1 Flanders Interuniversity Institute of Biotechnology (VIB6), Laboratory of Microbial Interactions, Vrije Universiteit Brussel, Building E, room 6·6, Pleinlaan 2, B-1050 Brussels, Belgium
2 Institute for Biomedical Research, Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom
3 Epidemiology and Bio-statistics Division, Department of Well-being, Queen Astrid Military Hospital, B-1120 Brussels, Belgium
4 Department of Microbiology, University of Colorado Health Sciences Center, Denver, CO 80262, USA
Correspondence
Pierre Cornelis
pcornel{at}vub.ac.be
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ABSTRACT |
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INTRODUCTION |
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Gram-negative bacteria have therefore developed numerous strategies for acquiring iron. A common mechanism is the production of low-molecular-mass iron-chelating compounds named siderophores (Guerinot, 1994), which are secreted to scavenge iron outside the cell. Besides siderophores, cell surface receptors are produced, which function as gated porin channels that recognize and internalize the ferri-siderophore complexes in concert with the TonB protein that energizes the receptor protein (Ratledge & Dover, 2000
).
These proteins are characterized by a large C-terminal domain of 22 antiparallel -strands, which form a so-called
-barrel that spans the outer membrane (Koebnik et al., 2000
). Unlike outer-membrane porins, TonB-dependent outer-membrane proteins also contain an additional domain known as cork or plug that transiently blocks the
-barrel domain and by using energy transduced by TonB, allowing selective uptake of cognate siderophore/ion complexes (Ferguson et al., 1998
).
Fluorescent pseudomonads form a group of Gram-negative bacteria which respond to iron-deficiency by secreting yellow-green siderophores termed pyoverdines or pseudobactins. All characterized pyoverdines and pseudobactins comprise a conserved dihydroxyquinoline chromophore linked to an acyl group and a short (612 amino acids) type-specific peptide chain (Ravel & Cornelis, 2003). Pyoverdines of a single strain have the same peptide but may differ in the nature of the acyl group. Typing methods exist to classify fluorescent pseudomonads according to the pyoverdine they produce, so-called siderotyping (Meyer et al., 2002a
).
The type species of the group, Pseudomonas aeruginosa, is an opportunistic human pathogen associated with infections of compromised individuals and a notorious hospital pathogen. Experiments in animal models have shown the importance of pyoverdine for P. aeruginosa virulence (Meyer et al., 1996; Takase et al., 2000
). The contribution of pyoverdine to P. aeruginosa virulence is not restricted to its siderophore activity. Recent work has shown that pyoverdine can be considered a signal molecule which orchestrates a synergic action between itself and other virulence determinants [e.g. exotoxin A (ToxA); endoprotease (PrpL)] to retrieve iron from host cells and proteins (Lamont et al., 2002
; Shen et al., 2002
). Different branches of this pyoverdine signalling pathway thereby regulate expression of virulence determinants and pyoverdine synthesis on the one hand and pyoverdine uptake via its receptor on the other hand (Beare et al., 2003
).
Three siderotypes of P. aeruginosa can be distinguished, producing three structurally different types of pyoverdine (types I, II, III) (Cornelis et al., 1989; Meyer et al., 1997
; De Vos et al., 2001
; Spencer et al., 2003
; Ernst et al., 2003
), each being recognized at the level of the outer membrane by a specific receptor (Cornelis et al., 1989
; De Chial et al., 2003
).
Even before its complete genome sequence became available, P. aeruginosa PAO1 (Stover et al., 2000), which produces type I pyoverdine, was already the most intensively studied P. aeruginosa isolate. Genetics and physiology of pyoverdine biosynthesis and uptake are therefore also the best characterized for the P. aeruginosa type I pyoverdine of P. aeruginosa PAO1 (Ochsner et al., 2002
; Ravel & Cornelis, 2003
; Lamont & Martin, 2003
; Palma et al., 2003
; Heim et al., 2003
). The FpvA receptor for P. aeruginosa PAO1 pyoverdine has been intensively characterized using physiological, immunological, and molecular approaches (Poole et al., 1993
; Schalk et al., 2001
, 2002
).
Recently, we have cloned the receptors for P. aeruginosa type II and type III pyoverdines and developed a multiplex PCR method for fast siderotyping of P. aeruginosa strains based on their specific fpvA sequences (De Chial et al., 2003). In this work we present evidence for an additional type I pyoverdine receptor in P. aeruginosa PAO1 and in other strains producing type II and type III pyoverdines.
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METHODS |
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Physiological characterization of the mutants.
Growth stimulation by the different pyoverdines on CAA plus EDDHA was done on agar plates by spreading 200 µl of a saturated P. aeruginosa CAA culture of the mutant on the agar surface on top of which filter discs impregnated with a 10 µM solution of the pyoverdines were deposited. Growth stimulation was recorded after 1 day, and the plates were photographed using a Fuji Digital Camera (Finepix S1 Pro). For more accurate analysis, growth was assessed in microtitre plates (300 µl CAA medium with EDDHA as described in the text), which were incubated for 48 h at 37 °C in a Bio-Screen incubator (Life Technologies), using the following parameters: shaking for 30 s per 3 min and readings recorded every 10 min (De Vos et al., 2001).
Analysis of outer-membrane proteins.
Outer-membrane proteins from bacteria grown under iron-limiting conditions (CAA) were prepared as described by Mizuno & Kageyama (1978). The protein content of the outer-membrane preparations was determined by the Lowry method, and analysed by SDS-PAGE (10 % polyacrylamide).
Generation of fpvA
fpvB double mutants in P. aeruginosa PAO1 by allelic exchange.
The P. aeruginosa PAO1 2429 bp genome fragment from position 4 663 835 to 4 666 264, which includes the PA4168 ORF with its putative ribosome-binding sites, was PCR-amplified with primers PA4168 F and PA4168 R, and cloned into pCR2.1 using the TA cloning kit (Invitrogen). The cloned insert was restricted with EcoRV and BamHI and ligated to EcoRV/BamHI-restricted pBR325, thereby replacing part of the tetracycline (Tc) resistance gene. Clones were selected by resistance for chloramphenicol and lost resistance to Tc. Restriction analysis confirmed insertion of the PA4168 ORF in pBR325. A SacISacI Tc cassette from the pTnModoTc plasposon (Dennis & Zylstra, 1998) was then inserted in the SacI site, 1·5 kb from the start of the ORF. Tc-resistant clones were picked up and additional screening was done by restriction analysis. The selected clones were used to transform Escherichia coli GJ23 cell before mobilization of the disrupted PA4168 ORF by conjugation into P. aeruginosa fpvA pvdDpchEF. Recombinants were selected by their resistance to both Tc (100 µg ml1) and gentamicin (Gm) (100 µg ml1). The fpvA II and fpvA III mutants were constructed as described by De Chial et al. (2003)
. The fpvA mutant in P. aeruginosa PAO1 was obtained as described by Lamont et al. (2002)
using the pEX18Gm vector (Hoang et al., 1998
). All mutations were confirmed by PCR amplification using the appropriate primers (Table 2
: PA4168 GR Fw and PA4168 GR Rv, FpvAII-2F and FpvAII-2R, FpvAIII-3F and FpvAIII-3R), except for the fpvA mutation in P. aeruginosa PAO1, which was confirmed by Southern blotting.
Generation of a Tn5-pyoverdine biosynthetic mutant in 59.20, a type III pyoverdine-producing P. aeruginosa strain.
Mutagenesis was done by biparental mating of 59.20 with the donor strain E. coli SM10( pir) containing the suicide delivery system pUT (de Lorenzo et al., 1990
) and the transposon miniTnphoA3, as described by Pattery et al. (1999)
and De Chial et al. (2003)
. Transconjugants were selected on CAA medium plates supplemented with appropriate antibiotics (100 mg Gm l1; 10 mg Tc l1). Candidate pyoverdine biosynthetic mutants were first selected on CAA for their lack of fluorescence, then for absence of growth in the presence of 0·5 mg EDDHA ml1 and finally on CAA-EDDHA-pyoverdine to assure that the mutant was not pyoverdine-uptake deficient. The localization of the mutation was done by inverse PCR (IPCR) using primers PhoA, GM1, PhoA4 and GM2 as described previously (De Chial et al., 2003
). The sequence of the DNA flanking the transposon revealed that the insertion occurred in a gene with high similarity to the pyoverdine synthetase gene pvdI from P. aeruginosa PAO1 (Lehoux et al., 2000
; results not shown).
Generation of mutants in fpvB homologues of type II and type III pyoverdine-producing P. aeruginosa strains by allelic exchange.
The P. aeruginosa PAO1 disrupted fpvB gene was also mobilized into a type II (7NSK2) and type III (59.20) pyoverdine-producing P. aeruginosa by conjugation to inactivate fpvB homologues in these strains.
RT-PCR detection of fpvA and fpvB transcripts.
Total RNA was isolated from P. aeruginosa PAO1 and P. aeruginosa PAO1pvdD grown overnight in CAA medium (iron-limited growth condititon) and CAA medium with 100 µM FeCl3 (high-iron growth condition) with the high pure RNA purification kit (Roche). To avoid DNA contamination, the kit protocol was slightly adapted. The purified RNA sample was mixed with binding buffer and again applied on the column so that DNase treatment could be repeated and the DNase easily removed in the following steps according to the manufacturer's instructions.
Complementary DNA, using as template these total RNAs, was synthesized with the first strand cDNA synthesis kit (Amersham Biosciences) following the manufacturer's instructions. The cDNAs served as template for a PCR with primers FpvAI RT F and FpvA I-1R (fpvA expression detection), PA4168 GR Fw and PA4168 GR Rv (fpvB expression detection) and PAL 1 and PAL 2 (oprL expression detection as housekeeping gene control; Lim et al., 1997).
Microarray analysis.
The expression of fpvA and fpvB were examined from microarray experiments as previously described (Ochsner et al., 2002). The PAO1 fur mutant contains a point mutation in Fur (A10G), which abrogates its ability to bind Fur boxes (Barton et al., 1996
). Strains were grown in dialysed trypticase soy broth (DTSB) or M9 minimal media as described by Ochsner et al. (2002)
.
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RESULTS |
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Conservation of fpvB in different P. aeruginosa strains
Primers PA4168 SC Fw and PA4168 SC Rv were developed in order to amplify a 562 bp fragment of PA4168. An amplification product was obtained in 20 out of 27 independent clinical and environmental P. aeruginosa isolates, including seven type II strains, eight type III strains and three type I strains as typed by amplified fragment-length polymorphism (AFLP) (Pirnay et al., 2002; results not shown), multiplex PCR for the presence of the fpvA gene (type I, II or III; De Chial et al., 2003
) and/or IEF (Pirnay et al., 2002
).
Ability of type II and type III producing P. aeruginosa strains to use heterologous type I ferripyoverdine
The fpvA genes of type III pyoverdine-producing P. aeruginosa clinical isolates 59.20 (positive for the amplification of fpvB), A15 (negative for the amplification of fpvB) and the type II pyoverdine-producing plant rhizosphere isolate 7NSK2 (also positive for the fpvB gene; Höfte et al., 1990) were inactivated by allelic exchange using a Gm cassette. With their fpvA genes inactivated, these strains were unable to use their cognate pyoverdines as a source of iron (Fig. 3
a, b). However, on EDDHA-containing CAA medium, growth of 59.20 fpvA and 7NSK2 fpvA was stimulated by the heterologous type I pyoverdine (Fig. 3a
) while the growth of strain A15 fpvA was not (Fig. 3b
). This result suggests that the homologues of FpvB in other P. aeruginosa strains could serve as functional receptors for type I pyoverdine, allowing these strains to utilize this heterologous siderophore.
|
The fpvB gene of P. aeruginosa PAO1 was cloned in the plasmid vector pBBR 1 MCS. Conjugational transfer of the construct into A15fpvA conferred the capacity for this mutant to grow in the presence of EDDHA when type I pyoverdine was provided (Fig. 3b
). Therefore, we can conclude that the provision of fpvB in trans confers the capacity to utilize type I pyoverdine. Interestingly, expression of fpvB also conferred some capacity to utilize type III pyoverdine (Fig. 3b
).
Demonstration of the functionality of fpvB homologues in type II and type III pyoverdine-producing P. aeruginosa strains 59.20 and 7NSK2
7NSK2 and 59.20 fpvA mutants are both capable of using the heterologous type I pyoverdine as source of iron. Homologues of fpvB were detected in these strains by PCR and were inactivated using the same construct that was used to inactivate fpvB in PAO1. Double fpvA fpvB mutants of both 7NSK2 and 59.20 completely lost the ability of utilizing type I pyoverdine (Fig. 3a). A mutant of 59.20 was obtained that carries a transposon insertion in a homologue of the PAO1 pvdI gene (Lehoux et al., 2000
) and is therefore unable to produce pyoverdine and to grow in the presence of EDDHA. However, growth can be restored by each of the three types of P. aeruginosa pyoverdines. Inactivation of the fpvB homologue in this mutant abolished growth stimulation by type I pyoverdine, but not by type II or type III (Fig. 3
). When fpvA is inactivated in 59.20, utilization of type II and III pyoverdines, but not of type I, is abolished. Likewise, inactivation of fpvB in 7NSK2 totally compromises the utilization of type I pyoverdine in this mutant, but does not affect the use of the cognate pyoverdine.
Regulation of fpvB expression
RT-PCR experiments show that iron limitation induces the transcription of fpvA and fpvB in CAA medium (Fig. 4).
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DISCUSSION |
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In recent articles on the response of P. aeruginosa to iron limitation, Heim et al. (2003) demonstrated the production of the protein encoded by PA4168 using a proteomic analysis while Palma et al. (2003)
, using a microarray approach, described the early induction of PA4168 by iron limitation. In this study, we detected close homologues of fpvB in several clinical and environmental isolates of P. aeruginosa, including type II and type III pyoverdine-producing strains. It was already previously reported that certain type II and type III pyoverdine-producing strains are able to utilize type I pyoverdine (De Vos et al., 2001
; Pirnay et al., 2002
). Here we demonstrate that this capacity to utilize type I ferripyoverdine as the sole source of iron is conferred by FpvB. Previous reports on heterologous pyoverdine uptake in fluorescent pseudomonads are often explained by structural identity with the homologous pyoverdine leading to promiscuous uptake by the cognate pyoverdine receptor (Meyer et al., 2002b
). Our results also clearly suggest that FpvA III is such a promiscuous receptor since it confers to the bacterium the ability to grow in the presence of type II and type III pyoverdines (Fig. 3
). FpvB also confers some ability to utilize type III pyoverdine (Fig. 3b
). Conversely, the type II pyoverdine receptor seems to be more specific for type II pyoverdine.
The discovery of FpvB, a previously undescribed pyoverdine receptor, has implications for P. aeruginosa biology that are not necessarily restricted to pyoverdine uptake alone. A second pyoverdine receptor in P. aeruginosa could also be involved in entry of pyocins as demonstrated for the type II pyoverdine receptor and pyocin S3 (Baysse et al., 1999). In the case of pyocin Sa there are indications (Smith et al., 1992
) that an fpvA mutant shows a reduced sensitivity, but not a complete resistance to the killing by Sa pyocin, suggesting that this bacteriocin could use a second type I pyoverdine receptor (perhaps FpvB) as gate of entry into the cell.
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ACKNOWLEDGEMENTS |
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Received 16 January 2004;
revised 19 March 2004;
accepted 25 March 2004.
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