic DNA from strain M18-T510 was digested with the restriction enzyme PstI, and the DNA fragment containing the Kmr cassette from the transposon mini-Tn5 lacZ-tet/1 and the partial downstream flanking sequence was visualized via routine Southern hybridization, as described (Sambrook et al., 1989). The probe used was a 1.7 kb XbaI fragment containing the Kmr cassette from plasmid pVDL24 (de Lorenzo et al., 1993). The Kmr cassette was located on a 2.3 kb PstI fragment from the genomic DNA of strain M18-T510. This PstI fragment was cloned into pBLS, creating the plasmid pBLST510 (Table 1). The nucleotide sequence of chromosomal DNA downstream of the insertion junction was determined using the primer PSRMTN5 (Table 1).
The RhlI coding region was amplified with Pfu polymerase from 1 ng Pseudomonas sp. M18 chromosomal DNA using primers PRSMRHLI1 and PRSMRHLI2 (Table 1), under the following cycling conditions: one initial step of 5 min at 94 °C; 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 3 min; followed by one final step of 5 min at 72 °C. The resulting 2.0 kb PCR product was purified and digested with XbaI and HindIII, and cloned into plasmid pBLS to create pBLSRHLI (Table 1). A 1.6 kb PCR product including rhlR and partial flanking sequences was obtained in a similar manner using primers PRSMRHLR1 and PRSMRHLR2 (Table 1). The 1.6 kb PCR product was purified and digested with EcoRI and PstI, and cloned into the plasmid pEX18Tc, resulting in plasmid pEXTcRHLR (Table 1). Subsequently, a 1.9 kb BamHI–HindIII fragment containing the intact rhlI gene, and a 1.6 kb EcoRI–PstI fragment containing an intact rhlR gene, were cloned into pME6000 to produce plasmids pME6000rhlI and pME6000rhlR, respectively (Table 1).
To construct the rhlI mutant, a 557 bp BglII–Eco91I fragment from pBLSRHLI, including the partial sequence of the rhlI gene with its promoter region, was replaced by an 850 bp SmaI fragment containing the aacC1 gene from plasmid pUCGM, to create plasmid pBLSRHLIG (Table 1, Fig. 2). Next, a 2.2 kb BamHI–HindIII fragment from pBLSRHLIG was cloned into pEX18Tc to generate the plasmid pEXTcRG (Table 1). After conjugation with strain M18 as the recipient, and E. coli SM10/pEXTcRG as the donor, we selected Tc-sensitive, Gm-resistant and sucrose-resistant transconjugants with an inactivated rhlI gene in the chromosome. One isolate (M18IG) was further confirmed by PCR for the additional 123 bp fragment in the rhlI gene using primers PRSMRHLI1and PRSMRHLI2. The rhlI gene in the Plt-negative strain M18T was inactivated by inserting the 
Km Km-resistance cassette of pDSK519 into the PstI site of the ORF of the rhlI gene (mutant M18TI). The plasmid pME6000rhlI was introduced into strain M18IG, forming the complemented rhlI mutant M18IG/pME6000rhlI.
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Km cassette was inserted into the rhlR gene at the SacI site in pEXTcRHLR, resulting in pEXTcRK (Table 1). Analogous to the rhlI mutant construction described above, we constructed the rhlR mutant M18RK (Fig. 2), and the M18T derivative M18TR. Successful inactivation of rhlR was further verified by PCR. Plasmid pME6000rhlR was introduced into the strain M18RK, forming the complemented rhlR mutant M18RK/pME6000rhlR.
RNA extraction and reverse transcription.
RNA was extracted from M18, M18IG, M18RK, and the complemented mutants M18IG and M18RK. Under normal growth conditions, each culture was harvested in early exponential phase, at an OD600 of 2.0–2.4, or in late exponential phase, at an OD600 of 5.0–6.0. RNA extraction and reverse transcription were performed using the EZ spin column total RNA isolation kit (Sangon), and the First Strand cDNA synthesis kit (MBI Fermentas), according to manufacturers' recommendations.
Real-time PCR.
The amounts of cDNA obtained by reverse transcription were quantified utilizing the MiniOpticon Real-Time PCR System (Bio-Rad), with a SYBR green I stain. We measured the transcription of the pltA gene, which is the first gene of the Plt biosynthetic gene cluster in Pseudomonas sp. M18 (Huang et al., 2004; Nowak-Thompson et al., 1999). Transcription of the endogenous housekeeping gene rpoD (Savli et al., 2003) was used as a reference. The primers used for amplifying the pltA gene (PRSMPLTA1 and PRSMPLTA2) and the rpoD gene (PSRMRPOD1 and PSRMRPOD2) were designed from their sequences in Pseudomonas sp. M18 (Table 1). The PCR fragments of the rpoD gene and pltA gene were 173 and 157 bp, respectively. Each 25 µl of reaction mixture contained: 2 µl of the cDNA sample diluted 1 : 5; 1x PCR buffer; 1x SYBR green I; 200 µM dATP, dGTP, dCTP and dTTP; 1 µM of each primer in water; and 2.5 U Taq DNA polymerase. Negative controls consisting of distilled water, or total RNA instead of the cDNA, were included in each test to check for DNA contamination. The target cDNA (pltA) and reference cDNA (rpoD) were amplified in separate wells. PCRs were run in an MJ Mini Personal Thermal Cycler (Bio-Rad), with the following program: one step of 5 min at 94 °C; 30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. The evolution of fluorescence intensity of each reaction mixture was recorded continuously using a MiniOpticon detector (Bio-Rad). The PCR products amplified from cDNA were further confirmed by sequencing. PCR analyses for each strain in different growth phases were repeated three times.
For analysis of the data, the comparative threshold cycle (CT) method was adopted as a mathematical model, as described by Livak & Schmittgen (2001), to calculate the differences of pltA gene expression among different strains grown in KMB broth. The amount of pltA cDNA, normalized to levels of the reference rpoD, and calibrated relative to the M18 strain, was expressed as a 
value, which represents the fold change of pltA gene expression in a given strain relative to strain M18. The 
CT value was the CT value of a certain strain (M18, M18IG, M18RK, or the complemented M18IG or M18RK) subtracted from the CT value of strain M18, where CT is the CT value of cDNA (pltA gene) subtracted from the CT value of the cDNA (rpoD gene) in each strain. Statistical significance was computed by an unpaired Student's t test; P<0.05 was considered statistically significant.
Assays for BHL, N-hexanoylhomoserine lactone, Plt and 
-galactosidase.
For extraction of AHL, a 1 ml culture of M18 strain or one of its derivatives (M18-T510, M18IG or M18IG/pME6000rhlI) was harvested at an OD600 of 5.0–6.0. After centrifugation (8000 g), the supernatant was extracted three times with 1 ml ethyl acetate. The ethyl acetate was evaporated with a rotary evaporator, and dried extracts containing AHL were resuspended in 20 µl ethyl acetate. BHL and N-hexanoylhomoserine lactone (HHL) were analysed employing a TLC bioassay, as described by McClean et al. (1997) and Shaw et al. (1997). Four samples, and standard AHLs (Fluka), were spotted (2 µl) onto TLC silica Gel RP-C18 plates (Merck) for migration, and Chromobacterium violaceum CV026 was used as the reporter strain (McClean et al., 1997; Shaw et al., 1997). The quantities of BHL and HHL in each sample were estimated in comparison with standards, i.e. 0.25, 0.5, 1 and 2 nmol BHL, and 0.01, 0.1, 0.2 and 0.5 nmol HHL.
The extraction and quantification of Plt from the culture suspension were performed using the methods described by Huang et al. (2004). -Galactosidase assays were done according to the method of Miller (Sambrook et al., 1989).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In previous experiments, we obtained a mini-Tn5 lacZ-tet/1 mutant (M18-T510) that could not produce observable levels of this red pigment (Fig. 1). The fragment containing the inserted Km cassette of the mini-Tn5 lacZ-tet/1, along with downstream chromosomal DNA, was cloned and sequenced. For strain M18-T510, sequence analysis of plasmid pSK-T510 revealed that the partial sequence downstream of the transposon shared 98 % identity with the rhlI gene (accession no. PA3476) of P. aeruginosa PAO1. In strain PAO1, rhlI encodes an enzyme related to the synthesis of BHL and HHL (Jiang et al., 1998). Considering such a remarkable level of identity, we gave the name rhlI to the gene from M18-T510. A pair of conservative primers was designed according to the nucleotide sequence flanking the rhlI gene in P. aeruginosa PAO1 (www.pseudomonas.com). A 2.0 kb PCR fragment from the chromosomal DNA of the wild-type M18 strain was purified, sequenced and cloned (see Methods). The full sequence of the 2.0 kb DNA fragment shared 99 % identity with a region of P. aeruginosa PAO1. The M18 rhlI gene was flanked upstream by sequence similar to rhlR, and downstream by pheC; this arrangement is identical to that in P. aeruginosa PAO1 (Ochsner & Reiser, 1995). The transposon was located at position 46 in the rhl1 ORF (Fig. 2). The deduced product (201 aa) of the M18 strain rhlI gene shared 98 % similarity with the RhlI protein of P. aeruginosa PAO1. The intact rhlR gene was also sequenced and cloned (see Methods), and the predicted protein sequence of RhlR showed 100 % identity to the transcriptional regulator RhlR (PA3477) of P. aeruginosa PAO1.
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). However, some distinct characters, such as plt genes, red pigment production (Fig. 1), and notable high levels of PCA production (Hu et al., 2005), were markedly different from strain PAO1. Together, these data indicate that Pseudomonas sp. M18 is an unusual pseudomonad that is closely related to P. aeruginosa PAO1, but not identical to it (Hu et al., 2005).
Effects of the rhlI null mutation on the synthesis of AHLs and red pigment
The effect of the rhlI mutation on AHL production in Pseudomonas sp. M18 was measured. A null mutant with an inactivated chromosomal rhlI gene was constructed by inserting a single Gm-resistance cassette by homologous recombination. The partial nucleotide sequence and promoter region of the rhlI gene from the wild-type strain M18 was deleted, and replaced with a fragment containing the aacC1 gene, which is responsible for Gm resistance, to generate an rhlI mutant designated M18IG (Fig. 2). TLC assays indicated that strain M18IG, as compared with wild-type M18, could not produce detectable amounts of BHL or HHL in KMB broth when grown to an OD600 of 5.0–6.0; these results are similar to those for strain M18-T510 (Fig. 3). The inactivated rhlI in M18IG resulted in the disappearance of red pigment (data not shown), which is also similar to the rhlI mutant M18-T510 (Fig. 1). In contrast, the complemented mutant M18IG/pME6000rhlI restored the ability to produce the two AHLs (Fig. 3) and the red pigment (Fig. 1).
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Km cassette (Fig. 2) into the SacI site of the rhlR gene, resulting in a mutant that was named M18RK. Inactivation of either rhlI or rhlR resulted in a dramatic increase of Plt production in strains M18IG and M18RK compared with the wild-type strain. The quantities of Plt produced by strains M18IG and M18RK were nearly identical, and were both greater than strain M18 over the same time course of growth (Fig. 4a). The expression of the Plt biosynthetic genes in the M18, M18IG and M18RK strains had to be determined in parallel. To this end, the plasmid pMEAZ harbouring a pltA'–'lacZ translational fusion was introduced into each of these three strains, and -galactosidase activity was assayed in each of them. The expression of the pltA'–'lacZ fusion increased fivefold after 24 h growth in KMB broth for both M18IG and M18RK compared with strain M18 (Fig. 4b).
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). These complementation experiments demonstrated that the increased Plt production was indeed due to inactivation of either the rhlI or the rhlR gene in these mutant strains, and not to any other genetic event.
Effect of the rhl QS system on pltA expression occurs at the transcriptional level
Genetic evidence has revealed that RhlR regulates certain QS-controlled genes at the transcriptional level (Fuqua et al., 2001). This evidence suggests that the rhl QS system may also regulate expression of Plt biosynthetic genes at the transcriptional level.
To confirm this hypothesis, the mRNA levels of pltA in M18, M18IG and M18RK were assessed by real-time PCR in two different growth phases. The housekeeping gene rpoD was assayed in parallel to normalize transcript levels of pltA. As PltA is a key component of Plt biosynthesis (Huang et al., 2004; Nowak-Thompson et al., 1999), the mRNA level of pltA can be taken as representative of the transcriptional expression of the plt gene cluster. The relative mRNA expression levels of the pltA were analysed by the 
method, as described in Methods. According to Table 2, the level of pltA transcription increased in strains M18RK and M18IG, as compared with strain M18 and the complemented mutant, in both the early exponential phase (P<0.05) and the late exponential phase (P<0.05). In the early exponential phase, the normalized pltA levels in strains M18IG and M18RK were 1.61 and 1.92, respectively, relative to strain M18. Even greater amounts were observed when cells reached an OD600 of 5.0–6.0 in late exponential phase, and the normalized pltA levels in M18IG and M18RK reached 5.03 and 4.41, respectively.
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), we predicted the putative promoter and transcriptional start site to be 254 bp upstream of the translational start site (ATG) of pltL (data not shown). Thus, the plasmid pMEAZ-12, harbouring the transcriptional plt'–lacZ fusion, which contains the nucleotides –470 upstream and +209 downstream of the transcriptional start site (Fig. 4e), was introduced into M18, M18RK and M18IG. We then assayed -galactosidase activity in each strain. The transcriptional expression of the plt'–lacZ fusion in strains M18RK and M18IG was significantly enhanced, and was 10-fold higher than strain M18 in KMB broth during some growth phases (Fig. 4c). These data, together with the results from real-time PCR, show that the expression of the Plt biosynthetic genes is negatively controlled by the rhl QS system at the transcriptional level. Another plasmid, pMEAZ-13, carrying a different transcriptional plt'–lacZ fusion, containing 470 bases upstream, but only 30 bases downstream of the transcriptional start site (Fig. 4e), was constructed, and introduced into the M18, M18RK and M18IG strains. It is interesting that the transcription of the plt'–lacZ fusion in these three strains was similar (Fig. 4d).
Differential contributions of BHL and HHL to the regulation of Plt production
RhlI in strain M18 was able to synthesize BHL and HHL at a ratio of approximately 10 : 1 in KMB broth; the concentrations of BHL and HHL, estimated by comparison with standards, reached maximum concentrations of 10 and 1 µM, respectively, during late exponential phase (OD600 between 5.0 and 6.0).
An AHL-supplementation experiment was carried out to investigate the respective contributions of BHL and HHL to Plt production in Pseudomonas sp. M18 in KMB broth. BHL and HHL were added to separate M18IG cultures at an OD600 of 2.4 before synthesis of AHL (BHL and HHL) is normally initiated in strain M18 (data not shown). The final concentrations of BHL and HHL in the M18IG cultures were 15 and 1.5 µM, respectively; both values were 1.5-fold higher than the estimated maximum values produced in M18 cultures, as described above. At the same time, another M18IG culture was treated with an equal volume of ethyl acetate vehicle to dissolve the AHLs, as a negative control. When cells entered late exponential phase (OD600 between 5.0 and 6.0), Plt production in the three different M18IG cultures was measured, and the results are presented in Fig. 5. In the M18IG culture supplemented with BHL, the concentration of Plt declined to 57 µg ml–1 compared with 230 µg ml–1 in the M18IG culture treated with ethyl acetate alone. Similarly, addition of HHL to the M18IG culture led to decreased Plt production (119 µg ml–1), but the extent of the reversion was much less than in the BHL-treated culture. Furthermore, increasing the concentration of each AHL to threefold more than in the wild-type caused a similar trend of differential decreases in Plt production (52 µg ml–1 for BHL, and 108 µg ml–1 for HHL).
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), and the deduced peptide sequence (343 aa) exhibited 63 % identity over 313 aa to the homologous PltR protein in P. fluorescens Pf-5 (Nowak-Thompson et al., 1999).
The pltR mutant M18TRG was constructed (data not shown), and the levels of Plt in the culture of the wild-type M18 and mutant M18TRG were quantified at an OD600 of 5.0–6.0 (late exponential phase). The inactive pltR mutant (M18TRG) showed significantly decreased Plt production (Fig. 6b). The empty plasmid pME6032, and the pltR overexpression plasmid pME6032pltR (Fig. 6a), were introduced into the mutant M18TRG. Following induction with IPTG, the quantity of Plt in culture of mutant M18TGR harbouring pME6032 plasmid was 9.0 µg ml–1, which was similar to that of the mutant alone (12 µg ml–1, Fig. 6b). In contrast, Plt production by M18TGR harbouring the plasmid pME6032pltR increased appreciably. After IPTG-induction, the Plt expression of M18TGR, harbouring the pME6032pltR plasmid, increased to 389 µg ml–1, which was 13-fold more than wild-type strain M18 (Fig. 6b). In addition, disruption of pltR in the mutant M18TRG resulted in a notable decline in the expression of the transcriptional plt'–lacZ fusion carried by plasmid pMEAZ-12, for which -galactosidase activity was 172 Miller units, as compared with M18 (450 Miller units) after 18 h. This confirms that PltR acts as a transcriptional activator of the Plt biosynthetic gene cluster in Pseudomonas sp. M18, and it is consistent with results found in P. fluorescens Pf-5 (Nowak-Thompson et al., 1999).
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). -Galactosidase activity of the pltR'–'lacZ fusion expressed in either M18RK or M18IG reached a peak of about 90 Miller units after 24 h, while the peak activity in the wild-type was less than 50 Miller units. These data indicate that inactivation of the rhl QS system enhances expression of pltR.
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-Galactosidase activity in strains M18IG and M18RK was higher than that in the wild-type during the growth process (Fig. 7b). The increase was consistent with the trend of increased Plt production in the mutant M18IG and M18RK strains, as described above. In addition, it has been reported previously that the Plt molecule itself induces expression of the ABC transport gene cluster in Pseudomonas sp. M18 (Huang et al., 2006). These results suggest that Plt might participate in this regulation. To examine this possibility, expression of a pltH'–'lacZ translational fusion was measured in the Plt-negative strain M18T, the rhlI Plt-negative mutant M18TI, and the rhlR Plt-negative mutant M18TR. -Galactosidase activity was almost identical in each of these three mutants, and was approximately 50 % of that in the wild-type M18 (Fig. 7b). The promotional effect of the rhlI mutation on the pltH'–'lacZ reporter was clearly suppressed by the absence of Plt in the M18TI and M18TR strains.
Effect of the rhl QS system on cell division and death
Survival of the rhlR mutant M18RK was better than strain M18. The growth rates of strains M18 and M18RK were indistinguishable before the stationary phase of growth (Fig. 8a, b). However, after entering stationary phase, the cell density of strain M18 declined more quickly than that of strain M18RK after 48 h incubation, and the cell density of the former was only 50 % of the latter at 72 h during the late stationary phase (Fig. 8a). Unlike the trend of decreased cell density, the population size [mean log (c.f.u. ml–1)] of strain M18 declined, while it increased for strain M18RK, reaching 15.0 log (c.f.u. ml–1) after 66 h incubation. This is much higher than the peak value of 13.0 log (c.f.u. ml–1) attained by the wild-type M18 at 48 h (Fig. 8b). In the complementation experiment, the growth rate and population size for the complemented mutant (M18RK/pME6000rhlR) were similar to those of the wild-type M18 strain (Fig. 8a, b). We also found similar results using strain M18IG (data not shown). This suggests that rhl QS is involved in long-term survival in Pseudomonas sp. M18.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In this study, we identified an rhl QS system (rhlI and rhlR) in Pseudomonas sp. M18, and this provided an excellent model to study the regulatory functions of AHL-dependent QS on Plt production.
Our results demonstrate clearly that Plt production, and expression of the Plt biosynthetic genes, are negatively controlled by the rhl QS system (RhlR and RhlI) in Pseudomonas sp. M18. In P. aeruginosa, RhlR can act as either an activator or a repressor for different target genes (Medina et al., 2003). In this study, we demonstrated that RhlR acts as a repressor of Plt production by showing that chromosomal inactivation of rhlR leads to increased expression of the Plt biosynthetic genes (Fig. 4b) and, subsequently, to increased Plt production (Fig. 4a). Similar results occurred in the rhlI mutant M18IG. Furthermore, an rhlI and rhlR double mutant was phenotypically similar to the M18IG and M18RK mutant strains, with respect to both Plt production and pltA'–'lacZ fusion expression (data not shown). These data suggest that rhlI and rhlR comprise an rhl QS system, and that they function together in the negative regulation of Plt production in Pseudomonas sp. M18.
The rhlI gene was shown to be essential for the production of BHL and HHL in Pseudomonas sp. M18, based on TLC analysis results (Fig. 3). The AHL-supplementation experiment confirmed that both BHL and HHL can take part in the negative regulation of Plt production; however, it was obvious that BHL was more effective than HHL in this regulation (Fig. 5). Perhaps the explanation for this might be that RhlR interacts specifically with the BHL molecule. It seems possible that the RhlR(HHL) complex may be a redundant component in strain M18 because there is no compelling mechanism to explain its presence when another more effective RhlR(BHL) system exists. Whether the RhlR(HHL) complex controls additional genes is unknown. Furthermore, enhancement of exogenous AHL in the M18IG culture, from 1.5- to 3.0–fold more than that produced by the wild-type, failed to result in a proportional decrease in Plt production. This disproportionate effect might be explained by the degradation of exogenous AHL during the process of culturing (Chen et al., 2005), and/or the hypothesis that the relatively limiting amounts of RhlR protein were already saturated by excessive AHLs.
Our findings provide evidence that an rhl QS system negatively controls the expression of the Plt biosynthetic genes at the transcriptional level. This conclusion was drawn from two different sets of experiments. First, the transcriptional levels of Plt biosynthetic genes in strains M18IG and M18RK were enhanced as compared with the wild-type M18 strain or the complemented mutants (Table 2). Second, the expression of a transcriptional plt'–lacZ fusion in strains M18IG and M18RK increased as compared with the wild-type strain (Fig. 4c). More intriguingly, the -galactosidase activity displayed a marked difference between the different transcriptional fusions (the plt'–lacZ carried by pMEAZ-12, and the plt–lacZ fusion carried by pMEAZ-13) when they were expressed in the same rhl-disrupted strain, such as M18IG or M18RK. The difference between the plt–lacZ and plt'–lacZ fusions is that the latter has an additional 176 bp sequence (Fig. 4e), indicating that the additional 176 bp region may be essential for regulation of the rhl QS system on Plt production, though further investigation will be required to detail the function of this region.
A pathway-specific transcriptional activator, PltR, was also identified upstream of the Plt biosynthesis genes. An inactive mutant of this protein significantly decreased both Plt production and pltA'–'lacZ fusion expression, while overexpression of pltR led to increased Plt production. Furthermore, the expression of pltR in the M18RK and M18IG strains was increased over the wild-type strain grown in KMB broth (Fig. 7a). We encountered preliminary evidence suggesting that the rhl QS system may function as a repressor of pltR, through which the rhl QS system negatively regulates Plt production indirectly.
In previously published work, we identified a putative ABC (ATP-binding cassette) transport gene cluster (pltHIJKN) required for Plt production, and this was characterized within a 7.5 kb genomic region downstream of the antibiotic Plt biosynthetic gene cluster in Pseudomonas sp. M18 strain. Overexpression of pltHIJKN led to increased Plt production (Huang et al., 2006), suggesting that the ABC transport system may be involved in the regulatory function of the rhl QS system on Plt production. Using a translational pltH'–'lacZ fusion, we obtained preliminary evidence that mutation of either rhlI or rhlR enhanced pltH'–'lacZ fusion expression (Fig. 7b). However, this enhancement disappeared when another pltB mutation was introduced. -Galactosidase activity of the pltH'–'lacZ fusion was similar in the Plt-negative strains M18T, M18TI and M18TR (Fig. 7b), and was approximately 50 % of that of the wild-type strain M18. Consistent with studies reporting that Plt could act as a signalling molecule (Brodhagen et al., 2004) to induce expression of ABC transport gene cluster (Brodhagen et al., 2005; Huang et al., 2006), we also found that the rhl QS system could directly regulate this Plt-specific ABC transporter alone, but did so in a Plt-dependent manner. However, this must be distinct from the regulation of the QS system on pltR expression, because our evidence showed that expression of pltR'–'lacZ fusion in Plt null mutant strains M18TI and M18TK was maintained at a high level (Fig. 7a).
Mutants defective in the rhl QS system survived longer than wild-type cells, suggesting retard cell death (Fig. 8a, b), which might confer a selective advantage on Pseudomonas sp. M18 in KMB broth. The mechanism behind this phenomenon may be related to a similar result that was recently discovered in a lasR mutant of P. aeruginosa (Heurlier et al., 2005). Whether the las system exists in strain M18 will need to be further investigated. The fact that the defective rhl QS system led to enhanced cell viability, along with increased Plt production, may provide some hints for understanding the ecological roles of QS (Manefield & Turner, 2002). However, the effect of a defective rhl QS system on root colonization and bio-control capacity should be investigated further.
In summary, we have presented preliminary results, and posited several suggestions, that may explain how the rhl QS system functions on several significant cell activities, including antibiotic biosynthesis, cell growth and pigment production. Future studies will determine the detailed mechanism of this RhlR–AHL complex on the regulation of these processes, and broaden the search for the putative regulators and pathways at work in strain M18.
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ACKNOWLEDGEMENTS |
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Edited by: J. Alfano
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REFERENCES |
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), M18IG (
), M18RK (
), the complemented M18IG (
) and the complemented M18RK (
), in KMB broth. 
), M18IG (
) and M18TR (
) grown in KMB broth. Values are the means (±SD) for triplicate cultures.