BIOMERIT Research Centre, Microbiology Department, National University of Ireland, Cork, Ireland
Correspondence
Fergal O'Gara
f.ogara{at}ucc.ie
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ABSTRACT |
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The GenBank accession number for the sequence reported in this paper is AJ542662.
Present address: Cork Institute of Technology, Cork, Ireland.
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INTRODUCTION |
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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 KyteDoolittle 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.
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METHODS |
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Construction and complementation of phlE mutant.
A 0·7 kb BamHISphI 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. ml1. 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. ml1. 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 ml1. 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.
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RESULTS AND DISCUSSION |
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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 -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
-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
-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).
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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.
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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.
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ACKNOWLEDGEMENTS |
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Received 15 January 2004;
accepted 7 April 2004.
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