Leiden University, Institute of Biology, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands
Correspondence
Guido V. Bloemberg
bloemberg{at}rulbim.leidenuniv.nl
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ABSTRACT |
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Supplementary microarray data are available with the online version of this paper.
These authors contributed equally to this paper.
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INTRODUCTION |
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PCL1391 produces the antifungal metabolite phenazine-1-carboxamide (PCN), which inhibits hyphal growth (Chin-A-Woeng et al., 1998). Using a PCN biosynthetic mutant it was shown that PCN is essential for the biocontrol ability of PCL1391 (Chin-A-Woeng et al., 1998
). PCN production is regulated by the quorum-sensing regulatory genes phzI and phzR, which are homologous to luxI and luxR (Chin-A-Woeng et al., 2001b
). In addition, biosynthesis of PCN is dependent on the two-component regulatory system formed by GacS and GacA and is regulated by PsrA and the stationary-phase sigma factor encoded by rpoS (Chin-A-Woeng et al., 2005
; G. Girard, unpublished).
Plant-pathogenic and non-pathogenic Fusarium spp. produce fusaric acid (FA; 5-butylpicolinic acid) (Bacon et al., 1996; Notz et al., 2002
; Schouten et al., 2004
). FA is toxic for eukaryotes and prokaryotes (Bochner et al., 1980
; Wang & Ng, 1999
). In addition, FA was shown to be involved in fungal defence against Pseudomonas spp. biocontrol strains by repressing the production of antifungal metabolites. FA represses the production of 2,4-diacetylphloroglucinol in Pseudomonas fluorescens CHA0 (Duffy & Défago, 1997
) and the synthesis of PCN in P. chlororaphis PCL1391 (van Rij et al., 2004
). The latter repression is correlated with a reduction of the level of the auto-inducer N-hexanoyl-L-homoserine lactone (C6-HSL) (van Rij et al., 2004
).
In this study we investigated the repression of PCN biosynthesis by FA in more detail with the aim of (i) analysing the molecular mechanisms and environmental conditions which influence PCN production in the presence of FA and (ii) identifying new genes of which the expression is affected by the presence of FA.
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METHODS |
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DNA manipulation.
PCRs were carried out with Super Taq enzyme (Enzyme Technologies). The primers were synthesized by Isogen Life Science. Restriction enzymes were purchased from New England BioLabs and ligase from Promega. To PCR the phzI gene under the Plac promoter, primers oMP812 (5'-ATATATGAATTCAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCCTAAAATGATGCACATGGAAGAGCACACACTGAACGG-3') and oMP813 (5'-ATATATGAATTCCGGCGTGATCATGGGGGTGTGCACCG-3') were used. The resulting PCR fragment was digested with EcoRI and ligated into EcoRI-digested pBBR1-MCS5 to obtain pMP7450. A PCR fragment with the phzR gene under control of the Ptac promoter was constructed (G. Girard, unpublished) by using primers oMP777 (5'-ATATATCTCGAGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTTCACACAGGAAACAGCTAAATGGAGTTAGGGCAGCAGTTGGGATGGG-3') and oMP778 (5'-ATATATGAATTCCCCCTCAGATATAGCCCATCGCAACTGCG-3'). The resulting PCR fragment was digested with XhoI and EcoRI and ligated into XhoI- and EcoRI-digested pBBR-MCS5 to obtain pMP7447.
Analysis of PCN production.
For monitoring PCN production in time, PCN was extracted according to Chin-A-Woeng et al. (1998) with minor modifications. Culture samples (250 µl) were centrifuged and the culture supernatants were acidified to pH 2 using 6 M HCl. They were subsequently extracted with an equal volume of toluene by shaking on an Eppendorf mixer 5432 for 5 min. After centrifugation, the toluene phase was collected and dried in a rotary evaporator. The dry residue was dissolved in 100 µl acetonitrile and the solution obtained was mixed with 400 µl water.
PCN concentrations were determined by HPLC (DIONEX, Chromeleon software version 6.20) using a calibration curve. HPLC was performed using an Econosphere C18 5u, 259 mmx4·6 mm column (Alltech Associates) at 30 °C with a linear gradient of 2080 % (v/v) acetonitrile acidified with 0·1 % (v/v) trifluoroacetic acid in water and a flow rate of 1 ml min1.
Analysis of N-acylhomoserine lactone production.
Culture supernatants were adjusted to pH 9·0 with NaOH. The supernatants were extracted with 0·5 vol. ethyl acetate, shaken for 30 min and the solvent phase was collected. Subsequently the solvent phase was dried by rotary evaporation. The dried residue was dissolved in 25 µl acetonitrile and analysed by TLC. Samples spotted on C18 TLC plates (Merck) were developed in methanol/water (60 : 40, v/v). After development, the TLC was overlaid with LB 0·8 % (w/v) agar containing a 10-fold diluted overnight culture of the C. violaceum indicator strain CV026 (McClean et al., 1997) and kanamycin (50 µg ml1). After incubation for 48 h at 28 °C, chromatograms were analysed for appearance of violet spots.
Construction of a chromosomal microarray of P. chlororaphis PCL1391.
Microarray construction, RNA isolation, and data analyses were developed by G. Girard. Microarray analyses were performed according to the MIAME standards (Brazma et al., 2001). The chromosomal microarray consists of approximately 12 000 random chromosomal SauIIIA DNA fragments from PCL1391 between 0·4 and 2 kb in size. After an initial cloning of the chromosomal fragments into pBluescript, each insert was amplified with amino-labelled primers annealing to the multicloning site flanking the insert. PCR products were spotted on poly-L-lysine-coated glass slides with a Genemachines Omnigrid 100 spotter (Genomic Solutions). The microarrays were designed to contain the following controls: empty spots (neither DNA, nor buffer), spots with only 50 % (v/v) DMSO, a negative control with
phage DNA (Westburg), and PCR products of several known genes of PCL1391: phzB, phzH, phzR, phzI, sss, gacS and psrA (Chin-A-Woeng et al., 2000
, 2001a
, b
, 2005
).
RNA preparation and microarray processing.
RNA was extracted from cultures at an OD620 of 2·0 with an adapted protocol developed by Jon Bernstein (http://bugarrays.stanford.edu/protocols/rna/Total_RNA_from_Ecoli.pdf). After phenol/chlorophorm extraction, the supernatant was applied to 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 % (w/v) agarose gel following the protocol of the RNeasy Midi kit. RNA was immediately used for cDNA probe generation using the CyScribe post-labelling kit (Amersham Biosciences). Each reaction was performed with 30 µg of total RNA and random nanomers. After purification, the efficiency of Cy label incorporation into the cDNA and the quality and amounts of labelled cDNA were verified with a Ultrospec 2100 Pro spectrophotometer (Amersham Biosciences). The amounts of Cy-labelled cDNA were calculated using http://www.pseudomics.com/percent_inc.html. Equal amounts of cDNA for both Cy3 and Cy5 labels, with a minimum of 40 pM of each dye, were hybridized. Before hybridization, the microarrays were rehydrated by H2O steam at 50 °C and snap-cooled on a hot plate. Then they were UV-cross-linked at 250 mJ cm2 (Amersham Life Sciences UV cross-linker). They were subsequently prehybridized with 100 µl prehybridization buffer [0·4 µg µl1 herring sperm DNA (Gibco, Invitrogen), 0·4 µg µl1 yeast tRNA (Gibco, Invitrogen), 5x Denhardt's (Denhardt, 1966), 3·2x SSC (sodium chloride/sodium citrate buffer, 3 M NaCl/0·3 M trisodium citrate), 0·4 %, w/v, SDS] for 2 min at 80 °C and 30 min at 65 °C. Finally, the slides were washed at room temperature in 2x SSC, twice in 70 % (v/v) ethanol, once in 90 % (v/v) and 100 % (v/v) ethanol (5 min per wash step) and air-dried. The Cy-labelled cDNA was hybridized overnight at 65 °C in a GeneTAC Hybstation (Genomic Solutions). After hybridization, the slides were washed in 2x SSC, 0·1 % (w/v) SDS for 5 min at 30 °C, in 0·5x SSC for 5 min at 25 °C and in 0·2x SSC for 5 min at 25 °C. The slides were dehydrated by washing in 70 %, 90 % and 100 % (v/v) ethanol (1 min per wash step), dried by compressed air and scanned in a G2565AA microarray scanner (Agilent). The experiment was repeated at least four times, including dye swaps.
Microarray data analysis.
After scanning, the microarrays were analysed in GenePix Pro version 5.0 (Molecular Devices). The following criteria were implemented to select spots corresponding to genes assumed to be affected significantly in their expression by FA: spots were considered as interesting if the mean of the ratio of red and green laser intensities was higher than 2 [ratio of medians (650/550)>2] or lower than 0·5, but positive [ratio of medians (650/550)<0·5 and ratio of medians (650/550)>0]. 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 channel [(%>B550+2SD)>80 and (%>b650+2SD)>80]. Results of four identical microarray experiments were compared and spots were categorized into three groups: (i) expression unaltered by FA; (ii) more than twofold upregulated by FA; (iii) more than twofold downregulated by FA. The values were normalized assuming that most genes of the array are not differentially expressed. Results of four microarray experiments were used in our analyses. Raw microarray data, microarray image, and sequences of clones are available as supplementary data with the online version of this paper.
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RESULTS |
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Effect of FA on the expression of the PCN biosynthetic and regulatory genes
Microarray analysis allowed the identification of changes in gene transcription of the phz operon and the regulatory PCN genes phzI, phzR, gacS, rpoS and psrA after the addition of FA. RNA was isolated from a PCL1391 culture at OD620 2·0, at which density the start of PCN production is detected in the medium, and was used for cDNA synthesis and fluorescent labelling. The labelled cDNA was hybridized with PCL1391 microarrays. Expression profiling showed that the expression of PCN biosynthetic genes and the quorum-sensing regulatory genes phzI and phzR was severely reduced in the presence of 1 mM FA (Table 4). The highest reduction (sixfold) was observed for the PCN biosynthetic genes phzB, phzC and phzH. The expression of phzI was reduced fourfold and the expression of the transcription factor phzR was reduced twofold (Table 4
). Expression of the PCN regulators rpoS, gacS and psrA was not affected by FA (Table 4
). Transcription of the PCN regulators psrA and gacS, and the biosynthetic gene phzB, was followed during growth with the use of Tn5luxAB reporter mutants (Fig. 5
). Transcription of psrA in PCL1111 (psrA : : Tn5luxAB) and transcription of gacS in PCL1969 (gacS : : Tn5luxAB) were not influenced by FA. Transcription of the biosynthetic operon (phzB : : Tn5luxAB) in PCL1119 increased with increasing optical density whereas it did not increase in the presence of FA (Fig. 5
).
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DISCUSSION |
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When the C6-HSL concentration was increased by constitutive expression of phzI (strain PCL1999), PCN levels were no longer reduced by the addition of FA (Figs 3 and 4). Constitutive expression of phzR also led to an increased production of HSLs (Fig. 3
). Increasing concentrations of the transcriptional regulator PhzR stimulate expression of phzI and therefore increase C6-HSL production. Transcription studies with Tn5luxAB reporter mutants and microarray analyses showed that the expression levels of the phenazine biosynthesis operon and of the quorum-sensing regulators phzI and phzR are reduced by FA (Table 4
, Figs 1 and 5
). This reduction in gene expression explains the reduced levels of PCN in the presence of FA and suggests that repression of PCN production occurs upstream of the biosynthetic phz operon and of phzI. FA could directly or indirectly repress phzR expression and thereby reduce the expression of phzI and biosynthesis of C6-HSL. Expression of the PCN regulators gacS, rpoS and psrA was not altered by FA (Table 4
, Fig. 5
), which suggests that FA acts in parallel to or downstream of these genes.
The microarray allowed identification of genes differentially expressed by FA. Because the genome of PCL1391 has not been sequenced, a microarray containing only random fragments of genomic DNA and control genes was available (see Methods). Sequencing revealed that the same genes were present in different selected clones spotted several times on the microarray, thereby internally validating the analyses (Table 4).
Comparison of our data with those of the literature suggests a correlation between treatments with FA and iron limitation. Genes upregulated by FA in P. chlororaphis were induced by iron limitation in P. aeruginosa. Clones of class one are homologous to outer-membrane ferripyoverdin receptors fpvA (PA2398) and fpvB (PA4168) (Ghysels et al., 2004) of P. aeruginosa and are upregulated by iron limitation (Ochsner et al., 2002
; Palma et al., 2003
). These outer-membrane ferripyoverdin receptors have similarity with the TonB system that was shown to be involved in tolerance to solvents and drugs and thought to play a role in drug exclusion (Godoy et al., 2001
). This could explain the upregulation of the clones in this class in response to FA. The pyoverdin synthetase genes found to be upregulated by FA are homologous to the non-ribosomal peptide synthetases pvdI (PA2402) pvdJ (PA2400) and pvdD (PA2399) and the pyoverdin modification gene pvdA (PA2386) (Ochsner et al., 2002
; Palma et al., 2003
). All of these genes are induced by iron limitation in P. aeruginosa. Upregulation of these genes involved in the biosynthesis and uptake of pyoverdin can be explained in relation to iron starvation but is less obvious in relation to FA stress. Iron limitation increased fumC1 (PA4470) and decreased hemN (PA1546) expression in P. aeruginosa (Ochsner et al., 2002
; Palma et al., 2003
) and FA treatment also increased fumarase (fumC1) and decreased oxygen-independent coproporphyrinogen III dehydrogenase (hemN) expression in P. chlororaphis. Remarkably, clone 14_C10 is repressed by FA but has similarity with PA2406 and PA2407, which are also part of the pyoverdin cluster of P. aeruginosa and are upregulated by iron limitation (Ochsner et al., 2002
). This is the only sequenced clone that is regulated oppositely when comparing the expression profile of FA stress and iron limitation. Interestingly, FA does not seem to stimulate the transcription of all the genes induced by iron limitation, but appears to stimulate the pyoverdin system more specifically.
Although FA induces many genes involved in the uptake of iron, an increased production of siderophores was detected only during iron limitation and not during FA stress of P. chlororaphis PCL1391 (data not shown). This suggests that FA stress does not result in a complete iron starvation response. FA has ferric ion chelating characteristics (Bochner et al., 1980) which could explain the repression of PCN by FA since iron limitation represses PCN production. However, an excess of ferric ions (2 mM FeCl3) did not restore PCN production. A second experiment also contradicts this hypothesis, since the high-affinity iron chelator EDDHA (0·1 and 1·0 mM) did not mimic FA stress and did not repress PCN biosynthesis (Table 2
). FA also chelates other divalent cations including zinc and this could cause PCN repression by FA. However, an excess of 2mM ZnSO4 or ZnCl2 does not restore PCN production in the presence of FA (Table 3
), refuting this possibility.
FA and iron limitation reduce PCN production in P. chlororaphis PCL1391 (van Rij et al., 2004) and have partially overlapping expression profiles, but FA is not causing or mimicking iron limitation. This suggests that the two stress conditions FA and iron limitation trigger partially overlapping regulatory cascades that result in the repression of PCN biosynthesis.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 23 March 2005;
revised 27 May 2005;
accepted 6 June 2005.
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