The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance

Abdelhamid Abbas, John E. McGuire, Delores Crowley{dagger}, Christine Baysse, Max Dow and Fergal O'Gara

BIOMERIT Research Centre, Microbiology Department, National University of Ireland, Cork, Ireland

Correspondence
Fergal O'Gara
f.ogara{at}ucc.ie


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
2,4-Diacetylphloroglucinol (PHL) is the primary determinant of the biological control activity of Pseudomonas fluorescens F113. The operon phlACBD encodes enzymes responsible for PHL biosynthesis from intermediate metabolites. The phlE gene, which is located downstream of the phlACBD operon, encodes a putative permease suggested to be a member of the major facilitator superfamily with 12 transmembrane segments. PhlE has been suggested to function in PHL export. Here the sequencing of the phlE gene from P. fluorescens F113 and the construction of a phlE null mutant, F113-D3, is reported. It is shown that F113-D3 produced less PHL than F113. The ratio of cell-associated to free PHL was not significantly different between the strains, suggesting the existence of alternative transporters for PHL. The phlE mutant was, however, significantly more sensitive to high concentrations of added PHL, implicating PhlE in PHL resistance. Furthermore, the phlE mutant was more susceptible to osmotic, oxidative and heat-shock stresses. Osmotic stress induced rapid degradation of free PHL by the bacteria. Based on these results, we propose that the role of phlE in general stress tolerance is to export toxic intermediates of PHL degradation from the cells.


Abbreviations: PHL, 2,4-diacetylphloroglucinol; MAPG, monoacetylphloroglucinol; TMS, transmembrane segments

The GenBank accession number for the sequence reported in this paper is AJ542662.

{dagger}Present address: Cork Institute of Technology, Cork, Ireland.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Many Pseudomonas fluorescens strains produce 2,4-diacetylphloroglucinol (PHL) (Bangera & Thomashow, 1996; Boruah & Kumar, 2002; Nowak-Thompson et al., 1994; Reddi & Borovkov, 1970; Sharifi-Tehrani et al., 1998). This phenolic molecule has broad-spectrum antifungal (Levy et al., 1992; Schoonbeek et al., 2002; Tomas-Lorente et al., 1989; Weller & Cook, 1983), antibacterial (Levy et al., 1992), antihelminthic (Bowden et al., 1965; Harrison et al., 1993) and phytotoxic (Keel et al., 1992; Reddi et al., 1969) activities. PHL is the primary determinant of biological control by P. fluorescens F113 (Shanahan et al., 1992). Synthesis of PHL is directed by the phlACBD genes, which are believed to constitute an operon (Bangera & Thomashow, 1996, 1999; Delany, 1999). PhlD shows structural similarities with plant chalcone synthase, also called polyketide synthase type III, and is necessary but not sufficient for the synthesis of monoacetylphloroglucinol (MAPG) (Bangera & Thomashow, 1999). PhlA, PhlC and PhlB are necessary and sufficient for the transacetylation of MAPG to produce PHL (Bangera & Thomashow, 1999; Shanahan et al., 1992). The available data strongly suggest that PhlA, PhlC, PhlB and PhlD function in a multienzyme complex (Bangera & Thomashow, 1999; Delany, 1999). Expression of the phlACBD operon is subject to complex regulation (Aarons et al., 2000; Abbas et al., 2002; Bangera & Thomashow, 1999; Chou et al., 1993; Corbell & Loper, 1995; Delany, 1999; Delany et al., 2000; Duffy & Defago, 1999; Schnider-Keel et al., 2000). In addition, PHL is an autoregulator, positively influencing its own biosynthesis (Abbas et al., 2002; Schnider-Keel et al., 2000).

Micro-organisms have developed various ways to resist the toxic effects of the secondary metabolites they produce. These mechanisms include export of the metabolite out of the producing cell. The phlE gene, located at the 3' end of the phlACBD operon in P. fluorescens strains (Bangera & Thomashow, 1996, 1999; Delany, 1999) encodes a putative transmembrane permease with 12 predicted transmembrane segments (TMS) (Bangera & Thomashow, 1999). The hydrophobicity profile predicted by the Kyte–Doolittle model indicates that PhlE is structurally similar to NorA of Staphylococcus aureus. NorA is a multidrug transporter mediating resistance to a range of structurally dissimilar drugs (Paulsen & Sukurray, 1993; Yoshida et al., 1990). PhlE also has structural similarity with integral membrane proteins associated with resistance which are encoded within clusters responsible for polyketide biosynthesis (Brautaset et al., 2000; Fernandez-Moreno et al., 1991; Guillfoile & Hutchinson, 1992; Marger & Saier, 1993; Molnar et al., 2000). It has been reported that phlE mutants produce less PHL than the parent strain (Bangera & Thomashow, 1996, 1999). The phlE mutants are also affected in production of a red pigment, usually associated with PHL production. The genetic arrangement of phlE with phlACBD, the reduction of PHL production in the phlE mutant and the structural similarity with other export proteins strongly suggest that PhlE might have a role in the export of the PHL. However, the role of PhlE in PHL export has not been directly examined.

Beside their role in drug resistance, many efflux pumps play an important role in stress tolerance. Consequently, in this report we have examined the role of PhlE not only in PHL resistance but also in general stress tolerance in P. fluorescens F113. We show that PhlE is important for F113 survival at high concentrations of PHL and that it plays an important role in protecting F113 from a range of environmental stresses, where it may export toxic intermediates of PHL degradation from the bacterial cell.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, plasmids and culture conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. P. fluorescens F113 and derivatives were routinely grown at 28 °C in minimal medium (SA) (Scher & Baker, 1982) with sucrose (50 mM) and asparagine (17·5 mM) as carbon and nitrogen sources, respectively. SA medium was supplemented with 100 µM FeCl3. Escherichia coli strains were grown at 37 °C in Luria–Bertani broth (Sambrook et al., 1989). Antibiotics were used at the following concentrations (µg ml–1): for P. fluorescens, tetracycline 75 and kanamycin 50; for E. coli, tetracycline 25, kanamycin 30 and ampicillin 100.


View this table:
[in this window]
[in a new window]
 
Table 1. Strains and plasmids used

 
Recombinant DNA techniques.
Plasmid DNA isolations were performed using the Qiagen plasmid mini kit according to manufacturer's specifications. Genomic DNA isolation was performed using standard methods (Sambrook et al., 1989). Plasmids were introduced into E. coli and P. fluorescens by electroporation (Farinha & Kropinski, 1990) or mobilized into P. fluorescens by triparental mating using the helper plasmid pRK2013 (Figurski & Helinski, 1979).

Construction and complementation of phlE mutant.
A 0·7 kb BamHI–SphI fragment of pMPS2 was subcloned into BamHI/SphI sites of the narrow-host-range vector pK18 (Pridmore, 1987) to produce pKE7. A 0·275 kb BanII fragment within the phlE gene was isolated from pKE7, blunt-ended and subcloned into the SmaI restriction site of the suicide vector pK18 to produce pKD-3. This construct was mobilized by triparental mating into F113. Cells with chromosomally integrated pKD-3 were selected for kanamycin resistance. The position of the mutation was confirmed by Southern blotting. The mutant was named F113-D3. To complement the F113-D3 mutant, a PCR fragment containing the full sequence of the phlE gene and 50 bp extra on both sides was obtained using pHLE1 (5'-CCGCTCGAGAGAGGGCTTCGAAAGCGCT-3') and pHLE2 (5'-CCCAAGCTTTGGCGAGTCCAGCAACAT-3'). The PCR product was digested using HindIII and XhoI and cloned into the broad-host-range cloning vector pBBR1MCS (Kovach et al., 1994), restricted by the same enzymes to produce pCUD4. This places the expression of phlE under control of the lacZ promoter.

Sequencing and sequence analysis.
The phlE gene was sequenced using the pMPS2 plasmid containing the entire phlE gene as template and the primers 5'phlE (5'-CCCGGCGCGGACTCAACC-3'), SEQE2 (5'-CAGCTACCTGACAGACATCC-3') and SEQE3 (5'-CCGGGCTGGTGGTGGGCTG-3') as well as the M13 universal primers. The sequence data were analysed using the MEGALIGN DNASTAR software package (Madison, WI, USA), the MFOLD program (Mathews et al., 1999; Zuker et al., 1999) and the Sossui system at www.expasy.ch.

Stress response assays.
To measure the ability of the wild-type and the mutant variants to survive heat shock, cells were grown overnight in LB at 28 °C with shaking (150 r.p.m.). The cells were then washed twice with LB and resuspended to a density of approximately 5000 c.f.u. ml–1. One millilitre of the diluted cell suspension was transferred to prewarmed Eppendorf tubes at 50 °C. Viable counts were determined by plating 100 µl from each tube per time point on LB plates. To measure the sensitivity to osmotic shock, cells were grown and washed as described above and resuspended to a density of approximately 50 000 c.f.u. ml–1. One millilitre of this cell suspension was inoculated into 10 ml LB or SA containing NaCl or sucrose. After incubation at 28 °C with shaking (200 r.p.m.), aliquots of 100 µl were taken at 0, 2, 4 and 5 h and plated on LB plates to determine the c.f.u. Sucrose concentrations of 1 and 2 M, and NaCl concentrations of 0·5 and 0·75 M were tested. The concentrations of sucrose used in this analysis are 20 and 40 times higher than the concentrations found under normal growth conditions. Sensitivity to hydrogen peroxide (H2O2) was measured on LB plates. Cells from 17 h growth in LB medium were plated on LB plates at a density of 106 c.f.u. per plate and sterile Whatman 3MM filter disks impregnated with 20 µl 30 % (v/v) H2O2 were placed on the plate. The plates were incubated for 36 h at 28 °C and the zone of inhibition was measured. To measure the cell sensitivity to PHL, cells were grown overnight on SA at 28 °C with shaking (150 r.p.m.), washed twice and diluted in Ringer's solution (Oxoid). A set of solutions with different concentrations of HPLC-purified PHL was prepared in ethanol (70 %) and added to SA medium before solidification to give final concentrations of 0, 50, 100, 150, 200, 300, 400 and 500 µg ml–1. Cells of F113 and F113-D3 were plated and incubated for 36 h at 28 °C.

Measurement of PHL levels.
P. fluorescens F113 and variants were assayed for the level of PHL production by HPLC using the method described by Shanahan et al. (1992). The free PHL was extracted from the culture supernatant. To extract cell-associated PHL, cells were broken by ultrasonic treatment. The broken cells were centrifuged at 10 000 g for 5 min and PHL was extracted from the supernatant.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Sequence analysis of the phlE gene from P. fluorescens F113
The phlE gene is located at the 3' end of the phlACBD operon. The 5' end of phlE of P. fluorescens F113 was recently sequenced along with the entire phlACBD operon (GenBank accession no. AF497760). In the present study, we determined the full sequence of the gene (AJ542662). To examine whether phlE constitutes an independent transcriptional unit, the DNA sequence of the phlE-phlD intergenic region was analysed for the presence of any secondary structure using the MFOLD program (Mathews et al., 1999; Zuker et al., 1999). A 22 bp palindrome, consisting of two GC-rich (73 %) inverted repeats of 11 bp separated by 4 bp, was identified downstream of the phlD translation stop codon. The sequence upstream of the palindrome is rich in C (41 %) and poor in G (12·5 %). These features are characteristic of the {rho}-dependent terminators. Additional evidence that the stem–loop formed by the two inverted repeats could function as a terminator comes from its calculated free energy, {Delta}G=–14·7 kcal (61·5 kJ), indicating that it could form a stable hairpin structure and function as a {rho}-dependent terminator of phlD transcription. Furthermore, the sequence (5'-CAGGGGCTTCGAAAGCGCT-3') located between positions –37 and –23, relative to the ATG translational start of phlE, shows similarity with the consensus sequence (5'-MRNRYTGGCACG-N4-TTGCWNNW-3') recognized by {sigma}54. The most important feature of this consensus is the perfectly conserved GC and GG elements (shown in bold type) positioned 12 and 24 nt downstream from the transcriptional start of genes under the control of {sigma}54 (Barios et al., 1999). This structure is not conserved in P. fluorescens Q2-87 (Bangera & Thomashow, 1999). This suggests that the phlE gene of P. fluorescens F113 may be transcriptionally independent of phlD and could be transcribed from a {sigma}54-dependent promoter.

The phlE gene consists of an ORF of 1269 bp, corresponding to a predicted protein of 423 aa with a predicted molecular mass of 45·2 kDa. PhlE proteins from F113 and Q2-87 are highly related and share 88·2 % identity and 97·4 % similarity throughout their entire amino acid sequence. It is noteworthy that the newly identified phlE of P. fluorescens HP-72 (AB125214) encodes a predicted protein showing two deletions of 3 aa each located between amino acids 270 and 300. Hydropathy analysis predicts that PhlE is organized in two sets of six hydrophobic {alpha}-helices, of 23 aa each, separated by a central hydrophilic loop. The central region, which is predicted to have a periplasmic location, corresponds to the most divergent amino acid sequence between the PhlE proteins of different P. fluorescens strains. Interestingly, PhlE of HP-72 has only eight hydrophobic {alpha}-helices of 23 aa each and a long predicted periplasmic C-terminal region. It is not known if these structural differences reflect functional differences with other PhlE proteins. Homology searches show that PhlE has similarity with Staphylococcus aureus NorA (Paulsen & Sukurray, 1993; Yoshida et al., 1990), E. coli Bcr (Bentley et al., 1993) and EmrD (Naroditskaya et al., 1993), and Bacillus subtilis Bmr (Neyfakh et al., 1991) and Blt (Ahmed et al., 1995). These proteins are members of the major facilitator superfamily having 12 TMS. We analysed the primary amino acid sequence of 10 randomly chosen members of the 12-TMS family. Interestingly, the analysed proteins share strong similarity in molecular mass (43·2±2·23 kDa) and in hydrophobic residue content (198±12 aa). The molecular mass and the hydrophobic residue content of PhlE from both F113 and Q2-87 are within these ranges. The 12-TMS family members have suggested functional motifs containing conserved amino acid residues. Motif D2 (LgxxxxxPvxP) and motif G (GxxxGPL) are specific to the 12-TMS family. Motif B (IxxxRxxqGxgaa) and motif C (gxxxGPxxGGxI) are common to both 12 and 14 TMS (Putman et al., 2000). Motif D2, found at the N-terminal region of 12-TMS family members, was found in the same location in PhlE. PhlE also has a G motif in {alpha}-helix 11; this motif is present in the same region of 12-TMS family members (Putman et al., 2001). Motifs B and C are also present at conserved locations. Overall these results suggest that PhlE is a transmembrane protein likely to be a member of the major facilitator superfamily having 12 TMS.

Effect of mutation of phlE on PHL synthesis and PHL resistance
Plasmid pKD-3, which contains an internal 0·275 kb BanII fragment of the phlE gene and a kanamycin resistance cassette, was integrated into the F113 chromosome via a single homologous recombination within phlE. Southern blotting analysis confirmed that F113-D3 had a disrupted phlE allele (data not shown). Colonies formed by F113-D3 are lighter in colour than the ones formed by F113 due to the absence of the red pigment, as reported by Bangera & Thomashow (1996, 1999). To investigate whether PhlE is implicated in PHL transport, we performed a time-course experiment monitoring the production of both free and cell-associated PHL in P. fluorescens F113 and in the phlE mutant derivative. Fig. 1 shows that mutation of phlE does not affect F113-D3 growth. F113-D3 produces less total PHL than the wild-type; levels of both free and cell-associated PHL are lower than in the wild-type strain. When we compared the ratio of cell-associated to free PHL, there were no significant differences between the wild-type and the mutant at any of the time points (Fig. 1). Overexpression of phlE in the F113-D3 mutant by introduction of pCUD4 (Table 1), which has approximately 15 copies per cell, restored PHL production to levels that were up to threefold higher than the wild-type. This complementation excludes the possibility that the phenotypic alteration of PHL production seen in the phlE mutant is due to polar effects of the mutation in phlE on downstream genes. Overall these data suggest that under normal growth conditions PhlE is not the only factor determining the distribution of PHL between the cells and the medium. In a study of indole synthesis in E. coli, Kawamura-Sato et al. (1999) reported that mutation of the acrEF genes, encoding a multidrug efflux pump, causes a reduction in indole production. This defect can be corrected if the mutant strain is cultured in a high concentration of tryptophan, suggesting the existence of an alternative indole efflux pump. The findings are thus superficially similar to our own on PHL production in P. fluorescens F113. They suggest that bacteria producing toxic compounds have multiple systems for their transport from the cell and that they engage a mechanism(s) for reducing the level of metabolite production when the transport is impeded. The mechanism by which mutation of phlE leads to a reduction of PHL synthesis is not known. The impact of PhlE on PHL production is not at the level of transcription of the phlACBD operon. A phlACBD : : lacZ transcriptional fusion revealed no differences in transcriptional activity between wild-type and phlE mutant backgrounds (data not shown).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1. Role of PhlE in PHL production. Samples from cultures of F113 (circles) and F113-D3 (phlE) (triangles) grown in SA, were taken at the indicated times and the cell-associated and free PHL content was quantified by HPLC. The concentration of PHL is expressed in nmol PHL per OD600 unit. The panel labelled ‘Growth’ shows the growth curve of F113 and F113-D3 in SA. Values for PHL levels are the means±SD of triplicate independent measurements.

 
P. fluorescens F113 produces and exports PHL to a maximum concentration of 50–70 µg per OD600 unit ml–1. F113 is resistant to this level of PHL, but is sensitive to higher levels. To study the influence of PhlE on PHL resistance, P. fluorescens F113 and its phlE mutant derivative were cultured on SA agar supplemented with increasing concentrations of PHL. Fig. 2 shows that when cells were grown at a PHL concentration of 300 µg ml–1 or higher, the survival of both strains was markedly decreased. The number of surviving cells of the phlE mutant strain was 32- and 62-fold less than the wild-type at PHL concentrations of 300 and 400 µg ml–1, respectively. These findings directly implicate PhlE in PHL resistance in P. fluorescens F113.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. PHL resistance test. F113 (circles) and F113-D3 (triangles) were grown to stationary phase and then plated on SA plates containing different concentrations of PHL. The number of surviving cells is expressed as a percentage of the number of c.f.u. on control plates not supplemented with PHL. Values given are the means±SD of triplicate independent measurements.

 
Role of PhlE in general stress tolerance
Both eukaryotic and prokaryotic cells increase expression of some multidrug transporter genes in response to environmental shock caused by heat (Miyazaki et al., 1992), osmotic (Ma et al., 1995) or oxidative (Chou et al., 1993; George & Levy, 1983) stresses. However, the role that transporters have in stress tolerance has not been extensively examined. To determine whether PhlE contributes to the tolerance of P. fluorescens F113 against different stresses, F113 and F113-D3 were compared for their sensitivity to increased osmolarity, heat shock and oxidative stress provided by the addition of exogenous H2O2.

To measure the effect of heat shock, cells were grown at 28 °C to stationary phase and were exposed to 50 °C. The phlE mutant was more sensitive to this heat shock than the wild-type F113 (Fig. 3); after 4 min at 50 °C, the number of viable cells was 20-fold higher in the wild-type compared to the phlE mutant. There was, however, no difference in behaviour between the wild-type and complemented mutant F113-D3/pCUD4.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. Effect of mutation of phlE on tolerance to heat shock. Stationary-phase cultures of F113 (squares), F113-D3 (phlE) (triangles) and F113-D3/pCUD4 (circles) grown in LB were washed, diluted in LB and transferred to pre-warmed tubes at 50 °C. Cells were heat-shocked at 50 °C for indicated time and the number of surviving cells counted on LB plates. Values presented are the means±SD of triplicate independent measurements.

 
To test whether PhlE has a role in protecting F113 against oxidative stress caused by H2O2, cells were grown to stationary phase in LB, seeded on LB plates at 106 cells per plate and a disk of Whatman 3MM filter paper impregnated with 20 µl 30 % (v/v) H2O2 was deposited on the plate. The diameter of the zone of inhibition, which indicates the relative sensitivity to H2O2, was 5·4±0·2 cm for F113, 6·8±0·1 cm for F113-D3 (phlE) and 4·3±0·1 cm for F113-D3/pCUD4 (n=3). H2O2 was thus more effective against F113-D3 than the wild-type, whereas overexpression of phlE, in F113-D3/pCUD4, increased the level of tolerance.

To analyse the effect of osmotic shock, F113, F113-D3 (phlE) and F113-D3/pCUD4 strains were grown in SA or LB medium and then subjected to osmotic shock by the addition of NaCl (to 0·75 M) or sucrose (to 2 M) to the medium. The effect of 0·75 M NaCl on the viability of these strains is shown in Fig. 4. The phlE mutant F113-D3 was considerably more sensitive to this osmotic stress than the wild-type strain F113, whereas strain F113-D3/pCUD4 has an increased tolerance compared to wild-type. Similar results were seen when 2 M sucrose was used to induce stress (data not shown). When bacteria were grown in LB medium rather than SA, the impact of these osmotic stresses was less pronounced, but the same differential behaviour of the strains was observed (data not shown). Overall the differential responses of the wild-type and the phlE mutant to heat, oxidative and osmotic stress indicate a role for PhlE in general stress tolerance in P. fluorescens F113. The restoration of stress tolerance of F113-D3 to wild-type or higher levels by the introduction of phlE also indicates that the increased sensitivity of the mutant to stress is solely due the phlE mutation.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Effect of mutation of phlE on tolerance to osmotic stress. To assay survival of exposure to high concentrations of NaCl, F113 (circles), F113-D3 (squares) and F113-D3/pCUD4 (triangles) cells were grown to stationary phase in SA, washed, diluted and transferred to tubes containing SA supplemented with 0·75 M NaCl. Values given are the means of three independent measurements, which differed from the mean by less than 10 %.

 
Possible relationship between response to stress and PHL metabolism
The observed role of phlE in both tolerance to exogenous PHL and in general stress tolerance prompted us to ask whether there is any relationship between these two phenomena. A decreased capacity to export PHL from the cell could account for the increased sensitivity of F113-D3 to exogenous PHL. An attractive hypothesis is that the effect of stress is to increase PHL production and that the increased sensitivity of the phlE mutant to stress is due to the enhanced accumulation of PHL within the mutant cells. We tested this hypothesis by analysis of the effects of osmotic stress (0·75 M NaCl) on PHL levels in the wild-type. Application of osmotic stress led to a rapid disappearance of PHL from the medium together with a concomitant appearance of MAPG (Fig. 5). As well as acting as the precursor of PHL synthesis, MAPG is also known to be a degradation product of PHL (Schnider-Keel et al., 2000; A. Abbas, unpublished results). Control experiments established that loss of PHL was not due to precipitation from the medium and required the presence of the bacteria. These findings, which indicate a hitherto unsuspected relationship between stress and PHL degradation, lead us to propose that one role of phlE in general stress tolerance is to export toxic intermediates of PHL degradation from the cells. PHL production and stress tolerance are functions that must contribute to the biocontrol ability of P. fluorescens F113, so that further work on the mechanism of PHL and MAPG transport and the role of PhlE in regulation of PHL synthesis and stress tolerance is clearly warranted.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5. Effect of osmotic shock on the kinetics of PHL and MAPG production. F113 cells were grown for 17 h on SA, diluted and transferred to tubes containing SA supplemented with 0·75 M NaCl. The control tubes were not supplemented with NaCl. Samples from control (solid lines) and shocked (dashed lines) cultures were taken at the indicated times and the free PHL (filled symbols) and MAPG (open symbols) content quantified by HPLC. The concentration of PHL is expressed in nmol PHL per OD600 unit. Values given are the means of three independent measurements, which differed from the mean by less than 10 %.

 


   ACKNOWLEDGEMENTS
 
The authors thank Dr Paul O'Toole for valuable discussions, Dr Gerard O'Donoghoue for critical reading of the manuscript, and Liam Burgess and Pat Higgins for advice and technical assistance. This work was supported in part by grants awarded by the Higher Education Authority of Ireland (PRTL 2/3 Programmes), Enterprise Ireland (SC/02/520), HRB (RP76/2001) and the European Commission (QLK5-CT-2002-0091; QLK3-CT-2001-0010; QLK3-CT-2000-31759).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Aarons, S., Abbas, A., Adams, C., Fenton, A. & O'Gara, F. (2000). A regulatory RNA (PrrB RNA) modulates expression of secondary metabolite genes in Pseudomonas fluorescens F113. J Bacteriol 182, 3913–3919.[Abstract/Free Full Text]

Abbas, A., Morrissey, J. P., Carnicero-Marquez, P., Sheehan, M. M., Delany, I. R. & O'Gara, F. (2002). Characterization of interactions between the transcriptional repressor PhlF and its binding site at the phlA promoter in Pseudomonas fluorescens F113. J Bacteriol 184, 3008–3016.[Abstract/Free Full Text]

Ahmed, M., Lyass, L., Markham, P. N., Taylor, S. S., Vásquez-Laslop, N. & Neyfakh, A. A. (1995). Two highly similar multidrug transporters of Bacillus subtilis whose expression is differentially regulated. J Bacteriol 177, 3904–3910.[Abstract]

Bangera, M. G. & Thomashow, L. S. (1996). Characterization of a genomic locus required for synthesis of the antibiotic 2,4-diacetylphloroglucinol by the biological control agent Pseudomonas fluorescens Q2-87. Mol Plant–Microbe Interact 9, 83–90.[Medline]

Bangera, M. G. & Thomashow, L. S. (1999). Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudomonas fluorescens Q2-87. J Bacteriol 181, 3155–3163.[Abstract/Free Full Text]

Barios, H., Valderrama, B. & Morett, E. (1999). Compilation and analysis of {sigma}54-dependent promoter sequences. Nucleic Acids Res 27, 4305–4313.[Abstract/Free Full Text]

Bentley, J., Hyatt, L. S., Ainley, K., Parish, J. H., Herbert, R. B. & White, G. R. (1993). Cloning and sequence analysis of an Escherichia coli gene conferring bicyclomycin resistance. Gene 127, 117–120.[CrossRef][Medline]

Boruah, H. P. & Kumar, B. S. (2002). Biological activity of secondary metabolites produced by a strain of Pseudomonas fluorescens. Folia Microbiol 47, 359–363.

Bowden, K., Broadbent, J. L. & Ross, W. J. (1965). Some simple antihelminthics. Br J Pharmacol 24, 714–724.[Medline]

Brautaset, T., Sekurova, O. N., Sletta, H., Ellingsen, T. E., Strøm, A. R., Valla, S. & Zotchev, S. B. (2000). Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455, analysis of the gene cluster and deduction of the biosynthetic pathway. Chem Biol 7, 395–403.[CrossRef][Medline]

Chou, J. H., Greenberg, J. T. & Demple, B. (1993). Posttranscriptional repression of Escherichia coli OmpF protein in response to redox stress, positive control of the micF antisense RNA by the soxRS locus. J Bacteriol 175, 1026–1031.[Abstract]

Corbell, N. & Loper, J. E. (1995). A global regulator of secondary metabolite production in Pseudomonas fluorescens Pf-5. J Bacteriol 177, 6230–6236.[Abstract]

Delany, I. (1999). Genetic analysis of the production of the antifungal metabolite 2,4-diacetylphloroglucinol by the biocontrol strain Pseudomonas fluorescens F113. PhD thesis, University College Cork.

Delany, I., Sheehan, M. M., Fenton, A., Bardin, S., Aarons, S. & O'Gara, F. (2000). Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of phlF as a transcriptional repressor. Microbiology 146, 537–543.[Abstract/Free Full Text]

Duffy, B. K. & Defago, G. (1999). Environmental factors modulating antibiotic and siderophore biosynthesis by Pseudomonas fluorescens biocontrol strains. Appl Environ Microbiol 65, 2429–2438.[Abstract/Free Full Text]

Farinha, M. A. & Kropinski, A. M. (1990). High efficiency electroporation of Pseudomonas aeruginosa using frozen cell suspensions. FEMS Microbiol Lett 58, 221–225.[Medline]

Fernandez-Moreno, M. A., Caballero, J. L., Hopwood, D. A. & Malpartida, F. (1991). The act cluster contains regulatory and antibiotic export genes, direct target for translational control by the bldA tRNA gene of Streptomyces. Cell 66, 769–780.[Medline]

Figurski, D. H. & Helinski, D. R. (1979). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 1648–1652.[Abstract]

George, A. M. & Levy, S. B. (1983). Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid determined efflux of tetracycline. J Bacteriol 155, 351–362.[Medline]

Guillfoile, P. G. & Hutchinson, C. R. (1992). Sequence and transcriptional analysis of the Streptomyces glaucescens tcmAR tetracenomycin C resistance and repressor gene loci. J Bacteriol 174, 3651–3658.[Abstract]

Harrison, L. A., Letendre, L., Kovacevich, P., Pierson, E. & Weller, D. M. (1993). Purification of an antibiotic effective against Gaeumannomyces graminis var. tritici produced by biocontrol agent, Pseudomonas aureofaciens. Soil Biol Biochem 25, 215–221.[CrossRef]

Kawamura-Sato, K., Shibayama, K., Horii, T., Iimuma, Y., Arakawa, Y. & Ohta, M. (1999). Role of multiple efflux pumps in Escherichia coli in indole expulsion. FEMS Microbiol Lett 179, 345–352.[CrossRef][Medline]

Keel, C., Schnider, U., Maurhofer, M., Viossard, C., Laville, J., Burger, U., Wirthner, P., Haas, D. & Defago, G. (1992). Suppression of root diseases by Pseudomonas fluorescens CHA0: importance of the bacterial secondary metabolite 2,4-diacetylphloroglucinol. Mol Plant–Microbe Interact 5, 4–13.

Kovach, M. E., Phillips, R. W., Elzer, P. H., Roop, R. M. & Peterson, K. M. (1994). pBBR1MCS: a broad-host-range cloning vector. BioTechniques 16, 800–802.[Medline]

Levy, E., Gough, F. J., Berlin, D. K., Guiana, P. W. & Smith, J. T. (1992). Inhibition of Septoria tritici and other phytopathogenic fungi and bacteria by Pseudomonas fluorescens and its antibiotics. Plant Pathol 41, 335–341.

Ma, D., Cook, D. N., Alberti, M., Pon, N. G., Nikaido, H. & Hearst, J. H. (1995). Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol Microbiol 16, 45–55.[Medline]

Marger, M. D. & Saier, M. H., Jr (1993). A major facilitator superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci 18, 13–20.[CrossRef][Medline]

Mathews, D. H., Sabina, J., Zuker, M. & Turner, D. H. (1999). Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol 288, 911–940.[CrossRef][Medline]

Miyazaki, M., Kohno, K., Uchiumi, T., Tanimura, H., Matsuo, K., Nasu, M. & Kuwano, M. (1992). Activation of human multidrug resistance-1 gene promoter in response to heat shock stress. Biochem Biophys Res Commun 187, 677–684.[Medline]

Molnar, I., Schupp, T., Ono, M. & 13 other authors (2000). The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum Soce90. Chem Biol 7, 97–109.[CrossRef][Medline]

Naroditskaya, V., Schlosser, M. J., Fang, N. Y. & Lewis, K. (1993). An E. coli gene emrD is involved in adaptation to low energy shock. Biochem Biophys Res Commun 196, 803–809.[CrossRef][Medline]

Neyfakh, A. A., Bidnenko, V. E. & Chen, L. B. (1991). Efflux-mediated multidrug resistance in Bacillus subtilis: similarities and dissimilarities with the mammalian system. Proc Natl Acad Sci U S A 88, 4781–4785.[Abstract]

Nowak-Thompson, B., Gould, S. J., Kraus, J. & Loper, J. E. (1994). Production of 2,4-diacetylphloroglucinol by the biocontrol agent Pseudomonas fluorescens Pf-5. Can J Microbiol 40, 1064–1066.

Paulsen, I. T. & Sukurray, R. A. (1993). Topology, structure and evolution of two families of proteins involved in antibiotic and antiseptic resistance in eukaryotes and prokaryotes – an analysis. Gene 124, 1–11.[CrossRef][Medline]

Pridmore, R. D. (1987). New and versatile cloning vectors with kanamycin resistance marker. Gene 56, 309–312.[CrossRef][Medline]

Putman, M., van Veen, H. W. & Konings, W. N. (2000). Molecular properties of bacterial multidrug transporters. Microbiol Mol Biol Rev 64, 672–693.[Abstract/Free Full Text]

Putman, M., van Veen, H. W., Degener, J. E. & Konings, W. N. (2001). The lactococcal secondary multidrug transporter LmrP confers resistance to lincosamides, macrolides, streptogramins and tetracyclines. Microbiology 147, 2873–2880.[Abstract/Free Full Text]

Reddi, T. K. & Borovkov, A. V. (1970). Antibiotic properties of 2,4-diacetylphloroglucinol (2,4-diacetyl-1,3,5-trihydroxybenzene) produced by Pseudomonas fluorescens strain 26-o. Antibiotiki 15, 19–21 (in Russian).[Medline]

Reddi, T. K., Khudiakov, Y. P. & Borovkov, A. V. (1969). Pseudomonas fluorescens strain 26-o, producing phytotoxic substances. Mikrobiologiya 38, 909–913 (in Russian).

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Scher, F. M. & Baker, R. (1982). Effect of Pseudomonas putida and a synthetic iron chelator on induction of soil supressiveness to Fusarium wilt pathogens. Phytopathology 72, 1567–1573.

Schnider-Keel, U., Seematter, A., Maurhofer, M. & 8 other authors (2000). Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin. J Bacteriol 182, 1215–1225.[Abstract/Free Full Text]

Schoonbeek, H. J., Raaijmakers, J. M. & De Waard, M. A. (2002). Fungal ABC transporters and microbial interactions in natural environments. Mol Plant–Microb Interact 15, 1165–1172.[Medline]

Shanahan, P., O'Sullivan, D., Simpson, P., Glennon, J. & O'Gara, F. (1992). Isolation of 2,4-diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production. Appl Environ Microbiol 58, 353–358.[Abstract]

Sharifi-Tehrani, A., Zala, M., Natsch, A., Moenne-Loccoz, Y. & Defago, G. (1998). Biocontrol of soil-borne fungal plant diseases by 2,4-diacetyphloroglucinol-producing fluorescent pseudomonads with different restriction profiles of amplified 16S rDNA. Eur J Plant Pathol 104, 631–643.[CrossRef]

Tomas-Lorente, F., Iniesta-Sanmartin, E., Tomas-Barberan, F. A., Trowitzsch-Kienast, W. & Wray, V. (1989). Antifungal phloroglucinol derivative and lipophilic flavenoids from Helichrysum decumbens. Phytochemistry 28, 1613–1615.[CrossRef]

Weller, D. M. & Cook, R. J. (1983). Suppression of take-all of wheat by seed treatment with fluorescent pseudomonads. Phytopathology 73, 463–469.

Yoshida, H., Bogaki, M., Nakamura, S., Ubukata, K. & Konno, M. (1990). Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones. J Bacteriol 172, 6942–6949.[Medline]

Zuker, M., Mathews, D. H. & Turner, D. H. (1999). Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. In RNA Biochemistry and Biotechnology (NATO ASI Series), pp. 11–43. Edited by J. Barciszewski & B. F. C. Clark. Dordrecht: Kluwer.

Received 15 January 2004; accepted 7 April 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Abbas, A.
Articles by O'Gara, F.
Articles citing this Article
PubMed
PubMed Citation
Articles by Abbas, A.
Articles by O'Gara, F.
Agricola
Articles by Abbas, A.
Articles by O'Gara, F.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2004 Society for General Microbiology.