Department of Microbiology, University of Colorado Health Sciences Center, 4200 E Ninth Avenue, Campus Box B175, Denver, CO 80262, USA1
Author for correspondence: Urs Ochsner. Tel: +1 303 315 5093. Fax: +1 303 315 6785. e-mail: urs.ochsner{at}UCHSC.edu
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ABSTRACT |
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Keywords: Pseudomonas, iron transport, haem receptor, ferric uptake regulator
Abbreviations: ABC, ATP-binding cassette; EDDHA, ethylenediamine di(o-hydroxyphenylacetic acid)
The GenBank accession numbers for the sequences reported in this paper are AF055999, AF127222, and AF127223.
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INTRODUCTION |
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These known haem-acquisition systems can be separated into three categories, based on the factors they involve. The largest category, into which the P. aeruginosa phu system falls, comprises uptake systems similar to those for the uptake of ferrisiderophores, colicins and vitamin B12 (Braun & Hantke, 1991 ). They involve a specific TonB-dependent receptor at the cell surface and a periplasmic binding-protein-dependent transport machinery (PBT) required for the passage through the cytoplasmic membrane. The PBT system belongs to the larger family of so-called ABC transporters and typically consists of a periplasmic substrate-binding protein, one or two hydrophobic integral membrane-spanning proteins, and one or two hydrophilic proteins with ATPase activity (Ames et al., 1990
; Higgins, 1990
; Linton & Higgins, 1998
). The best studied haem-uptake systems in this category include the hemRhemSTUV system of Y. enterocolitica (Stojiljkovic & Hantke, 1992
) and the hmuRSTUV system of Y. pestis (Hornung et al., 1996
).
The haem-uptake systems in the second category consist of an outer-membrane receptor, an extracellular haem-binding protein and a type I secretion apparatus, referred to as an ABC export system. The ABC protein-mediated exporters are an inner-membrane ATPase, an inner-membrane fusion protein, and an outer-membrane component (Pugsley, 1993 ; Linton & Higgins, 1998
). Such a haem-acquisition system has been demonstrated for Ser. marcescens (Binet & Wandersman, 1996
; Ghigo et al., 1997
) and was also postulated for P. aeruginosa after the isolation and characterization of an extracellular haem-binding protein, HasA, from this organism (Létoffé et al., 1998
). HasA is an iron-regulated protein required for the utilization of haemoglobin in P. aeruginosa. In this paper, we provide further evidence for such a system in P. aeruginosa through the genetic and biochemical characterization of the corresponding outer-membrane receptor, HasR.
The third category of haem-uptake systems involves a haem-binding outer-membrane lipoprotein. The best studied species containing such a lipoprotein are H. influenzae type b (Hanson et al., 1992 ) and H. influenzae Rd (Reidl & Mekalanos, 1996
).
Haem from intracellular host haemoglobin becomes available as an iron source after lysis of erythrocytes, and it is thus not surprising that several haem-utilizing pathogens have been shown to produce efficient haemolysins. Well-studied examples include V. cholerae El-Tor, which produces a cytotoxic haemolysin (HlyA) under iron deprivation (Stoebner & Payne, 1988 ; Menzl et al., 1996
). Similarly, iron starvation triggers the secretion of ShlA haemolysin in Ser. marcescens (Poole & Braun, 1988
). Besides lysing erythrocytes, purified ShlA haemolysin was also shown to cause lysis of human epithelial cells at nanomolar concentrations (Hertle et al., 1999
). P. aeruginosa secretes several factors that have the potential to facilitate the acquisition of haem from exogenous sources. A heat-labile haemolysin, phospholipase C, was purified from P. aeruginosa culture supernatants (Berka & Vasil, 1982
) and the corresponding gene, plcH, was subsequently cloned (Pritchard & Vasil, 1986
). In addition, P. aeruginosa secretes a heat-stable glycolipid haemolysin composed of rhamnose and ß-hydroxydecanoate; the genes required for the production of these rhamnolipids are regulated by quorum sensing and iron starvation (Ochsner et al., 1994
; Ochsner & Reiser, 1995
). Furthermore, iron depletion activates the production of exotoxin A, which has a broad cytotoxic activity toward eukaryotic cells (Vasil et al., 1977
). Lysis of host cells makes wide sources of haem and free iron accessible for P. aeruginosa, especially in concert with extracellular proteases that degrade the host haem-containing proteins and siderophores that immediately scavenge free iron (Wolz et al., 1994
).
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METHODS |
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RNase protection analyses were performed using the Riboprobe system (Promega). Radiolabelled riboprobes from suitable cloned DNA fragments were generated by run-off transcription from the T7 promoter of linearized plasmids using T7 RNA polymerase and [-32P]CTP as described in detail elsewhere (Barton et al., 1996
). A 699 bp EcoRVHincII fragment containing the phuRphuS divergent promoters with the first 195 bp of the phuR coding region and the first 314 bp of the phuS coding region was cloned in either orientation into the EcoRV site of pBluescript SK+, resulting in plasmids pPHU699-R and pPHU699-S, which served to generate riboprobes antisense to phuR and to phuS, respectively. Plasmid pPHU-R1 contained a 346 bp PCR fragment obtained with primers 5'-GATCGAAGGCATGGCCCG-3' and 5'-GACAAGCCGGTTCTCATTC-3' and was used to generate a riboprobe to detect T1 and T2 transcripts. pPHU359- and pPHU359+ were constructed by cloning an internal XhoIStuI phuV fragment into pBluescript SK (XhoIEcoRV) and into pBluescript KS (XhoIEcoRV), and served to generate riboprobes for the detection of sense and antisense phuV transcripts.
Isolation of the phu and has loci.
The repeats R1 and R2 that were originally isolated in a cycle-selection procedure for Fur targets (Ochsner & Vasil, 1996 ) were used as probes to obtain overlapping larger DNA clones by Southern blot analysis followed by colony hybridization. First, a 0·85 kb DdeI fragment containing the 3' end of repeat R2 was cloned and used as a probe to isolate a 1·3 kb StuI fragment containing the phuW gene, which was cloned behind the lac promoter in pUCP19, resulting in pPHU3. Using phuW as a probe, a 5·5 kb SphI fragment harbouring the phuV, phuU, phuT and phuS genes, and the 5' portion of phuR was isolated and cloned into pUCP19, yielding pPHU2. Subsequently, a 6 kb EcoRV fragment containing the entire phuR gene was cloned into pUCP19, yielding pPHU1. The DNA sequence of a total 7·7 kb portion of the phu locus was determined. The DNA sequence of the hasR gene was pulled out from the unfinished P. aeruginosa genome project (Pathogenesis Corporation) using a TBLASTN search with the Ser. marcescens HasR amino acid sequence. The P. aeruginosa hasR homologue was subsequently isolated by PCR using the primers 5'-CCTCGTTGCAGTCGATCAG-3' and 5'-aagcttGCTCATGCCAAAACTCCAA-3' (lower-case letters indicate a non-complementary HindIII site). The 3·05 kb PCR product was cloned into pCRII-2.1, partially sequenced and directionally cloned as an EcoRIHindIII fragment into pUCP19, yielding pHAS3051. Plasmid pETW999 contained a 999 bp PCR fragment of the phuW gene and was generated with the primers 5'-catatgCGCATTCGCCTGTTCGC-3' (lower-case letters indicate a non-complementary NdeI site) and 5'-AGATTATCTCCTCATCAGGCT-3'. The phuW gene was then directionally cloned as an NdeIHindIII fragment from pCRII-2.1 into pET23a. In the resulting plasmid, pETW999, phuW was under T7 promoter control and also translational initiation signals were plasmid derived.
Generation of mutant strains affected in the phu and has loci.
Specific deletion mutants were obtained by replacing portions of the relevant genes with a gentamicin-resistance (GmR) or a tetracycline-resistance (TcR) cassette. The mobilizable suicide plasmid pSUP203 (Simon et al., 1983 ) containing the selection marker with suitable cloned DNA flanking regions served as the donor plasmid and E. coli HB101/pRK2013 (Figurski & Helinski, 1979
) served as the helper strain in triparental matings using P. aeruginosa PAO1 as the recipient, as described in detail elsewhere (Ochsner et al., 1996
). The successful gene replacement in the transconjugants was verified by Southern blot analysis. In brief, the deletion mutants created for this study had the following genotypes. In the
phuR mutant, an internal 0·4 kb HincII fragment of the 5' part of the phuR gene was replaced with a GmR cassette. Similarly, the
phuSTUV mutant had a replacement of a 2·4 kb StuI fragment covering the 3' end of phuS, the entire coding regions of phuT and phuU and the 5' half of phuV with a GmR cassette. The
phuW mutant harboured a 0·22 kb MluISphI deletion in the 3' portion of the phuW gene and carried a GmR cassette in that location. Mutant
phu-R1R2 had a 0·3 kb NheIDdeI fragment replaced with a GmR cassette, thereby deleting the entire R1 repeat and the 5' portion of the R2 repeat, including the promoter located within R2. In the
hasR mutant, two adjacent internal SmaI fragments of 0·45 kb and 1·05 kb of the hasR gene were deleted and replaced with a TcR cassette. The double mutant
phuR
hasR was obtained by deleting the hasR gene in the
phuR background using the same donor plasmid constructs as for the generation of the single mutants.
Translational fusions to the lacZ reporter gene.
pHAS1, pHAS2 and pHAS3, containing translational fusions to the lacZ reporter gene, were constructed as follows. A 0·32 kb PCR product was generated using the primers 5'-CCTCGTTGCAGTCGATCAG-3' and 5'-aagcttGCGACCGCCTCGCACG-3' (lower-case letters indicate a non-complementary HindIII site), cloned into pCRII-2.1, sequenced and directionally cloned as an EcoRIHindIII fragment into pPZ20 (Schweizer, 1991a ), resulting in pHAS1 harbouring a hasR::lacZ fusion. Similarly, a 0·23 kb PCR product was generated with the primers 5'-TGAGCGATGAACTGACCCT-3' and 5'-aagcttGCTCATGCCAAAACTCCAA-3' (includes HindIII site), and yielded pHAS2 harbouring a hasA::lacZ fusion. A 0·29 kb PCR fragment comprising the hasR promoter without the hasR translational signals was obtained with the primers 5'-CCTCGTTGCAGTCGATCAG-3' and 5'-aagcttCATGACAGCGCCTACACT-3' (includes HindIII site) and cloned into the EcoRI site of pHAS2. In the resulting plasmid, pHAS3, the hasR promoter fragment was upstream of the hasA promoter and had the same orientation.
Biochemical procedures.
Outer-membrane proteins were isolated by EDTA/lysozyme treatment of osmotically stabilized cells followed by differential centrifugation, as published elsewhere (Ochsner et al., 1999 ). The proteins in the crude outer-membrane preparation were separated on an 8% SDS-polyacrylamide gel and stained with Coomassie blue. High-molecular-mass proteins and the Benchmark ladder (BRL) were used as size standards. The Bradford assay (Bio-Rad) was used to measure protein concentrations. ß-Galactosidase activities were determined spectrophotometrically using ONPG (Sigma) as the substrate with 1 U corresponding to a substrate conversion of 1 µmol min-1. The ß-galactosidase activities given as U mg-1 were normalized to the amount of soluble protein.
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RESULTS |
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Utilization of haem and haemoglobin by P. aeruginosa wild-type and by mutants affected in the phu and hasR loci
The proteins encoded by the phu genes exhibited a high degree of identity to the factors involved in haem uptake in other micro-organisms. However, their role in iron acquisition from haem-containing compounds remained to be formally demonstrated, since different iron-uptake systems were often found to share a similar genetic organization and had substantial homologies among different systems. In particular, the known outer-membrane receptors for iron chelates such as haem, haemoglobin, transferrin, lactoferrin, ferrisiderophores and ferric citrate appear to be highly conserved among Gram-negative bacteria in spite of their distinct specificities.
The phu locus was not essential for P. aeruginosa and separate deletions in the phuR gene, in the phuSTUV operon, in the phuW gene and in the repeat elements phu-R1R2 were constructed (Fig. 1a). Growth curves and pigmentation of these mutant strains were indistinguishable from PAO1 wild-type cells in low-iron tryptic soy broth or M9 medium. Iron-starved wild-type and mutant cells were placed on iron-restricted M9 agar and scored for growth stimulation around filter disks onto which various iron-containing compounds have been spotted. In this assay, 103104 c.f.u. were mixed with top agarose and poured onto the iron-restricted agar plates. This method allowed qualitative growth monitoring by colony counting and discriminating between slow-growing small colonies still embedded in the top agarose and faster-growing colonies that had reached the plate surface. While all strains grew equally well with exogenously supplied transferrin or lactoferrin (data not shown), significant growth stimulation differences were observed when haemin or haemoglobin was used as the sole iron source (Fig. 3
). Both a
phuR and a
phuSTUV mutant were markedly impaired for utilization of either haem or haemoglobin and formed only very few small colonies around the disks. Growth of these mutants was partially restored by supplying functional copies of the phuR gene or the phuSTUV operon in trans on the plasmids pPHU1 and pPHU2, respectively (Fig. 3
). A
phuW mutant also exhibited reduced growth zones, although the effect was less dramatic. The
phu-R1R2 mutant strain lacking the tandem repeats downstream of the phuW gene was only mildly affected.
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Fur-mediated iron regulation of the phuR gene and of the phuSTUVW operon
Expression of phuR and phuS, as measured with translational fusions to the lacZ reporter gene, occurred preferentially under iron-limiting conditions and was repressed 12-fold and 25-fold, respectively, in high-iron media (data not shown). Two tandem Fur boxes spaced 5 bp apart were detected in the intergenic region between the divergently transcribed phuR and phuS genes (Fig. 4a). Fur box 1 had the sequence GATAATTATTTGCATTAGC, which matched the Fur-binding consensus sequence in 15 of 19 residues. Fur box 2 was less obvious and its sequence CAAAACGCATATCTGAATC matched the consensus in only 11 of 19 residues. DNase I footprint reactions were carried out to verify the binding of purified Fur to the phuRphuS intergenic region (data not shown). A Fur concentration of 30 nM resulted in a primary footprint of 34 bp which covered the stronger Fur box 1. At Fur concentrations of 100 nM or higher, an additional 17 bp were protected; this extended footprint also covered the weaker Fur box 2 (Fig. 4a
). The transcriptional-start sites for phuR and phuS were roughly mapped by RNase-protection analysis. Plasmids pPHU699-R and pPHU699-S containing a 699 bp EcoRVHincII phuSphuR DNA fragment cloned in either direction with respect to the T7 promoter served to generate riboprobes specific for phuR and phuS (Fig. 4a
). Transcripts of both phuR and phuS were detected in RNA isolated from cells grown to stationary phase in low-iron media, while transcripts were absent in RNA from cells cultivated under high-iron conditions (Fig. 4b
). The phuR-specific riboprobe protected a 245±5 nt portion of phuR mRNA, and the phuS-specific riboprobe protected a 380±10 nt portion of the phuS transcript (Fig. 4b
). These experimentally determined transcriptional-start sites correlated very well with the hypothetical start sites obtained through a promoter-prediction program and are indicated in Fig. 4(a)
. The phuR and phuS transcription-start sites were located 50 and 54 nt upstream of the phuR and phuS translation-initiation sites. Potential -35 promoter elements in the proper distance of the mRNA start sites were located within Fur box 1, leaving only 3 bp between the -35(phuS) and -35(phuR) elements (Fig. 4a
). Additional riboprobes specific for phuV and phuW transcripts resulted in hybridization patterns identical to the pattern obtained with the phuS-specific probe as far as growth phase and iron-dependent expression were concerned (data not shown). This finding, together with the tight spacing of the genes in the phuSTUVW operon and the lack of any potential promoter besides the phuS promoter, strongly suggested that these five genes were indeed organized in an operon. Taken together, it was evident that the phuR gene and the phuSTUV operon were co-regulated by Fur through binding to the strong Fur box 1 that overlapped their promoters.
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DISCUSSION |
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Factors encoded in the phu and has loci
Most of the systems involved in the uptake of iron chelates consist of an outer-membrane receptor and an ABC transport system. In P. aeruginosa the phu-encoded ABC transport system consisted of five proteins, while only four proteins are involved in the homologous haem-uptake systems hem of Y. enterocolitica (Stojiljkovic & Hantke, 1994 ), hmu of Y. pestis (Hornung et al., 1996
) and in the fec ferric dicitrate uptake machinery of E. coli (Staudenmaier et al., 1989
). In contrast, the Shi. dysenteriae shu haem-transport locus consists of seven genes, one of which is a pseudogene and two others do not have any known function (Wyckoff et al., 1998
). The cellular locations and functions of the components have been studied in some of these systems. The receptors, including PhuR, contain a typical signal sequence, a TonB box, and are found in the outer membrane. The PhuS component of the uptake system exhibited striking homology to the HemS, HmuS, FecB and ShuS components of the ABC transporters mentioned above, for one of which (FecB) a periplasmic location has been demonstrated (Staudenmaier et al., 1989
). HemS has been suggested to have haemin-binding activity and to prevent the accumulation of haem to toxic levels in the cell (Stojiljkovic & Hantke, 1994
). However, its homologue in Shi. dysenteriae, ShuS, was not required for protection against haem toxicity (Wyckoff et al., 1998
). PhuT and the homologues HemT, HmuT and ShuT are assumed to be localized in the periplasm, although this has not been directly demonstrated. The similarities of PhuU and PhuV to HemU and HemV suggest that they represent the permease and ATPase required for the haem transport across the inner membrane. Interestingly, an additional factor, the putative inner-membrane-bound PhuW protein encoded by the last gene of the phuSTUVW operon, was necessary for optimal efficiency of haem and haemoglobin utilization.
Mutations in the phu or has loci affect haem and haemoglobin utilization
The feeding of various iron-containing compounds to iron-starved phu mutant cells clearly indicated that the phu locus was required for haem and haemoglobin uptake, whereas the utilization of other iron chelates such as transferrin or lactoferrin was not affected. While the haem-uptake defect was very pronounced in the phuR and
phuSTUV mutants, the
phuW mutant was still capable of growing quite well compared to the
phuR and
phuSTUV mutants with haem as the sole iron source. In agreement with this was the finding that plasmid-borne copies of just the phuSTUV genes restored haem utilization to near wild-type levels in the
phuSTUV mutant in which expression of phuW was absent due to a polar effect of the deletion.
Surprisingly, a mutant strain affected in the second haem-receptor gene, hasR, did not grow efficiently with either haem or haemoglobin as the iron source in spite of a functional phu locus. Haem utilization of the hasR mutant was not restored with plasmid-borne copies of the hasR gene. As demonstrated by using translational lacZ fusions, hasR appeared to be the first gene in a Fur-regulated operon with hasA. Only low and constitutive expression of the hasA gene was detected from a weak hasA promoter. This was in good agreement with an earlier observation that the HasA protein accumulated in culture supernatants preferably under iron-limiting conditions (Létoffé et al., 1998
). The insertion of a gentamicin-resistance cartridge into the hasR gene had thus a predicted negative polar effect on hasA, resulting in very low production of HasA protein from its own promoter. Most likely, these low levels of HasA protein impaired efficient haem uptake even in the presence of a functional phu locus such as in the
hasR mutant. Also, the extent of incapacity to grow on haem of the
phuR and
phuSTUV mutants was somewhat surprising, since these mutants still harbour a functional has locus. It is intriguing to discuss the possibility that the phu and the has systems may be interdependent and to entertain a model in which the HasA haemophore may use either the PhuR or the HasR receptor for haem delivery to the cell. Although there is no direct experimental evidence for this model at this point, the phenotypes of the mutant strains studied support the hypothesis of such a cross-talk between the two systems. A
phuR
hasR double mutant grew very poorly with haem-containing compounds as the sole iron source, although some small colonies were formed around a disk containing haemoglobin. This remaining growth may be due to the combined action of extracellular proteases and siderophores as an indirect mechanism to acquire haemoglobin.
Regulation of the phu and has systems by Fur
Haem- and haemoglobin-acquisition systems in many bacterial species are directly regulated by Fur. Strong Fur boxes are present in the promoter regions of the Y. enterocolitica hemRSTUV operon (14 of 19 matches to the Fur consensus), of the Y. pestis hmuRSTUV operon (14 of 19), of the Ser. marcescens hasR gene (15 of 19) and of the Shi. dysenteriae shuA gene (17 of 19). The phuR gene and the phuSTUV operon were found to be co-regulated by Fur. Two tandem Fur boxes matching the consensus Fur-binding sequence in 15 of 19 and in 11 of 19 bp were detected and their significance was demonstrated in DNase footprints. The presence of two tandem Fur boxes has been associated with very tight repression of the affected genes. The P. aeruginosa pvdS gene encoding a Fur-regulated alternative sigma factor (Ochsner et al., 1996 ) also harbours two adjacent Fur boxes, resulting in a complete repression of pvdS in the presence of iron (H. Barton, unpublished results). The P. aeruginosa hasR gene was under direct control of Fur binding to a single weaker Fur box (13 of 19 matches) in the hasR promoter region, suggesting that the two haem-uptake systems of P. aeruginosa may be derepressed sequentially upon encountering iron limitation.
The role of the two Fur-regulated antisense phuSTUVW RNAs originating at the tandem repeats R1 and R2 downstream of the phuSTUVW operon was explored in terms of a regulatory mechanism in response to haem. Since haem binding of Fur has been observed in vitro for E. coli Fur (Smith et al., 1996 ), a model was tested in which the transcripts T1 and T2 would be repressed by haem. However, the addition of haem to otherwise iron-depleted medium did not result in a selective repression of T1/T2, but had a non-discriminative negative effect on T1/T2 as well as on phuS expression. An alternative role of the tandem repeats could be to function as excision sites for the haem-uptake locus as a mobile element. Although there is no direct evidence for such a mechanism, a horizontal gene transfer of the shu haem transport locus has been proposed for Shigella and pathogenic E. coli, based on the presence of two short repeats of 12 nt flanking the shu locus (Wyckoff et al., 1998
).
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ACKNOWLEDGEMENTS |
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Received 27 July 1999;
revised 26 September 1999;
accepted 5 October 1999.