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Leiden University, Institute of Biology (IBL), Clusius Laboratory, Wassenaarseweg 64, 2333AL Leiden, the Netherlands
Correspondence
Guido V. Bloemberg
bloemberg{at}rulbim.leidenuniv.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Bassler, 1999; Lugtenberg et al., 2002). In Pseudomonas chlororaphis PCL1391, the production of the antifungal metabolite phenazine-1-carboxamide (PCN) (Chin-A-Woeng et al., 2003) is synthesized through expression of the biosynthetic phzABCDEFGH operon (Chin-A-Woeng et al., 1998). Previous work led to a model of regulation of PCN production involving three different groups of genes: the phzI/phzR quorum-sensing system (Chin-A-Woeng et al., 2001b), gacS/gacA (global antibiotic and cyanide control), and the regulatory psrA gene (Pseudomonas sigma regulator) (Chin-A-Woeng et al., 2005).
The phzI gene is responsible for the synthesis of autoinducers, of which N-hexanoyl-L-homoserine lactone (C6-HSL) is the main product (Chin-A-Woeng et al., 2001b). C6-HSL is believed to bind to PhzR, thereby activating it. Subsequently, the PhzR–C6-HSL complex probably binds to the lux (or phz) box upstream of the phz biosynthetic operon, which results in initiation of the transcription of the phz operon. The PhzR–C6-HSL complex also upregulates phzI via a second lux box. A similar regulation of phenazine synthesis by quorum sensing was shown in Pseudomonas aureofaciens 30-84 (Pierson et al., 1994).
The GacS/GacA system is composed of a sensor kinase, responding to an unknown (possibly environmental) factor (Heeb et al., 2002; Zuber et al., 2003), and a response regulator belonging to the FixJ family. In Pseudomonas species, GacS and GacA are global regulators of secondary metabolism, since they are situated upstream of many regulatory cascades and seem to function as master regulators. GacS and GacA are involved in the regulation of a substantial set of genes and of multiple traits, such as production of metabolites like HCN and 2,4-diacetylphloroglucinol (Phl) in Pseudomonas fluorescens CHAO (Laville et al., 1992), of enzymes like exoprotease and phospholipase C in P. fluorescens CHAO (Sacherer et al., 1994) and of various phenazines in P. aureofaciens 30-84 and Pseudomonas aeruginosa PAO1 (Chancey et al., 1999; Reimmann et al., 1997). GacS and GacA exert their effect on secondary metabolism by modulating the expression of various regulators (Chatterjee et al., 2003; Haas & Defago, 2005), including quorum sensing (Bertani & Venturi, 2004; Chancey et al., 1999; Reimmann et al., 1997) and 
s (Schmidt-Eisenlohr et al., 2003; Whistler et al., 1998). In P. chlororaphis strain PCL1391, a mutation in gacS results in a severe decrease of PCN production to undetectable levels, while the N-acylhomoserine lactone (N-AHL) production is also much lower than in the wild-type (Chin-A-Woeng et al., 2005).
GacS/GacA is also required for psrA expression in P. chlororaphis PCL1391 (Chin-A-Woeng et al., 2005). The psrA gene of Pseudomonas putida was shown to regulate the transcription of the rpoS gene (Kojic & Venturi, 2001) by directly binding to the rpoS promoter (Kojic et al., 2002). rpoS encodes the stationary-phase alternative sigma factor s, which is responsible for the switch in gene expression occurring upon exposure of cells to starvation and/or various stresses (Lange & Hengge-Aronis, 1991). In Pseudomonas species, rpoS mutants are often affected in their secondary metabolism, and particularly in their antibiotic production (Sarniguet et al., 1995; Suh et al., 1999). However, the results are different depending on the species and the antibiotic considered. For example, an rpoS mutation results in a decrease of pyrrolnitrin production by P. fluorescens, but in an increase of pyoluteorin and 2,4-diacetylphloroglucinol production by the same strain (Sarniguet et al., 1995) and of pyocyanin in P. aeruginosa (Suh et al., 1999).
Here we describe rpoS in P. chlororaphis PCL1391 and its role in the synthesis of PCN. A significant number of PCL1391 derivatives were constructed that are affected in the expression of the psrA, rpoS and phzR genes. Quantification of PCN and C6-HSL, as well as preliminary microarray analyses, showed that the phz operon is regulated by a cascade involving GacS, PsrA, RpoS and PhzI/PhzR. In addition, the microarray survey allowed us to identify new genes of the psrA/rpoS regulon that might be involved in secondary metabolism.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Pseudomonas strains were cultured at 28 °C in liquid MVB1 (van Rij et al., 2004) and shaken at 195 r.p.m. on a Janke und Kunkel shaker KS501D (IKA Labortechnik). E. coli strains were grown at 37 °C in Luria–Bertani medium (Sambrook & Russell, 2001) under vigorous aeration. LC medium, used for some experiments as indicated in the text, contained 10 g Bactotryptone (Difco) l–1, 5 g yeast extract l–1, 137 mM NaCl, 51 mM MgSO4, 5 mM Tris. Media were solidified with 1·8 % Bacto agar (Difco). When appropriate, growth media were supplemented with kanamycin (50 µg ml–1), carbenicillin (200 µg ml–1) and gentamicin (10 µg ml–1 for Escherichia coli and 30 µg ml–1 for P. chlororaphis), and X-Gal (40 µl ml–1). To follow growth, the optical density of liquid cultures was measured at 620 nm.
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) in combination with oMP49 and oMP50, which anneal close to the multi-cloning site of pBlueScript. oMP770 in combination with oMP49 produced a fragment of 1·2 kb containing the flanking region upstream of rpoS. oMP771 in combination with oMP50 produced a PCR fragment of 1·8 kb that contained the flanking regions downstream of rpoS. A third primer was designed for further sequencing of the 3' downstream region with the same method (oMP772), which in combination with oMP50 produced a 1·2 kb PCR fragment. In order to construct an rpoS mutant derivative of PCL1391, two primers (oMP773 and oMP774) were used in PCR on the chromosomal DNA of PCL1391, and an internal fragment of 0·5 kb of the rpoS gene was obtained. This fragment was ligated into pGEM-T easy (Promega) to obtain pMP7425. An EcoRI digestion of pMP7425 produced an EcoRI internal fragment of rpoS which was cloned into the suicide plasmid pMP5285, resulting in pMP7418. This vector was transformed into PCL1391 for single homologous recombination by triparental mating using pRK2043 as helper. The rpoS mutant of PCL1391 that was obtained was checked by Southern blotting and by PCR with oMP686 primer (annealing on pMP5285 close to the multi-cloning site) and oMP776 (annealing on the rpoS 3' end which is not present in pMP7418) and named PCL1954.
Two primers were designed according to the sequence of the newly characterized rpoS gene of PCL1391 (oMP775 and oMP776), in order to produce a PCR fragment containing the whole rpoS under control of the Ptac promoter and a part of the 3' downstream region of rpoS including the putative terminator. The PCR fragment obtained had the expected size of 1·2 kb and was checked by restriction analysis and sequencing. Subsequently it was cloned into pGEM-T easy, which yielded pMP7424. The Ptac rpoS fragment was isolated from pMP7424 by EcoRI digestion and ligated into pBBR1MCS-5 to produce pM7420, which was transformed into PCL1391 and PCL1954 by triparental mating to produce PCL1958 and PCL1955, respectively. The control strains PCL1960 and PCL1957 were obtained by transforming the cloning vector pBBR1MCS-5 into PCL1391 and PCL1954, respectively. pMP7420 and pBBR1MCS-5 were also transformed into the psrA mutant PCL1111 to obtain PCL1961 and PCL1962, respectively.
Two primers (oMP777 and oMP778) were used with pMP4030 as template to produce a PCR fragment containing the phzR gene under Ptac control. This product was digested by XhoI and EcoRI and ligated into XhoI/EcoRI-digested pBBR1MCS-5 to obtain pMP7447, which was validated by sequencing and its ability to restore a wild-type PCN production in the phzR mutant PCL1104. The resulting strain PCL2000 was able to produce PCN (not shown), in contrast to the PCL1104 derivative PCL2001, which contained the cloning vector pBBR1MCS-5. pMP7447 was also transformed into PCL1391, PCL1954, PCL1111 and PCL1123 to obtain PCL1993, PCL1986, PCL1996 and PCL1998, respectively.
Primers oMP859 and oMP861 were used with chromosomal DNA as template to produce a PCR fragment containing the psrA gene. This fragment was used as template for PCR with oMP860 and oMP861 to obtain the psrA gene under Ptac promoter control. This fragment was digested with EcoRI and ligated into the EcoRI site of pBBR1MCS-5, to obtain pMP7465. pMP7465 was validated by sequencing. pMP7465 was subsequently transformed into PCL1391, PCL1111, PCL1123 and PCL1954 to obtain PCL2044, PCL2045, PCL2047 and PCL2048, respectively.
Three mutants were constructed in genes selected by microarray analyses. The genes chosen were a putative transcriptional regulator gene found in microarray clone 76_G2, a putative GGDEF/EAL regulator found in microarray clone 42_G8 and a hypothetical protein found in microarray clones 76_G2 and 47_F5. Primers oMP810 and oMP811, oMP972 and oMP973, oMP977 and oMP978 were used with clone 76_G2, clone 42_G8 and chromosomal DNA as a template, respectively, to produce an internal fragment of 0·4 kb for the putative transcriptional regulator gene, 0·4 kb for the putative GGDEF/EAL regulator gene and 0·5 kb for the hypothetical protein gene, respectively. The PCR products obtained were cloned in the EcoRI site of pMP5285, resulting in pMP7452, pMP7467 and pMP7470, respectively. These vectors were transformed into PCL1391 to obtain PCL2009, PCL2050 and PCL2052, respectively. The mutations were verified by PCR and/or sequencing.
Extraction and analysis of phenazine and N-AHL.
Phenazine extraction was carried out from 10 ml MVB1 liquid cultures in 100 ml Erlenmeyer flasks at regular time points during growth and/or after overnight growth of bacterial strains as described previously (van Rij et al., 2004).
For extraction of N-AHL, supernatants from 50 ml liquid MVB1 cultures in 500 ml Erlenmeyer flasks were mixed with 0·7 vol. dichloromethane, and shaken for 1 h, after which the organic phase was collected. Each supernatant was extracted twice and the pooled extracts were dried using a rotary evaporator. The dried residue was dissolved in 25 µl acetonitrile and spotted on C18 TLC plates (Merck). As a control, 0·5 µl synthetic C6-HSL (5 µM) (Fluka) was spotted on the TLC plate. The plates were developed in methanol/water (60 : 40, v/v). For detection of N-AHL, the TLC was overlaid with 0·8 % LC agar containing a 10-fold diluted overnight culture of the Chromobacterium violaceum indicator strain CV026 and supplemented with kanamycin (50 µg ml–1). After incubation for 48 h at 28 °C, chromatograms were judged for appearance of violet spots.
Western blot analysis.
Cells were grown after inoculation of 10 ml MVB1 from an overnight culture diluted to OD620 0·1. Cells were harvested at OD620 1·0 or 2·2 in volumes of culture corrected for their differences in OD620 to obtain similar amounts of cells. Cell pellets were suspended in 200 µl cracking buffer (50 mM Tris/HCl pH 6·8, 1 % SDS, 2 mM EDTA, 10 %, v/v, glycerol, 0·01 % bromophenol blue, 1 % 
-mercaptoethanol) and boiled for 3 min. The samples were subsequently loaded on a 10 % SDS-PAGE gel and proteins were separated and transferred on to a blot following a standard Western blot procedure (Ausubel et al., 1997). A dry aliquot of RpoS antibodies was kindly provided by Professor K. Tanaka (Tokyo, Japan). The pellet was suspended in 100 µl PBS and diluted 1000-fold for reaction with immobilized protein, as recommended. Peroxidase-labelled goat anti-rabbit antiserum (Amersham Biosciences) was subsequently incubated with the blots. Finally, blots were incubated in luminal solution [250 µM sodium luminol (Sigma), 0·1 M Tris/HCl, pH 8·6, 0·01 % H2O2] mixed with 60 µl enhancer solution [67 µM p-hydroxycoumaric acid (Sigma) in DMSO]. Hybridizing protein bands were visualized on Super R-X photographic film (Fujifilm) after chemiluminescence detection.
RNA preparation, cDNA probe generation and microarray processing.
The methods followed were described previously (van Rij et al., 2005). Briefly, 12 ml MVB1 medium was inoculated in 100 ml flasks to an OD620 of 0·1 from overnight cultures of P. chlororaphis PCL1391 or derivative strains. The cultures were shaken at 28 °C at a speed of 195 r.p.m. on a Janke und Kunkel shaker KS501D until the OD620 reached a value of 2·0. This optical density corresponds to the moment where PCN starts to be produced (see Fig. 2), which indicates that the genes regulating the phz operon are probably expressed. After phenol/chloroform extraction, the water phase was applied on columns from the RNeasy Midi kit (Qiagen), and the RNA was extracted following the protocol supplied by the manufacturer, including the DNase step. RNA purity was verified on 1·2 % agarose gel following the protocol of the RNeasy Midi kit (Qiagen). RNA was immediately used for cDNA probe generation using the CyScribe post-labelling kit (Amersham Biosciences). After purification, the efficiency of Cy label incorporation into the cDNA and the quality and amounts of labelled cDNA were verified with an Ultrospec 2100 pro spectrophotometer (Amersham Biosciences). Equal amounts of each dye were hybridized on the microarray. A minimum of 45 pmol of each dye was hybridized.
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). Before hybridization, the DNA on the microarrays was UV-cross-linked at 250 mJ cm–2 (Amersham LifeSciences UV cross-linker). After prehybidization and washing of the slides, the Cy-labelled cDNA was hybridized on the microarrays overnight at 65 °C in a GeneTAC Hybstation (Genomic Solutions). After washing and drying, the slides were scanned in a G2565AA Microarray Scanner (Agilent). Each experiment was repeated at least four times, including at least two independent experiments and a dye swap. Each experiment included as ‘test’ the Cy-labelled cDNA deriving from the RNA of a mutant, and as ‘reference’ the Cy-labelled cDNA deriving from the RNA of the wild-type.
Microarray data analysis.
After scanning, the microarrays were analysed in GenePix Pro version 4.0. The values were normalized assuming that most genes of the array are not differentially expressed. Several criteria were implemented to select spots corresponding to differentially expressed genes: spots were selected if the mean of the ratio of red and green laser intensities was higher than 2 [in GenePixPro: Ratio of Medians (650/550)>2] or lower than 0·5, but positive [in GenePixPro: Ratio of Medians (650/550)<0·5 and Ratio of Medians (650/550)>0]. These values of 2 and 0·5 were arbitrarily chosen to select genes of which the expression is increased or decreased at least twofold in the rpoS or psrA mutant as compared to the wild-type. In both cases, the spots were selected only if they had at least 80 % of their feature pixels more than two standard deviations above background in both the green and red channels [in GenePixPro: (%>B550+2SD)>80 and (%>b650+2SD)>80]. This condition prevents the selection of spots from which the feature intensity is too close to the background. As additional selection criteria, spots that had intensities lower than the intensity of the 
control in both the red and green channel were eliminated. This eliminates spots where labelled cDNA hybridized non-specifically to the spotted DNA. In order to avoid false positives due to problems of uniformity of the background and/or the feature, all the selected spots were finally controlled directly on the image of the scan.
Phenotypic analyses of mutants deriving from microarray analyses.
Bacteria were tested for protease production as described by Chin-A-Woeng et al. (1998), except that the concentration of milk was increased to 10 % in MVB1 agar plates.
To test swimming and swarming ability, the method described by Deziel et al. (2001) was used, in which 1/20 KB-0·3 % agar plates were used for the swimming, and 1/20 KB-0·5 % agar plates were used for swarming.
For measuring the production of chitinase, plates were poured with 2 % agar dissolved in 0·05 M sodium acetate and Cm-Chitin-RBV solution (Loewe Biochemica), following recommendations of the manufacturer. Samples (200 µl) of supernatant of 3-day-old LC cultures were applied in wells made in the plates. After overnight incubation at 28 °C, the formation of a halo was verified.
The production of hydrogen cyanide (HCN) was measured as described by Castric (1975). Whatman 3MM paper was soaked in a chloroform solution containing copper(II) ethyl acetoacetate (5 mg ml–1) and 4,4'-methylene-bis-(N,N-dimethylaniline) (5 mg ml–1), and subsequently dried and stored in the dark. A piece of paper was placed in the lid of a Petri dish in which bacteria had been plated on MVB1 agar (1 %). The Petri dishes were incubated overnight at 28 °C. Production of HCN by the bacteria was indicated by blue colouration of the paper.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), 97 % with rpoS of P. fluorescens PfO1 (accession no. ZP_00266495.1), 93 % with rpoS of P. putida (Kojic et al., 1999), and 93 % with rpoS of P. syringae pv. tomato DC3000 (accession no. NP_791390).
A putative Shine–Dalgarno sequence was detected starting 12 nt upstream of the start codon, and a putative rho-independent terminator sequence is present 22 nt downstream of the stop codon. In addition a sequence (GAAACTGCACTTTG) was identified close to the ATG codon in the promoter of the PCL1391 rpoS homologous gene, identical to the PsrA binding box consensus of P. putida (Kojic et al., 2002).
The ORF upstream of PCL1391 rpoS is homologous (98 % identity) to the lipoprotein gene nlpD of P. chlororaphis 06 (Kang et al., 2004). The ORF sequence identified downstream of rpoS shows homology (50 % identity) to a transposase gene of Ralstonia solanacearum (accession no. NP_520694.1).
In contrast, rpoS of other Pseudomonas strains is followed by the small RNA regulator rsmZ (regulator of secondary metabolites) and fdxA (ferredoxin A) (Heurlier et al., 2004). Neither a repetitive GGA motif (Heurlier et al., 2004) nor a conserved upstream element (Heeb et al., 2002) indicating the presence of an rsmZ homologue downstream of rpoS were found in PCL1391. Alignment analysis using Vector NTI with rsmZ sequences of several Pseudomonas species with a 500 nt sequence downstream of rpoS in PCL1391 did not show any homology (not shown).
Effect of rpoS on PCN and N-AHL production
A 500 bp internal fragment of rpoS was generated by PCR and used for single homologous recombination in strain PCL1391, resulting in PCL1954 (for details see Methods). Western blot analysis showed that the RpoS protein was absent in PCL1954 (Fig. 1, lane 6). The production of PCN by the rpoS mutant PCL1954 was decreased by 99 % compared to that by PCL1391 (Fig. 2a, b). Constitutive expression of rpoS was established by cloning rpoS under control of the tac promoter in the vector pBBRMCS-5, resulting in pMP7420 (for details see Methods). In the derivative PCL1955 (rpoS mutant with Ptac rpoS), the production of RpoS was shown using Western blot analysis (Fig. 1, lane 7) and PCN production was restored to between 35 % (Fig. 2b) and 70 % (Fig. 3a) of that of the control strain PCL1960 (wild-type+pBBR1MCS-5).
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). C6-HSL levels in an rpoS mutant background were restored by the constitutive production of RpoS in PCL1955 (Fig. 3a).
Since a defect in rpoS decreased PCN production, the effect of overexpression of rpoS in the wild-type strain PCL1391 was also analysed by transforming pMP7420 into PCL1391. No major difference was observed between the amounts of PCN produced by wild-type PCL1391 with constitutive expression of rpoS and wild-type PCL1391 containing the empty cloning vector (Fig. 2a).
Interactions between quorum sensing and rpoS and their influence on PCN production
The observations that the amounts of PCN and C6-HSL are decreased in an rpoS mutant, and restored by the constitutive expression of rpoS, indicate that RpoS regulates the phz operon via phzR and/or phzI. Therefore the effect of constitutive expression of phzR in the rpoS mutant was tested. For this purpose phzR was cloned under the control of the tac promoter, resulting in plasmid pMP7447, and transformed into the rpoS mutant PCL1954. The resulting strain PCL1986 showed complementation for PCN production of the rpoS mutation, as it produced 1·5-fold higher PCN than PCL1391 harbouring empty pBBR1MCS-5 (Fig. 2b) and showed increased C6-HSL production (Fig. 3b).
Regulation of RpoS synthesis by genes involved in PCN synthesis
The effect of mutations in gacS, psrA, phzI or phzB of PCL1391 on the production of RpoS protein was tested by Western blot analysis. The experiments were performed in MVB1 medium and samples for RpoS analysis were harvested during exponential phase (OD620 1·0) and at the beginning of the stationary phase (OD620 2·2). The amounts of RpoS appeared to be similar at the two time points. A blot of the results at OD620 1·0 is shown in Fig. 1. RpoS amounts were severely reduced as a result of mutations in psrA (PCL1111, lane 3) and gacS (PCL1123, lane 5). Mutations in phzI (PCL1103, lane 2) and in phzB (PCL1119, lane 4) did not affect the production of RpoS.
Relationship between psrA, rpoS and gacS
A psrA mutant of PCL1391 showed low production of PCN and N-AHL when grown in MVB1 medium as compared to PCL1391 (decrease of 99 %) (Figs 2c and 3a). It was shown that psrA regulates rpoS in other Pseudomonas species (Kojic & Venturi 2001), probably via binding to the promoter of the rpoS gene at a PsrA-binding box. Therefore, an attempt was made to complement the psrA mutant PCL1111 with constitutively expressed rpoS. For this purpose, the vector pMP7420 (Ptac rpoS) was transformed into PCL1111, which resulted in PCL1961. PCL1961 showed increased PCN and N-AHL levels compared to the psrA mutant and produced up to 55 % of the amount of PCN produced by the wild-type (Figs 2c and 3a). PCL 2048, the rpoS mutant overexpressing psrA, was unable to produce PCN (Fig. 2b) or N-AHL (Fig. 3c). As a control, we transformed pMP7465 (pBBR1MCS-5 harbouring Ptac psrA) into PCL1111 (psrA). The resulting strain, PCL2045, showed restored levels of PCN and C6-HSL (Figs 2c and 3c). Constitutive expression of phzR also restored production of PCN and N-AHL in a psrA background (strain PCL1996, Figs 2c and 3b).
Since it was shown that GacA/GacS regulate PCN and N-AHL production in KB medium, as well as psrA expression (Chin-A-Woeng et al., 2005), and that RpoS is severely decreased in the gacS mutant PCL1123, the relationship between GacS, PsrA/RpoS, quorum sensing and PCN was studied in more detail. The gacS mutant did not produce any detectable PCN or N-AHL in MBV1 (Figs 2d and 3d). Neither constitutive rpoS expression, nor constitutive expression of psrA (Figs 2d and 3d), was sufficient to compensate for the gacS mutation. Only the constitutive phzR gene restored PCN and AHL production in a gacS mutated background (Figs 2d and 3d). Surprisingly, after overnight growth (see Figure 1 in the supplementary materials available at
http://rulbim.leidenuniv.nl/girard/suppl material.htm), high amounts of PCA are present in strains PCL1986 and PCL1998 (rpoS and gacS mutant, respectively, both overexpressing phzR), but not in PCL1993 or PCL1996 (wild-type and psrA mutant, respectively, both overexpressing phzR).
Transcriptomics in psrA and rpoS mutants: a preliminary survey
In order to evaluate the pathways regulated by psrA and rpoS, the gene expression profiles of psrA (PCL1111) and rpoS (PCL1954) were compared with the gene expression of wild-type PCL1391 on microarrays. As an example for the reader and in order to comply with the MIAME standards (Brazma et al., 2001), one representative experiment among four, for both sets of microarrays, was selected for each mutant and the corresponding data, i.e. images, raw output of image analysis, normalized and flagged data, are available at
http://rulbim.leidenuniv.nl/girard/suppl material.htm. The data filter (see Methods for details of the filtering of the data) selected in total 190 spots for the experiments with the psrA mutant, of which 157 had a stronger intensity in the wild-type, and 33 a lower intensity. Two hundred and thirty-four spots were selected from the experiments involving the rpoS mutant, of which 211 had a strong intensity in the wild-type, and 23 a lower intensity. A total of 108 spots were common to the group of 190 spots from psrA arrays and the group of 234 spots from rpoS arrays. They were all more intense in the wild-type than in the psrA and rpoS mutants. Among these 108 spots, the 57 spots that were most strongly affected by both mutations were selected and the corresponding DNA was sequenced. The sequences of the clones are also available at http://rulbim.leidenuniv.nl/girard/suppl material.htm. The analysis of sequences and the variations of expression due to rpoS and psrA mutations are presented in Table 3. The clones were grouped according to the predicted function of the ORF in the insert.
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, and controls shown in the supplementary material). In addition, some clones sequenced after microarray analysis containing parts of the phzI/phzR genes (24_C5 and 93_G11) have decreased expression around fivefold in the psrA mutant and around sevenfold in the rpoS mutant.
Sequencing of 19 selected clones and homology studies identified the presence of hypothetical proteins. On several clones, two or three ORFs could be identified since the microarrays were constructed from a library of random PCL1391 chromosomal fragments of approximately 1 to 2 kb. Additional RT-PCR experiments should be performed to show which gene or operon is responsible for the ratio measured. For most genes, it was observed that they correspond to homologues that are also adjacent to each other in other sequenced Pseudomonas genomes. Several genes were sequenced that give homology to genes which cannot be obviously linked to rpoS and psrA functions, like an aminotransferase (clone 4_G11), or a deoxycytidylate deaminase (clone 4_C1) and a putative adhesin (Pflu3629) which is recurrent in the clones. However, many clones (12) show homology to genes that could be related to intermediary and secondary metabolism (see Table 3). Other interesting clones (4) show homology to regulators.
In order to test several of the genes that were selected by microarray analyses, three mutants were constructed. (i) PCL2009 is mutated in a putative transcriptional regulator gene identified in microarray clone 76_G2. (ii) PCL2050 is mutated in a putative GGDEF/EAL regulator identified in microarray clone 42_G8. (iii) PCL2052 is mutated in a hypothetical protein identified in microarray clones 76_G2 and 47_F5. Various phenotypic traits of these mutants were analysed (see Methods). The mutants showed wild-type production of HCN, chitinase and exoprotease. They were all able to swim and swarm, although PCL2052 showed decreased swimming ability and PCL2050 seemed to be also affected in its swarming (not shown). The PCN production of PCL2009 (465±28 µM) and PCL2052 (435±14 µM) appeared to be increased twofold compared to PCL1391 (237±9 µM).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Heeb & Haas, 2001; Kojic et al., 1999, 2002; Ramos-González & Molin, 1998). A substantial difference is the presence of a putative transposase downstream of rpoS in PCL1391, whereas in many other Pseudomonas spp. rpoS is followed by rsmZ and the ferredoxin gene fdxA (Heurlier et al., 2004). No indication could be found of the presence of an rsmZ gene downstream of rpoS in PCL1391. Measurements of the production of PCN and N-AHL in various derivatives (Figs 2 and 3) show that rpoS activates the synthesis of these two metabolites.
This study was started with the assumption that psrA and rpoS would constitute two components of a cascade regulating the phz operon, according to results in other strains (Kojic & Venturi, 2001). Previous work in rich growth medium indicated that PsrA inhibits N-AHL and PCN production in PCL1391 (Chin-A-Woeng et al., 2005). In our study using the poor MVB1 medium, psrA was shown to activate PCN and N-AHL production in PCL1391. The microarray data from cells grown in MVB1 medium confirmed that the expression of the phz genes is strongly reduced by the psrA and rpoS mutations (Table 3). Constitutively expressed rpoS strongly increased PCN and N-AHL production in a psrA mutant (strain PCL1961, Figs 2c and 3a). The fact that the complementation was only partial (also in the rpoS mutant) could be explained by two hypotheses. (i) PsrA regulates other genes not downstream of rpoS that are necessary for full activation of the phz genes. (ii) A fine-tuning of rpoS expression might be necessary for wild-type amounts of PCN, which is not possible when the gene is under the control of a constitutive promoter. It was previously shown for P. putida that PsrA regulates the expression of rpoS (Kojic & Venturi, 2001) by binding to its promoter (Kojic et al., 2002). Our results indicate a similar regulation in PCL1391. Our results show to our knowledge for the first time that this interaction is relevant for a particular phenotypic trait, the production of the secondary metabolite PCN.
Interestingly, our results show that the effect of a psrA mutation is dependent on the growth conditions. Additionally, it is remarkable that constitutive expression of rpoS does not restore PCN production in rpoS and psrA mutants to the same level in cultures grown in different volumes of medium (Figs 2 and 3). Although this looks peculiar, it is not unique, since conditional results were also reported for another phenazine regulator, RpeA (repressor of phenazine expression) (Whistler & Pierson, 2003). RpeA was shown to regulate PCN production mostly in minimal medium, not in complex medium. Similarly, RpoS could have a role in controlling secondary metabolism mostly under nutrient-limiting conditions. It could act as a controller of energy distribution in the cell when the nutritional conditions are more stringent, as indicated by the high sensitivity to external conditions of the strains constitutively expressing rpoS (see also below). It is also likely that other unidentified factors sensing environmental changes are involved in PCN regulation; this could explain the switch in the role of PsrA between KB medium and MVB1 medium. Conditions in the soil are known to be nutrient-limiting. Therefore the choice of a relatively poor medium as MVB1 seemed more relevant for this study.
A regulation cascade between gacS and the phz operon involves psrA, rpoS and the quorum-sensing system phzI/phzR
Under various growth conditions the amounts of C6-HSL present in PCL1391 spent culture medium were shown to be correlated with the amounts of PCN produced (Chin-A-Woeng et al., 2001b, 2003; van Rij et al., 2004). In our study, the correlation of PCN and C6-HSL levels among the various PCL1391 derivatives (Fig. 3) and the restoration of PCN production by constitutive phzR expression in the rpoS and psrA mutants (Fig. 2b, c) show that rpoS stimulates PCN production via phzI/phzR. Conversely, phzI does not regulate rpoS expression (Fig. 1). A role of rpoS in antibiotic production has been reported (Sarniguet et al., 1995; Suh et al., 1999), but not for regulating PCN production. The inhibitory effect of RpoS on quorum sensing and pyocyanin in P. aeruginosa (Whiteley et al., 2000) or of PsrA and RpoS on quorum sensing in P. putida WCS358 (Bertani & Venturi, 2004) is the opposite of what we observed for strain PCL1391. Surprisingly, RpoS was previously shown not to be involved in homoserine lactone production by P. putida WCS358 (Kojic et al., 1999). This is interesting for our study, because in the latter case P. putida was grown in minimal medium (Kojic et al., 1999), whereas in the most recent study P. putida was grown in the complex LB medium (Bertani & Venturi, 2004). In P. aeruginosa, the effect of quorum sensing on rpoS transcription in P. aeruginosa is mild (Schuster et al., 2004). Thus, very diverse relationships exist between PsrA/RpoS and quorum sensing/antibiotic production depending on the bacterial species, and on environmental conditions for any one species.
Western blot analysis showed that a mutation in phzB does not affect the RpoS level, which suggests that there is no feedback effect from PCN production on rpoS expression. A defect in the regulatory genes psrA or gacS results in a severe decrease of the amounts of RpoS. Similar observations were made in other strains (Kojic & Venturi, 2001; Schmidt-Eisenlohr et al., 2003; Whistler et al., 1998).
Our data confirm the key role of gacS for PCN synthesis (Fig. 2d). Constitutive rpoS expression did not restore PCN synthesis in a gacS mutant (PCL2010), which indicates that as well as rpoS in the regulatory cascade, other factors affected by gacS are necessary for PCN production (Fig. 4). However, constitutive expression of the phzR gene restores PCN and N-AHL synthesis in a gacS mutant (PCL1998). This could be surprising considering that in P. aureofaciens 30-84, which is closely related to P. chlororaphis PCL1391, GacS/GacA affect mostly the transcription of phzI and not that of phzR (Chancey et al., 1999). Additionally, phenazine synthesis is regulated in a comparable way in both strains by PhzI/PhzR/C6-HSL and GacS/GacA (Chancey et al., 1999; Pierson et al., 1994; Wood et al., 1997; Wood & Pierson, 1996). The role of PsrA and RpoS in phenazine synthesis has so far not been studied in strain 30-84. The following hypothesis would reconcile the results in both strains: GacS/GacA could regulate phzI at the transcriptional level and phzR at the post-transcriptional level, since it was shown that GacA acts at both levels (Blumer et al., 1999; Pessi & Haas, 2001). In our gacS mutant, the presence of constitutively expressed phzR would result in an excess of PhzR mRNA that would overcome negative post-transcriptional regulation. The PhzR protein produced in turn would bind the low amounts of C6-HSL resulting from leakage of the phzI promoter and restart the positive regulatory loop of PhzI/PhzR by binding to the lux box upstream of phzI. It would be of great interest to test if a constitutive expression of phzR could restore phenazine production in a gacS mutant of strain 30-84.
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in the supplementary material available at http://rulbim.leidenuniv.nl/girard/suppl material.htm). Conversely, only PCL1993 and PCL1996 (wild-type and psrA mutant, respectively, both overexpressing phzR) show a peak of PCN production (Fig. 2a, c). These observations could be explained by precipitation of PCN, indicated by the presence of numerous crystals in overnight cultures observed only in the case of PCL1993 and PCL1996. Since there is still a low amount of RpoS present in the psrA mutant (Fig. 1), an explanation for the high production of PCA could be that rpoS regulates (probably indirectly) the phzH gene, responsible for the conversion of PCA to PCN. The phzH gene is the last gene of the phz operon in PCL1391. It is remarkable that the distances between the phz genes phzA, B, C, D, E, F and G do not exceed 15 nt, whereas there are 111 nt between phzG and phzH (Chin-A-Woeng et al., 2001a), which could provide a binding site for regulatory proteins. Various computer analyses did not point to any particular sequence within these 111 nt.
Transcriptome analyses of psrA and rpoS mutants of P. chlororaphis PCL1391
Functional genomics provides a high-throughput analysis possibility to identify the genes of the cascade downstream of rpoS. However, the expected large amount of genes due to very downstream effects of rpoS would hamper the selection of genes of interest. rpoS and psrA are predicted to be close to each other in the regulatory cascade for PCN synthesis. Therefore the data of microarray analyses of psrA and rpoS were crossed. This approach increases the probability of selecting genes which are part of the psrA/rpoS regulatory cascade.
Our selection method revealed 13 clones containing parts of genes from the phz operon and phz quorum-sensing system (not all of them are shown in Table 3 for conciseness), which strongly validates our method. Besides, many of the genes sequenced from the positive clones were also present on other selected clones spotted elsewhere on the microarray (like phaG in clones 53_F2, 60_E1, 71_A4, 74_B4, 74_E7 or chiC in clones 11_G8 and 121_H3). These observations contribute to the validation of our microarray analyses.
Many clones carry genes that show homology to genes related to intermediary and secondary metabolism, such as phaC2, phaG, chiC, pyoverdine synthase and a probable dihydrorhizobitoxine desaturase (Table 3). phaC2 was reported to be involved in polyhydroxyalkanoic acid (PHA) synthesis (Nishikawa et al., 2002; Qi et al., 1997). phaG is also involved in PHA synthesis (Rehm et al., 1998). PHAs are polymers used for carbon and energy storage in bacteria in response to environmental stress, which would explain their regulation by rpoS. chiC encoding a chitinase was shown to be regulated by quorum sensing in P. aeruginosa PAO1 (Folders et al., 2001).
One clone (76_G2) contains a putative regulatory gene with a HTH-LuxR domain (SMART accession SM00421) and therefore might respond to N-AHLs. A mutation in this regulator, as well as in the hypothetical protein upstream of it, resulted in a twofold increase in PCN production. The function of these genes has to our knowledge not yet been characterized in other strains. Our data show that these genes affect PCN production in strain PCL1391. The third gene of interest located on clone 42_G8 contains GGDEF and EAL domains, which are found in two-component signalling systems (Galperin et al., 2001). A recent study shows the involvement of such a protein (RocS) in regulation of the rugose phenotype and biofilm formation in Vibrio cholerae (Rashid et al., 2003). A mutation in this putative regulatory gene did not change PCN production.
Our results show that a cascade involving GacS/GacA, PsrA, RpoS and quorum sensing regulates the phz operon and that several regulators downstream of GacS/GacA must exist in addition to PsrA/RpoS to activate expression of the phz operon. Preliminary microarray analyses, by allowing measurement of the effect of psrA and rpoS mutations on the phz genes, support our model of the regulation of PCN production. In addition, these data led to the identification of novel genes involved in regulatory fine-tuning of PCN production. The microarray analyses form a solid basis for future studies on identifying the role of other novel genes and their relation to psrA, rpoS and secondary metabolism, particularly PCN production.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 21 June 2005;
revised 27 September 2005;
accepted 30 September 2005.
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), derivative containing pBBR1MCS-5 (
), derivative containing Ptac rpoS (
), derivative containing Ptac phzR (
), derivative containing Ptac psrA (
). For panels (b), (c) and (d), the values for PCL1960 (PCL1391+pBBR1MCS-5) are plotted as a control (|). (a)Wild-type derivatives; (b) rpoS derivatives; (c) psrA derivatives; (d) gacS derivatives. The error bars represent SD.
