Biomerit Research Centre, Department of Microbiology, National University of Ireland, Cork, Ireland1
Author for correspondence: Fergal OGara. Tel: +353 21 272098. Fax: +353 21 275934. e-mail: f.ogara{at}ucc.ie
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ABSTRACT |
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Keywords: biocontrol, repressor, transcriptional control, Pseudomonas fluorescens
The GenBank accession number for the sequence reported in this paper is AF129856.
a Present address: Department of Molecular Biology, IRIS, Chiron, Via Fiorentina 1, 53100 Siena, Italy.
b Present address: Department of Agronomy, Lethbridge Research Centre, Agriculture and Agri-Food Canada, 5403 1st Avenue South, Lethbridge, Alberta T1J 4B1, Canada.
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INTRODUCTION |
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Previous work in this laboratory showed that phloroglucinol produced by Pseudomonas fluorescens F113 inhibits growth of Pythium ultimum in vitro (Shanahan et al., 1992 ) and protects sugar beet seedlings from damping-off disease, caused by Pythium ultimum in soil microcosms (Fenton et al., 1992
).
The widespread application of pseudomonads as biocontrol agents in agriculture is impeded, however, by their variable performance under field conditions (Weller, 1988 ; Cook, 1993
; Dowling & OGara, 1994
). An understanding of how biocontrol bacteria regulate the expression of genes involved in the inhibition of pathogens is likely to be a prerequisite for predicting the environmental conditions for the optimum performance of these bacteria.
The genes involved in the biosynthesis of the phloroglucinol molecule have been cloned from different strains (Vincent et al., 1991 ; Fenton et al., 1992
) and the sequence of the entire biosynthetic locus is now available for P. fluorescens strain Q2-87 in the EMBL database (accession no. U41818). Six ORFs have been identified within a 6·5 kb segment of DNA from strain Q2-87 which is sufficient to transfer phloroglucinol biosynthetic capability to recipient strains of Pseudomonas spp. that did not previously produce the antifungal metabolite (Cook et al., 1995
). The genes phlA, phlC, phlB and phlD have been reported to be contained within a large transcriptional unit, previously shown to be required for phloroglucinol production (Bangera & Thomashow, 1996
; Thomashow et al., 1997
). Their predicted products show similarities with proteins involved in fatty acid and polyketide biosynthesis (Cook et al., 1995
; Thomashow et al., 1997
). phlE is associated with the presence of the red pigment that is usually present in media when phloroglucinol is produced (Bangera & Thomashow, 1996
; Keel et al., 1996
) and is hypothesized to be involved in the transport of phloroglucinol out of the cell (Thomashow et al., 1997
). However, the precise role of each gene in the biosynthetic pathway has not yet been elucidated.
The phlF gene located upstream of the biosynthetic genes shows some similarity to transcriptional repressor genes from a wide variety of organisms and is the focus of this study. In this paper we show that the phlF gene product is a DNA-binding protein which binds specifically to the phlAphlF intergenic region. We also characterize phlF as a repressor specific for the biosynthesis of the phloroglucinol antifungal metabolite.
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METHODS |
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Deregulated biosynthetic clones were constructed as follows. The phloroglucinol biosynthetic region of pCU203 was cloned as a 6 kb BamHI fragment into the medium-copy-number broad-host-range vector pBBR1MCS. A 1·1 kb SacI deletion generated pBSc-8 containing a subclone of the biosynthetic region in which phlF was deleted. An 8·3 kb EcoRI DNA fragment containing the biosynthetic locus, including phlE but a truncated and inactive phlF gene, was cloned into the medium-copy-number vector pMP220, generating pMPE8.3.
The mobile phloroglucinol biosynthetic gene fusions carried by the plasmids pCU106 and pCU107 were constructed by cloning a BamHISphI fragment and an EcoRISphI fragment of the phloroglucinol biosynthetic locus of F113 from pCU203 into pMP220 such that the phlD gene forms a transcriptional gene fusion with the promoterless lacZ gene of pMP220 (Fig. 1). phlF is truncated and inactive in pCU107 but intact and functional in pCU106. A phlA::lacZ transcriptional fusion pCU102 was constructed by cloning a 1·8 kb KpnI fragment from pBSL-8 into pMP220, such that the phlA gene is fused to lacZ. phlF is truncated in this construct. The cloning of the 1·8 kb SalIKpnI fragment from pCU203 into pMP190 generated the phlF transcriptional fusion pCU109.
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phlF mutants were created by cloning the internal SacIKpnI fragment of phlF into narrow-host-range vector pK18. The resulting pKREG1 suicide construct was introduced into F113 by electroporation and the integrants were selected by their resistance to Km. The resulting phlF mutant (F113-phlF-), in which the pK18 plasmid had integrated into the chromosomal phlF copy, was verified by Southern blot hybridization.
Determination of nucleotide sequence and sequence analysis.
A series of subclones of the phlF locus were constructed (Table 1) and the nucleotide sequence was determined by primer walking using an Applied Biosystems 370 automated sequencer. The sequence data were assembled using DNASTAR software package (DNASTAR, Madison, WI, USA) and analysed using BLAST (Altschul et al., 1990
) at the National Centre for Biotechnology Information (http://www3.ncbi.nlm.nih.gov/). DNASTAR software was employed for multiple sequence alignments.
Construction of PhlF expression plasmid.
phlF was amplified by PCR using pBSL-8 as the DNA template and the oligonucleotide primers employed were HTID1 (5'-TTGATCCATGGCCCGTAAACCATC-3') and HTID3 (5'-ACCCGAAGATCTGGCTTCGGCG-3'). The restriction enzyme sites for NcoI and BglII, which were incorporated into the amplified DNA fragment by the primers, are indicated by single- and double-underlining, respectively. The NcoI site is incorporated at the putative ATG translational start site of phlF and the BglII site is positioned in the correct reading frame in the 5' orientation with respect to the putative stop codon which is modified and hence removed. The PCR product encompassing phlF was restricted with NcoI and BglII, isolated and purified, and subsequently cloned into the NcoIBglII sites of the expression vector pQE-60 of the QIAexpress system (Qiagen), generating the recombinant plasmid pQE-60.27 (Table 1). This construct places six consecutive histidine residues (6xHis-tag) at the C-terminus of the PhlF protein sequence without altering the folding of the resulting protein. Ni-NTA resin has specific affinity for proteins with an affinity tag of six histidine residues, allowing the one-step purification of PhlF with six histidine residues.
Transformant colonies containing the appropriate insertion were identified by restriction analysis and subsequently transformed into E. coli strain SG31009(pREP4) (Gottesman et al., 1981 ). Transformant strains expressing PhlF were initially analysed by SDS-PAGE. Freshly inoculated 10 ml cultures were grown at 37 °C with shaking to an OD600 of 0·5 and protein overexpression was induced from the E. coli phage T5 promoter in pQE-60 upon the addition of 2 mM IPTG. After 2 h further incubation, 1 ml samples were removed and centrifuged at 14000 r.p.m. in a microcentrifuge, resuspended in 50 µl Laemmli buffer (Laemmli, 1970
) and analysed by SDS-PAGE. The inserted DNA fragments from plasmids of strains which were seen to overproduce a protein of the appropriate size were sequenced to confirm the correct DNA fragment had been cloned.
Overexpression and purification of PhlF.
A 1% (v/v) inoculum of an overnight culture of E. coli SG31009(pREP4)(pQE-60.27), grown at 37 °C with shaking, was added to 1 litre LB medium containing 100 µg Ap ml-1 and 25 µg Km ml-1 and grown at 30 °C for 56 h. Overexpression of PhlF was induced by the addition of 0·1 mM IPTG. After 34 h further incubation at 30 °C, the cells were harvested by centrifugation (8000 g for 10 min), washed once and resuspended in 10 ml lysis buffer (50 mM Na2PO3, 300 mM NaCl, 20 mM imidazole, 5 mg lysozyme ml-1). Cell lysis was achieved by sonication (Sambrook et al., 1989 ). The serine protease inhibitor PMSF was added to 1 mM final concentration immediately after cell lysis, using a 100 mM solution in 2-propanol. Cell debris was removed from the crude extracts by centrifugation for 1 h at 20000 g at 4 °C.
The 6xHis-tag PhlF protein was partially purified from the cleared cell extract under non-denaturing conditions using Qiagen Ni-NTA affinity spin columns according to the manufacturers instructions. Protein concentrations were determined using the Bradford assay with bovine serum albumin (BSA) as standard (Bradford, 1976 ).
Gel retardation assays.
DNA fragments 2.9 and 10.6 were amplified using PCR with pBSc-8 as the DNA template and primers specific for the amplification of the intergenic promoter region of the biosynthetic locus: primer 2 (5'-GCTGGCAGAAAGCCGAGACAGG-3'), primer 6 (5'-ATTTATGGGCATGGGACCGC-3'), primer 9 (5'-AATAGGGAGATATGAGAAGG-3'), primer 10 (5'-CAAGAACGTCTAGATGACCCTTGA-3') (see Fig. 4). A third DNA fragment, 7.U, was amplified as above, using primer 7 (5'-CAATAGGTTTGTTTTCGTAC-3') and the universal forward primer (Genosys). Fragment 7.U of 280 bp was restricted with AluI which generates two DNA fragments of 180 and 100 bp; the 180 bp band was chosen and isolated. The DNA fragments were 5'-end-labelled with [
-32P]ATP using polynucleotide kinase (Boehringer Mannheim).
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Detection of phloroglucinol production and ß-galactosidase assays.
Pseudomonas test strains were assayed for phloroglucinol production by HPLC using the method described by Shanahan et al. (1993) . ß-Galactosidase assays were performed by the colorimetric procedure described by Miller (1972)
, using two drops of chloroform and 1 drop of 0·1% SDS to permeabilize the cells. All reported enzyme activities are the means of several replicate assays of at least three independently grown cultures and are expressed in Miller units. pMP220 and pMP190 were also mobilized into each strain and the background ß-galactosidase activity was measured as a control.
Detection of HCN and protease production.
Strains to be screened were grown overnight in triplicate in 10 ml SA broth amended with 100 µM FeCl3 and 10 µl aliquots were spotted onto appropriate plates. For the HCN assay, test strains were spotted on SA agar plates amended with 100 µM FeCl3 for 2448 h. The production of HCN by Pseudomonas strains was determined using HCN indicator paper (Castric & Castric, 1983 ). Production of HCN was visually recorded by the intensity of blue coloration of the indicator paper placed in the lid after 24 and 48 h growth. The protease assay was performed using 3% (w/v) skim milk agar plates. Protease production was measured as the diameter of the clear zones of proteolysis around protease-positive colonies after 24 and 48 h growth.
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RESULTS |
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The sequence of phlF is similar to known repressor genes
The phlF gene of F113 consists of an ORF of 627 bp, with a corresponding predicted protein of 209 aa with a predicted molecular mass of 23570 Da, which, interestingly, is 6 aa longer than the proposed phlF gene product of Q2-87 (Bangera & Thomashow, 1996 ). The deduced protein sequence was compared with those in the EMBL database by means of the BLAST program. At the amino acid sequence level, the predicted PhlF protein shows similarity to a group of transcriptional regulators, including six proteins implicated as DNA-binding transcriptional repressors (Fig. 2
) (Postle et al., 1984
; Shaw & Fulco, 1992
; Hansen et al., 1993
; Schwecke et al., 1995
; Yang et al., 1995
; GenBank accession no. M14641). The deduced PhlF protein displays the strongest similarity to a putative DNA-binding repressor protein of the rapamycin biosynthetic locus in Streptomyces hygroscopicus, sharing 24·5% identity with the proposed protein of orfY (Schwecke et al., 1995
). The most conserved region of the amino acid sequence in relation to the repressor proteins is significantly at the N terminus (Fig. 2
), which exhibits a helixturnhelix DNA-binding motif (Brennan & Matthews, 1989
). This strongly suggests that phlF encodes a DNA-binding protein. This region was also found to be conserved in the amino acid sequence of PhlF of P. fluorescens Q2-87 (Bangera & Thomashow, 1996
).
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Specific binding of PhlF to the phlAphlF intergenic region
Gel retardation assays were used to investigate the DNA-binding activity of PhlF. The phlAphlF intergenic region spans 421 bp between the proposed phlA and phlF translational start sites. Three shorter overlapping DNA fragments spanning parts of this region were amplified by PCR (Fig. 4) and used as substrates for gel retardation assays with extracts containing overexpressed PhlF (data not shown) and partially purified PhlF (Fig. 4
). Samples of 0·8 ng of each of the fragments of DNA migrated freely through the polyacrylamide gels. However, when the DNA fragments were incubated with 0·5 µg aliquots of Ni-NTA-enriched PhlF extract, prior to being applied to a polyacrylamide gel, the migration of the 2.9 and 7.U fragments was retarded but the 10.6 fragment migrated equidistant to the free DNA fragment. This suggests that PhlF had bound to the 2.9 and 7.U fragments but not to the 10.6 DNA fragment. As this apparent binding occurred in the presence of poly[d(I-C)] non-specific competitor DNA, the binding of PhlF was shown to be specific for the fragments. In the control reactions, a 125-fold excess of unlabelled DNA fragment was added along with the PhlF extract to the respective labelled fragments during incubation. When specific competitor DNA was added in excess to the binding reactions of fragment 2.9 and 7.U, no gel shift was observed. The labelled fragments were specifically out-competed for binding to PhlF by the unlabelled fragment in excess and migrated in a similar fashion to the free DNA fragments. In contrast, gel retardation studies with cell extracts of the IPTG-induced and -uninduced E. coli cultures harbouring pQE-60 did not reveal any DNA binding with the DNA fragments described (data not shown). This experiment showed that PhlF binds specifically to the phlAphlF intergenic region. Binding of PhlF to fragments 2.9 and 7.U but not to fragment 10.6 suggests that the PhlF-binding site is upstream of primer 6. The 2.9 and 7.U DNA fragments overlap by approximately 110 bp. The observation that PhlF retarded the migration of the 2.9 and 7.U fragments to approximately the same extent suggests that each fragment contains only one binding location. Therefore, the binding site of PhlF may be located in the overlapping region of the fragments (Fig. 4
). However, the possibility that more than one binding site for PhlF exists within the phlAphlF intergenic region cannot be overlooked.
Repression of phloroglucinol production and transcription of the biosynthetic genes by phlF in F113
The negative role of phlF on phloroglucinol production was further investigated by the mobilization of pBSL-8 into F113 by triparental mating. The introduction of multiple copies of phlF on pBSL-8 in F113 leads to a reduction of over 90% in phloroglucinol production after 20 h growth in SA medium, supplemented with chloramphenicol (Table 2). To further investigate the repressive effect of phlF, the ß-galactosidase activity of cells with pCU107, which contains the phlD::lacZ transcriptional fusion, was tested in strain F113 containing the phlF subclone and the control strain. Expression from the phlD::lacZ reporter plasmid, pCU107, was reduced by over 90% in the presence of multiple copies of phlF (Table 2
). This demonstrates that transcription of the phlD biosynthetic gene is repressed by phlF in F113.
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PhlF does not have a repressive effect on other secondary metabolite production
The secondary metabolites HCN and protease, as well as phloroglucinol, are known to be under similar global control from a two component system in P. fluorescens (Corbell & Loper, 1995 ; Dunne et al., 1996
). Strain F113(pBSL-8), in which phloroglucinol production was repressed, and the F113-phlF- mutant were assessed in their production of the HCN and protease metabolites by plate assays, as described in Methods. The F113-phlF- mutant appeared identical to the wild-type in its production of HCN and protease after 24 and 48 h. Also, the F113 strain carrying multiple copies of phlF [F113(pBSL-8)] did not differ in its production of HCN and protease after 24 and 48 h growth when compared to the control strain [F113(pBBR1MCS)]. This suggests that the increase in gene dosage of phlF did not affect the production of HCN or protease in this strain. As such the repressive ability of phlF appears to be specific for phloroglucinol production.
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DISCUSSION |
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During this study, we have compiled evidence which proves that phlF acts as a negative regulator of phloroglucinol production, which is responsible for repression of the phloroglucinol biosynthetic genes at the transcriptional level. High copy numbers of phlF in the wild-type background inhibits phloroglucinol production in F113 and reduces the level of transcription of the biosynthetic phlD gene fusion by over 90%. Second, a mutation of phlF leads to increased phloroglucinol production in vitro. Phloroglucinol production in the phlF mutant increased with respect to wild-type F113 levels in all three media tested, suggesting that phloroglucinol production was derepressed on inactivation of phlF. It is possible that in SA medium F113 phloroglucinol production is fully derepressed in late exponential phase and therefore does not exhibit any difference to the repressor mutant during later phases of growth.
The time-course experiment of phloroglucinol production from the phlF mutant seems to indicate that the repressive action of PhlF is time-dependent; phlF appears to play a role in preventing phloroglucinol production in the early stages of growth. High phloroglucinol production early in the growth cycle may be detrimental to the cell. It was indeed observed that growth of F113-phlF- was delayed compared to that of the wild-type. Similar effects of phloroglucinol on cell growth were reported previously for the phloroglucinol-overproducing strain Q2-87(pPHL5122) which was seen to attain lower cell density in broth culture and died more rapidly than the Q2-87 wild-type strain (Bonsall et al., 1997 ). The inhibitory mode of action of the phloroglucinol molecule has not yet been investigated and little is known about the inherent phloroglucinol resistance of the phloroglucinol-producing strains. The addition of synthesized phloroglucinol to liquid cultures of F113 in early exponential phase stops growth and replication of the culture (data not shown), indicating that the accumulation of phloroglucinol during this phase may be self-inhibitory. It also appears that production of phloroglucinol is fully derepressed at the maximum levels of induction for F113. It is tempting to speculate that PhlF may play an important role in preventing phloroglucinol accumulation early in the growth cycle, when it may be deleterious to the strain. While the evolutionary advantages of many antibiotics may be as agents of competition, some secondary metabolites with (perhaps incidental) antimicrobial activities may also have other functions, such as pamamycin which stimulates aerial mycelium formation in Streptomyces alboniger (Kondo et al., 1988
).
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ACKNOWLEDGEMENTS |
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Received 18 March 1999;
revised 4 October 1999;
accepted 20 October 1999.