Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa

Urs A. Ochsner1, Zaiga Johnson1 and Michael L. Vasil1

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A gene cluster similar to haem iron uptake loci of bacterial pathogens was identified in Pseudomonas aeruginosa. This phu locus (‘Pseudomonas haem uptake’) consisted of the phuR receptor gene and the phuSTUVW operon encoding a typical ABC transporter. Expression of phuR and phuSTUVW from mapped transcriptional-start sites occurred under iron-restricted growth conditions and was directly controlled by the Fur protein. Binding of Fur was demonstrated by DNase footprinting of two adjacent ‘Fur boxes’ that overlapped both the phuR and phuSTUVW promoters. Two tandem repeats of 154 bp were identified downstream of the phuSTUVW operon, each of which contained a strong Fur-dependent promoter driving expression of iron-regulated RNAs antisense to phuSTUVW. Mutant strains with deletions in phuR and phuSTUV showed greatly reduced growth with either haem or haemoglobin as the only iron source: the defects were complemented by plasmids harbouring the phuR or the phuSTUV genes, respectively. Deletions of phuW or of the tandem repeats had only minor effects on haem utilization. The remaining haem and haemoglobin uptake still observed in the {Delta}phuR or {Delta}phuSTUV deletion mutants was due to a second haem-acquisition system, has, which was also under the direct control of Fur. This second haem-receptor gene, hasR, was identified upstream of and in an operon with hasA, encoding a haem-binding extracellular protein. A {Delta}hasR mutant also exhibited decreased utilization of haem and haemoglobin, and a {Delta}phuR {Delta}hasR double mutant was virtually unable to take up either compound. Both the PhuR and HasR proteins were detected in the outer-membrane fraction of P. aeruginosa grown in low-iron media. Taken together, the evidence suggests that the phu and has loci encode two distinct systems required for the acquisition of haem and haemoglobin in P. aeruginosa.

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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Pseudomonas aeruginosa is an opportunistic pathogen and has the potential to severely infect injured or immunocompromised hosts such as patients with cystic fibrosis, emphysema, cancer or serious burns (Woods & Vasil, 1994 ). A large variety of potential virulence factors play a role in colonization, survival, tissue invasion and in the ultimate damage of the host (Nicas & Iglewski, 1985 ). P. aeruginosa prefers an aerobic metabolism, requiring respiratory enzymes that need iron for their function. Extremely low concentrations of free iron are encountered during growth in a human host because virtually all host iron is bound to haemoglobin, transferrin and lactoferrin (Crosa, 1997 ; Payne, 1993 ; Litwin & Calderwood, 1993 ). Iron restriction plays a central role in the stress response in P. aeruginosa, which has thus evolved numerous iron-acquisition systems. These include the release of siderophores, production of extracellular proteases, secretion of cytotoxic exotoxin A, and, as described in this report, the capacity to utilize haem and haemoglobin from exogenous sources. Typically, the factors relevant for these iron-uptake mechanisms are tightly regulated and only expressed during iron-limited growth. The ‘ferric uptake regulator’ protein (Fur) plays the central role in this iron-starvation response. Fur is a dimeric DNA-binding repressor which employs Fe(II) as a co-repressor and binds to a so-called ‘Fur box’ in the promoter regions of iron-regulated genes (Ochsner et al., 1995 ; Prince et al., 1993 ). Upon iron limitation, the Fur–Fe(II) complex dissociates and is displaced from the DNA, thereby allowing transcription of iron-regulated genes. A large number of Fur-regulated genes have been identified in P. aeruginosa and their role in iron acquisition has been characterized (Ochsner & Vasil, 1996 ). In this report, we present the isolation and characterization of the phu (‘Pseudomonas haem uptake’) gene cluster encoding an outer-membrane receptor and a specific ABC (ATP-binding cassette) transporter required for haem and haemoglobin uptake. The ability to use free haem or haem complexed to haemoglobin is characteristic for a growing number of pathogens, including Vibrio cholerae (Henderson & Payne, 1994 ), Shigella dysenteriae (Mills & Payne, 1997 ), enterohaemorrhagic Escherichia coli O157:H7 (Torres & Payne, 1997 ), Yersinia enterocolitica (Stojiljkovic & Hantke, 1992 ), Yersinia pestis (Hornung et al., 1996 ), Neisseria spp. (Stojiljkovic et al., 1996 ), Haemophilus influenzae (Cope et al., 1995 ; Maciver et al., 1996 ; Hanson et al., 1992 ) and Serratia marcescens (Ghigo et al., 1997 ).

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 ).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, media and growth conditions.
The strains and plasmids used in this study are shown in Table 1. Luria broth was used for strain maintenance. The low-iron medium was tryptic soy broth treated with Chelex 100 Resin (Bio-Rad), dialysed, and supplemented with 1% (w/v) glycerol and 50 mM glutamate; 50 µg FeCl3 ml-1 was also added for high-iron medium (Prince et al., 1993 ). For growth-stimulation experiments, P. aeruginosa wild-type and mutant strains were grown for 8 h at 37 °C in Chelex-treated M9 medium containing 0·2% glucose and 0·1 mM dipyridyl (Sigma) as a non-metabolizable iron chelator. The cultures were diluted to 104–105 cells ml-1, and 100 µl of the diluted cells were mixed with 3 ml Chelex-treated M9 medium, 0·2 % glucose, 0·8% low-melting-point agarose (Sigma) at 37 °C and poured onto Chelex-treated M9 medium, 0·2% glucose, 1·5% agarose plates containing 100 µg EDDHA [ethylenediamine di(o-hydroxyphenylacetic acid)] ml-1 (Sigma) as an iron chelator. Sterile filter disks were placed on top of the plates and spotted with 15 µl 5 mg ml-1 solutions of bovine haemin chloride in 10 mM NaOH, bovine haemoglobin in PBS, bovine lactoferrin in PBS or holo-transferrin in PBS; all these compounds were from Sigma. Antibiotics were used at the following concentrations: for E. coli, 100 µg ampicillin ml-1, 15 µg gentamicin ml-1, 100 µg kanamycin ml-1 and 15 µg tetracycline ml-1 were used; for P. aeruginosa, 750 µg carbenicillin ml-1, 75 µg gentamicin ml-1 and 150 µg tetracycline ml-1 were used.


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Table 1. Strains and plasmids

 
General genetic methods.
PCR was performed using Taq polymerase (BRL) and appropriate custom-made primers (BRL) in a Perkin Elmer Cetus thermal cycler, with 30 cycles of denaturing (1 min, 94 °C), annealing (1 min, 54 °C) and extending (1 min, 72 °C). The PCR products were purified in preparative agarose gels and cloned into pCRII-2.1 (Invitrogen). DNA sequencing of the phu genes and of all cloned PCR products was performed with Sequenase 2.0 (United States Biochemical) and M13 primers or custom-made 18-mer oligonucleotides. Published procedures were followed for Southern blot analysis (Sambrook et al., 1989 ) and colony hybridization (Forster et al., 1990 ).

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 [{alpha}-32P]CTP as described in detail elsewhere (Barton et al., 1996 ). A 699 bp EcoRV–HincII 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 XhoI–StuI phuV fragment into pBluescript SK (XhoI–EcoRV) and into pBluescript KS (XhoI–EcoRV), 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 EcoRI–HindIII 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 NdeI–HindIII 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 {Delta}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 {Delta}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 {Delta}phuW mutant harboured a 0·22 kb MluI–SphI deletion in the 3' portion of the phuW gene and carried a GmR cassette in that location. Mutant {Delta}phu-R1R2 had a 0·3 kb NheI–DdeI 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 {Delta}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 {Delta}phuR {Delta}hasR was obtained by deleting the hasR gene in the {Delta}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 EcoRI–HindIII 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Isolation of the P. aeruginosa haem uptake (phu) locus
A large number of P. aeruginosa iron-regulated genes (pigs) have been previously identified in a cycle-selection procedure based on the binding of the Fur protein to DNA sequences containing a ‘Fur box’ (Ochsner & Vasil, 1996 ). Two isolates, pig9 and pig20, mapped to the same region at 0·9–6·6 min on the P. aeruginosa chromosome by PFGE and resulted in identical Southern hybridization patterns (data not shown). Further DNA sequence analysis indicated that pig9 and pig20 were located adjacent to each other and contained 154 bp repeat sequences (R1 and R2) with 90% nucleotide sequence identity which were spaced 58 bp apart (Fig. 1a). Both repeat elements contained a strong Fur box matching 15 of the 19 residues of the conserved GATAATGATAATCATTATC Fur-binding consensus sequence (Fig. 1a), which resulted in DNaseI footprints that covered an area of 30 bp each (data not shown). Putative -35 and -10 promoter elements overlapping these Fur boxes correlated well with the mapped RNA start sites of strong iron-regulated transcripts originating within R1 and R2 (Fig. 1a). Possible roles of the R1 and R2 repeats are discussed below. The end of an iron-regulated transcript in the opposite direction was detected immediately downstream of repeat R2 (Fig. 1a). A 5·5 kb SphI DNA fragment containing this region and an overlapping 6 kb EcoRV DNA fragment extending even further downstream were cloned into pUCP19, yielding pPHU2 and pPHU1, respectively (Fig. 1a). The DNA sequence of pPHU2 and of parts of pPHU1 was determined and a 7702 bp portion has been deposited in GenBank (accession no. AF055999).



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Fig. 1. (a) Map of the P. aeruginosa haem uptake locus containing the phuR gene and the phuSTUVW operon. Relevant restriction sites and the composition of relevant plasmids are shown. Also indicated are transcripts (dashed arrows), tandem repeat elements (R1, R2), and the deleted parts in phu mutants (thick black lines). Four Fur-binding elements shown as white boxes were present within the phu locus. The Fur box sequences and the number of matching residues to the Fur box consensus (GATAATGATAATCATTATC) were as follows. Upstream of repeat R1, GGAAATGAGAATCATTATT (15 of 19); upstream of repeat R2, GATAATGAGAATAGTTATT (15 of 19); upstream of phuS, CAAAACGCATATCTGAATC (11 of 19 on the complementary DNA strand); and upstream of phuR, GATAATTATTTGCATTAGC (15 of 19 on the complementary DNA strand). (b) Phylogenetic tree based on bacterial haem and haemoglobin receptors. The percentages indicate the amino acid sequence matching at each branching point and are a measure of evolutionary distance. The protein primary structures were derived from GenBank entries for P. aeruginosa PhuR (accession no. AF055999), Pseudomonas fluorescens PfhR (accession no. AF127222), V. cholerae HutA (L27149), Shi. dysenteriae ShuA (U64516), E. coli ChuA (U67920), Y. eneterocolitica HemR (S49393), Y. pestis HmuR (U60647), N. meningitidis HmbR (U18558), H. influenzae HhuA (43198), Ser. marcescens HasR (Y08983) and P. aeruginosa HasR (AF127223).

 
Characterization of the PhuR and HasR outer-membrane receptors and of an ABC transporter encoded by phuSTUVW
The phu locus consisted of six ORFs, as depicted in Fig. 1(a). The characterization of the encoded proteins, such as biochemical properties, presumed cellular locations and homologies to other bacterial proteins, is summarized in Table 2. The phuR gene encoded an 84·6 kDa protein with a 25 aa signal sequence typical for exported proteins; the predicted size of the mature PhuR protein was 82 kDa (Table 2). The amino acid sequence of the PhuR protein had a high degree of overall identity to the family of outer-membrane receptors for haemin and haemoglobin, such as V. cholerae HutA (28%) (Henderson & Payne, 1994 ), enterohaemorrhagic E. coli O157:H7 ChuA (25%) (Torres & Payne, 1997 ), Shi. dysenteriae ShuA (25%) (Mills & Payne, 1997 ; Wyckoff et al., 1998 ), Y. enterocolitica HemR (24%) (Stojiljkovic & Hantke, 1992 ) and Y. pestis HmuR (24%) (Hornung et al., 1996 ). The latter two are encoded in an operon with the small hemP and hmuP genes, respectively;however, the phu locus lacked an ORF encoding a homologue of these hypothetical factors. PfhR was isolated from P. fluorescens and had 64% amino acid sequence identity with P. aeruginosa PhuR. These proteins appear to form a subfamily of haem receptors and have less homology to the members of the second subfamily which contains the Ser. marcescens HasR receptor (Ghigo et al., 1997 ). A HasR homologue was isolated from P. aeruginosa using the DNA sequence information of the unfinished P. aeruginosa genome project (Pathogenesis Corporation) and exhibited 58% amino acid sequence identity to Ser. marcescens HasR. A phylogenetic tree of selected receptors with the relative evolutionary distances given as percentages is shown in Fig. 1(b).


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Table 2. Characterization of the predicted proteins encoded in the P. aeruginosa haem-uptake (phu) locus

 
The PhuR protein contained the amino acid sequence motifs conserved among TonB-dependent receptors, previously designated regions I, II and III (Bitter et al., 1991 ). Region I is the ‘TonB box’ with the consensus sequence (D/E)T..V.A(A/S) and is typically located near the N terminus of the protein (Ankenbauer & Quan, 1994 ; Bitter et al., 1991 ). A somewhat weak ‘TonB box’-like motif, STVSVQTR, was located at amino acid positions 25–32 of the mature PhuR protein. Region II has the consensus sequence (F/I/L/M/V)... (I/L/V).NL.(D/N)(K/R).Y and is typically located in the C-terminal portion of the receptor protein. In PhuR, the motif LNAGLYNLTDKKY, completely matching this consensus, was detected at positions 686–698 of the mature PhuR sequence. Region III, typically located at a distance of roughly 100 aa from region I, is characterized by the consensus sequence R(V/I)(D/E)(I/V)(I/V/L)(K/R)G..(G/S/A)...G....G(G/A).(V/I) and the matching pattern RVEILRGPASALYGSNAIGGAV was found 110 aa C-terminal from region I. The finding of these three characteristic elements and the presence of an N-terminal signal sequence suggested that the PhuR protein was an outer-membrane receptor. Outer-membrane-protein profiles from iron-starved PAO1 wild-type and from {Delta}phuR or {Delta}hasR mutant strains were compared (Fig. 2a). SDS-PAGE analysis revealed the lack of a roughly 80 kDa protein in the {Delta}phuR mutant, which was in good agreement with the predicted size of 82 kDa for the mature PhuR receptor. A prominent protein band at that position was detected upon genetic complementation of the {Delta}phuR mutant with pPHU1, which contains a constitutively expressed functional copy of the phuR gene. Similarly, the second P. aeruginosa haem receptor, HasR, was detected by SDS-PAGE analysis of outer-membrane-protein preparations. In PAO1 wild-type extracts, a faint protein band was present at roughly 94 kDa, which was the predicted size of the HasR receptor. This band was missing in a {Delta}hasR mutant and was restored in {Delta}hasR by the plasmid pHAS1, which contained a copy of the hasR gene (Fig. 2a).



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Fig. 2. SDS-PAGE analysis of P. aeruginosa proteins involved in haem acquisition. (a) Outer-membrane proteins of PAO1 wild-type, single mutants {Delta}phuR, {Delta}hasR, double mutant {Delta}phuR {Delta}hasR and complemented mutants {Delta}phuR/pPHU1 and {Delta}hasR/pHAS3051. The positions of the putative 82 kDa PhuR and 94 kDa HasR outer-membrane receptors are indicated by arrows. (b) Overexpression and labelling of PhuW using a T7 expression system. Whole-cell extracts of E. coli BL21(DE3) containing plasmid pETW999 (pET23a-phuW) or the pET23a vector control were separated by SDS-PAGE and autoradiographed. The positions of the unprocessed and processed forms of the putative 32 kDa PhuW inner-membrane protein as well as of the plasmid-encoded Bla protein are indicated by arrows.

 
Immediately upstream and divergently transcribed of the phuR gene was an operon consisting of five genes: phuS, phuT, phuU, phuV and phuW. The putative proteins encoded by the first four of these genes were highly similar to the ABC transport components HemS, HemT, HemU and HemV required for haem acquisition in Y. enterocolitica (Stojiljkovic & Hantke, 1994 ); their properties are described in Table 2. PhuT contained a 33 aa signal sequence typical for exported proteins. PhuV contained two motifs common for ABC transporter ATPases, the Walker nucleotide-binding consensus motif GPNGAGKS at positions 34–41 and the motif VLLLDEP at positions 158–165 (Walker et al., 1982 ). PhuW harboured a possible signal sequence for export and an {alpha}-helical hydrophobic stretch in the centre part of the protein sequence at residues 148–163 which may function as a potential membrane-spanning domain. A PhuW homologue of 27% amino acid sequence identity was identified in an iron-acquisition locus of Campylobacter jejuni. (A. van Vliet, unpublished results). The hypothetical phuW gene was overexpressed in P. aeruginosa using pPHU3, which contained the phuW gene under lac promoter control, but no protein of the predicted size was detectable in total cell extracts (data not shown). However, T7 expression and specific labelling of PhuW in E. coli using plasmid pETW999 resulted in the detection of putative PhuW protein. A double band at roughly 34 and 32 kDa was present, which corresponded very well to the size of the unprocessed and processed forms of PhuW, respectively (Fig. 2b).

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, 103–104 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 {Delta}phuR and a {Delta}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 {Delta}phuW mutant also exhibited reduced growth zones, although the effect was less dramatic. The {Delta}phu-R1R2 mutant strain lacking the tandem repeats downstream of the phuW gene was only mildly affected.



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Fig. 3. Growth stimulation by haemin or haemoglobin as the sole iron source. PAO1 wild-type, single mutants {Delta}phuR, {Delta}phuSTUV, {Delta}phuW, {Delta}phu-R1R2 and {Delta}hasR; double mutant {Delta}phuR {Delta}hasR; and the complemented mutants {Delta}phuR/pPHU1, {Delta}phuSTUV/pPHU2 and {Delta}hasR/pHAS3051 were pre-grown in M9 medium containing dipyridyl. Roughly 103–104 such iron-starved cells were then embedded in a top layer of agarose onto M9 plates containing the iron chelator EDDHA. Growth of colonies around sterile filter disks impregnated with haemin or haemoglobin was monitored after 36 h at 37 °C. Note: colonies appeared big when they reached the surface of the plates; the small colonies were located within the top agar.

 
The faint yet detectable growth of the {Delta}phuR and the {Delta}phuSTUV mutants with haemin or haemoglobin as the sole iron source suggested the existence of a backup system for the acquisition of iron from these compounds. In fact, the extracellular haem-binding protein HasA, a so-called haemophore, has been recently described in P. aeruginosa, suggesting the presence of a haem-acquisition system analogous to the Ser. marcescens system (Létoffé et al., 1998 , 1999 ). Immediately upstream of, and contained in, an operon with the hasA gene, we identified the hasR gene, which encoded a second putative haem receptor. Downstream of hasA was a three-gene operon (data not shown) encoding factors with high homology to the Ser. marcescens HasD, HasE and HasF ABC-exporter proteins required for HasA secretion in S. marcescens (Binet & Wandersman, 1996 ; Ghigo et al., 1997 ). A {Delta}hasR mutant was affected in the utilization of haemin and haemoglobin as severely as the {Delta}phuR mutant and a {Delta}phuR {Delta}hasR double mutant did not grow significantly under these conditions (Fig. 3). Attempts to complement the {Delta}hasR mutant with pHAS3051, containing a functional hasR gene, failed, presumably due to polar effects on the downstream genes as discussed below.

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 EcoRV–HincII 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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Fig. 4. Coregulation of phuR and phuS by Fur. (a) Map of a 0·7 kb EcoRV–HincII DNA fragment containing the phuRphuS intergenic region. The 5' portions of the phuR and phuS genes, their promoters, Fur boxes and transcriptional- and translational-start sites are shown. Riboprobes to detect phuR and phuS transcripts were generated from plasmids pPHU699-R and pPHU699-S, respectively, as indicated by dashed arrows. Also shown is the DNA sequence between the divergent phuR and phuS start codons. The location of the two adjacent Fur boxes is indicated by the boxed sequences, and the areas protected by Fur in DNase I footprint analyses are emphasized with asterisks (primary Fur binding site) and primes (secondary Fur binding site). (b) RNase protection of phuR and phuS transcripts. RNA was isolated from PAO1 grown under low (-) or high (+) iron conditions for 12 h at 37 °C. Normalized samples (20 µg) were hybridized with excess anti-phuR riboprobe generated from pPHU699-R or anti-phuS riboprobe made from pPHU699-S. The sizes of the protected portions of phuR and phuS probes were estimated by comparison with the bands of radiolabelled RNA ladder on a 5% polyacrylamide/8 M urea gel, to allow the rough mapping of the corresponding transcriptional-start sites.

 
Mapping of Fur-regulated transcripts antisense to phuSTUVW
The tandem repeats R1 and R2 located downstream of the phuSTUVW operon were 154 bp long and 58 bp apart. Each repeat contained a strong Fur-regulated promoter at its 5' end, and a stem–loop structure of a calculated {Delta}G=-20 kcal mol-1 near its 3' end (Fig. 5a). The transcripts T1 and T2 driven from the promoters P1 and P2 were detected by RNase protection analysis using a 346 nt riboprobe spanning R1, generated by pPHU-R1 (Fig. 5a). The most prominent band of a protected RNA species was at 111±3 nt, followed by a weaker signal at 184±5 nt: both protected fragments were highly iron regulated (Fig. 5b, lanes 1 and 2). The protected 111 nt fragment corresponded to the expected sizes of T1 and T2 from their transcriptional-start site to the stem–loop structure (Fig. 5a), indicating that the stem–loop within the repeats R1 and R2 acted as transcriptional terminator. The protected 184 nt fragment corresponded to the expected size of a longer T1 transcript from its start site to the start of the riboprobe (Fig. 5a), providing evidence that transcriptional termination at the stem–loop was only partial and that a substantial amount of T1 transcripts extended beyond that site. A riboprobe from pPHU359+ consisting of the plus-strand of a 359 bp internal phuV fragment was completely protected, indicating that the T1 and/or T2 transcripts extended at least 1·6 kb further: that is, through the phuW and phuV genes (Fig. 5b, lanes 3 and 4). However, that protection pattern did not show any level of iron regulation. In contrast, the phuV message detected with the minus-strand of phuV as a riboprobe from pPHU359- showed iron repression as expected (Fig. 5b, lanes 5 and 6). Clearly, the T1 and/or T2 transcripts were antisense relative to the phuSTUVW polycistronic mRNA. However, the T1 or T2 transcripts lacked translational-initiation signals and did not encode any detectable protein when the relevant DNA portion was subjected to a T7 expression and labelling system in E. coli (data not shown). A possible regulatory role of the T1 and T2 transcripts in response to haem was explored, following a model in which expression of T1 and T2 RNAs would be haem repressible through Fur, possibly resulting in increased phuSTUVW translation in the presence of haem. RNA from PAO1 grown in the absence or presence of haem was analysed with riboprobes to detect T1/T2 RNA and phuS mRNA. Interestingly, the signal at 184 nt representing the longer T1 transcript was greatly reduced in the presence of haem (Fig. 5b, lanes 7 and 8). However, also the phuS mRNA signal was weaker when haem was present (Fig. 5b, lanes 9 and 10). PhuSlacZ activity was also reduced several-fold (data not shown), suggesting that haem was partially relieving the iron starvation.



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Fig. 5. Characterization of anti-phuSTUVW transcripts. (a) Map of the relevant portion of the phu locus. The two 154 bp repeats R1 and R2 (black boxes) each contained a Fur-binding site (white boxes) and a stem–loop structure (hairpin). Transcripts T1 and T2 were antisense to the phuSTUVW mRNA and partially terminated at the stem–loop structures (thick arrows), but a fraction of T1 and T2 transcripts extended beyond these sites, representing anti-phuSTUVW RNAs (thin arrows). The R1 riboprobe (dashed arrow) detected the indicated two major protected fragments of 111 nt and 184 nt, respectively. (b) RNase protection analysis. RNA was isolated from PAO1 grown under low (-Fe) or high (+Fe) iron conditions as indicated in lanes 1–6, or in low-iron medium containing 25 µg haemin ml-1 (+haem) as indicated in lanes 7–10. The arrows point to the signals of the relevant transcripts. Lanes 1 and 2, T1 and T2 RNA; lanes 3 and 4, anti-phuV RNA (T1 and T2 extended transcripts); lanes 5 and 6, phuV mRNA; lanes 7 and 8, T1 and T2 RNA; lanes 9 and 10, phuS mRNA.

 
Fur-mediated iron regulation of the hasR gene encoding a second haem receptor
Upstream of the hasA gene encoding an extracellular haemophore (Létoffé et al., 1998 ) we identified the hasR gene which encoded a second haem receptor of P. aeruginosa. The 85 bp region between the stop codon of the hasR gene and the start codon of the hasA gene had a lower G+C content, indicating a potential promoter, although any obvious strong promoter characteristics were undetectable. Also, the hasRhasA intergenic region contained a stem–loop structure (-25 kcal mol-1) as a potential transcriptional terminator for hasR. To address the question whether hasR and hasA form an operon, translational fusions of the putative hasA promoter in the absence or in the presence of the upstream hasR promoter to the lacZ reporter gene were constructed, along with a hasRlacZ fusion (Fig. 6a). The expression of hasR was found to be strongly iron regulated, and hasA gene expression followed that pattern when the upstream hasR promoter and the intergenic stem–loop motif were present, suggesting that hasR and hasA were co-transcribed. In the absence of the hasR promoter, hasA expression was low and constitutive, indicating a separate but very weak promoter for hasA (Fig. 6b). A putative Fur box that matched the Fur-binding consensus sequence in 13 of 19 bp was detected in the hasR promoter region, and a DNase I footprint of 29 bp covering that Fur box was obtained (data not shown).



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Fig. 6. Characterization of the alternate hasRhasA haem-acquisition system. (a) Map of the hasRhasA locus. Promoters for hasR and for hasA are shown as black boxes and the location of a Fur box upstream of the hasR gene is indicated. Also shown are the DNA portions used to construct the translational lacZ-fusion plasmids (pHAS1, pHAS2, pHAS3) for the measurement of hasR and hasA gene expression. (b) Expression of hasR::lacZ (pHAS1), hasA::lacZ alone (pHAS2) or in the presence of the upstream hasR promoter (pHAS3). The ß-galactosidase activities were measured in soluble protein extracts prepared from triplicate cultures grown for 15 h under low and high iron conditions at 37 °C.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Two distinct haem- and haemoglobin-uptake systems in P. aeruginosa
Haem and haemoglobin acquisition has been described for a growing number of Gram-negative micro-organisms. Here we present evidence that P. aeruginosa has two distinct haem-uptake systems which enable this opportunistic pathogen to utilize iron from these sources very efficiently. An extracellular protein, HasA, which acts as a haemophore, has recently been isolated from P. aeruginosa through immuno cross-reaction with polyclonal antibodies against HasA from Ser. marcescens (Létoffé et al., 1998 ). In that study, the addition of anti-HasA antibodies inhibited the use of haemoglobin iron at concentrations lower than 10-6 M. However, P. aeruginosa could use free haem iron at a higher concentration (10-5 M) and growth inhibition by the specific anti-HasA serum was only partial. These data suggested that the haemophore-dependent haem-uptake system was the unique P. aeruginosa high-affinity transport system for haemoglobin-bound haem and that a separate lower-affinity haem-acquistion system existed. The phu locus described here is unrelated to the has locus and encodes an alternative haem-uptake pathway. The six factors encoded by the phu genes show high similarity to the haem-uptake components of Y. enterocolitica and Y. pestis in terms of genetic organization and protein identities, while the factors encoded by the has system are more closely related to the homologous system in Ser. marcescens. To our knowledge, P. aeruginosa is now the second micro-organism besides H. influenzae type b (Cope et al., 1995 ; Hanson et al., 1992 ) known to have two distinct haem-acquisition systems. This finding further stresses the importance of iron for the preferred aerobic growth of P. aeruginosa and may indicate the importance of iron and haem in pathogenesis.

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 {Delta}phuR and {Delta}phuSTUV mutants, the {Delta}phuW mutant was still capable of growing quite well compared to the {Delta}phuR and {Delta}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 {Delta}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 {Delta}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 {Delta}hasR mutant. Also, the extent of incapacity to grow on haem of the {Delta}phuR and {Delta}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 {Delta}phuR {Delta}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 ).


   ACKNOWLEDGEMENTS
 
This work was supported by Grant AI15940 from the National Institute for Health to Michael L. Vasil. We thank Pathogenesis Corporation for the hasR DNA sequence made available through the unfinished P. aeruginosa genome project and Arnoud van Vliet for sharing unpublished DNA sequence information on a phuW homologue in C. jejuni.


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Received 27 July 1999; revised 26 September 1999; accepted 5 October 1999.