Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA1
Author for correspondence: Radheshyam K. Jayaswal. Tel: +1 309 438 5125. Fax: +1 309 438 3722. e-mail: drjay{at}ilstu.edu
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ABSTRACT |
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Keywords: Fur, iron, metal resistance, Staphylococcus aureus
Abbreviations: EMSA, electrophoretic mobility-shift assay; HTH, helixturnhelix
The GenBank accession numbers for the S. aureus fur gene and fhu operon reported in this paper are AF118839 and AF132117, respectively.
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INTRODUCTION |
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Intracellular iron concentrations in many bacteria have been reported to be under the control of a ferric-uptake regulator, Fur (Guerinot, 1994 ; Crosa, 1997
). Fur also regulates a variety of iron-dependent cellular processes, such as the acid-shock response (Foster, 1991
) and the oxidative-stress response (Tardat & Touati, 1993
) and it also regulates the genes involved in the biosynthesis and uptake of several siderophores (Guerinot, 1994
). However, the acid-shock response has been reported to be normal in a fur- mutant of Salmonella typhimurium that was no longer able to sense iron in the environment (Hall & Foster, 1996
). Fur has also been reported to regulate metabolic pathways. In E. coli, it regulates the genes involved in purine, pyrimidine and methionine biosynthesis (Stojiljkovic et al., 1994
).
In Gram-positive bacteria, several iron-dependent repressors have been reported. The DtxR of C. diphtheriae and IdeR of Mycobacterium tuberculosis show limited homology to the Fur protein of Gram-negative bacteria (Tao et al., 1994 ; Schmitt et al., 1995
). In Bacillus subtilis, three distinct Fur homologues designated Fur, Zur and PerR, regulating iron uptake, zinc uptake and the peroxide-stress response, respectively, have been reported (Bsat et al., 1998
; Gaballa & Helmann, 1998
). The presence of a DtxR homologue, SirR, has been detected in several staphylococcal species (Hill et al., 1998
) and a Fur-like protein has been reported in Staphylococcus epidermidis (Heidrich et al., 1996
).
One of the mechanisms facilitating iron uptake involves several small molecules called siderophores that chelate iron from the host and deliver it into the bacterial cells through specific uptake systems (Guerinot, 1994 ; Neilands, 1995
). Three different siderophores, aureochelin (Courcol et al., 1997
), staphyloferrin A (Meiwes et al., 1990
) and staphyloferrin B (Haag et al., 1994
), have previously been identified in staphylococci. However, very little is known about the mechanism by which these siderophores transport iron into the cells. The cloning of a novel iron-regulated operon consisting of three genes, sitA, -B and -C, encoding an ATPase, a cytoplasmic membrane protein and a lipoprotein, respectively, has been reported in S. epidermidis (Cockayne et al., 1998
). Whether this transport system is involved in either siderophore- or transferrin-mediated iron uptake in S. epidermidis remains to be determined. Recently, a locus from S. aureus containing three ORFs with a high sequence homology to the siderophore-acquisition genes of Erwinia chrysanthemi was identified (Heinrich et al., 1999
). The first ORF, which encodes a membrane-associated siderophore-binding protein SirA, is preceded by a sequence of 19 bp with dyad symmetry, similar to the Fur boxes of Gram-negative bacteria. It is thus believed that the biosynthesis and uptake of the siderophores among staphylococci are regulated by Fur (Trivier & Courcol, 1996
; Cockayne et al., 1998
; Heinrich et al., 1999
) but there is no direct evidence to support this hypothesis.
Here we report the cloning of a ferric-uptake regulatory gene (fur) and a putative high-affinity ferrichrome-uptake operon (fhu) from a genomic library of S. aureus. For functional analysis, fur was overexpressed in E. coli and purified to near homogeneity. Electrophoretic mobility-shift and DNase I footprinting assays revealed that Fur specifically interacted with the fhu promoter region and therefore possibly regulates the expression of the fhu operon in S. aureus.
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METHODS |
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Cloning of the ferric-uptake regulatory gene, fur, and a putative fhu operon of S. aureus.
Based on the DNA sequences of the fur gene, specifically of B. subtilis and E. coli, two oligonucleotide primers were designed using the program CODHOP (Rose et al., 1998 ). These primers were later modified to reflect the codon usage bias in S. aureus. The forward primer used from the highly conserved helixturnhelix (HTH) domain was 5'-GGCTTGGCGACAGTATACAGC-3' and the reverse primer from the metal-binding region was 5'-ACATTCCATACATACTAAATGATG-3'. PCR was performed using S. aureus RN450 chromosomal DNA as the template. The amplified PCR product was sequenced to determine its potential homology with fur genes of other bacteria and was later used as a probe to screen the genomic library of S. aureus RN450 to clone fur.
For functional characterization of the Fur protein, we searched for fhu-operon-like sequences in the S. aureus genome. The fhu operon in B. subtilis is regulated by Fur (Bsat et al., 1998 ; Mademedis & Koster, 1998
). A fragment with strong homology to the fhuC gene of the fhu operon of B. subtilis was found in the S. aureus genome database of the University of Oklahomas Advanced Center for Genome Technology (www.genome.ou.edu/staph.html). This fragment was PCR amplified and used as a probe to screen the S. aureus genomic library. A representative cosmid clone was subsequently sequenced by gene walking to locate the entire fhu operon.
Cloning, overexpression and purification of Fur.
Two oligonucleotide primers (upstream 5'-GGATCCTTGGAAGAACGATTAA-3') and (downstream 5'-AAGCTTTTCTATCCTTTACCTTT-3') were designed to incorporate a BamHI restriction site at the 5' end and a HindIII site at the 3' end of the gene during the amplification of fur (restriction enzyme sites underlined). The PCR product was cloned into pCR2.1 (Invitrogen), and then digested with BamHI and HindIII. The fragment corresponding to the fur coding region was gel purified and subcloned into the BamHI and HindIII sites of pRSETa (Invitrogen) to generate plasmid pSA-fur. pSA-fur was used to transform E. coli BL21(DE3)(pLysS). The overnight culture of these transformants was used to inoculate 500 ml fresh LB. At an OD600 of 0·4, the cells were induced for the expression of Fur by the addition of 2·5 mM IPTG for 2·5 h. All subsequent procedures were performed at 4 °C unless stated otherwise. The cells from the culture were harvested and resuspended in 5 ml 50 mM Tris/HCl buffer (pH 7·0). Cell suspensions were subjected to sonication (seven pulses of 30 s spaced 30 s apart, with settings at an output control of 5 and 50% duty cycle) with a Sonifier Cell Disrupter (Branson Ultrasonic). Cell debris and unbroken cells were removed by centrifugation (30000 g for 15 min at 4 °C). The supernatant was applied to a nickel-charged agarose affinity column and eluted with 200400 mM imidazole using the Xpress Purification System (Invitrogen). Eluted fractions were subjected to a 15% SDS-PAGE analysis. Fractions containing the overexpressed His-tagged Fur were pooled and dialysed in a dialysis cassette with molecular-mass cut off of 7 kDa (Pierce) against the dialysis buffer (10 mM MgCl2, 0·1 mM DTT, 5%, v/v, glycerol in 20 mM Tris/HCl, pH 7·9). The enterokinase Cleavage Capture Kit (Novagen) was used to cleave the histidine tag from this preparation. A second round of chromatography on a Ni2+-affinity column retained the histidine tag peptide and any undigested recombinant protein and the His-tag-free Fur was recovered from the flow-through fraction. The final Fur protein was stored in 20 mM Tris/HCl, pH 7·0, containing 50 mM NaCl, 2 mM CaCl2 and 33%, v/v, glycerol, at -20 °C for future use.
Electrophoretic mobility-shift assays (EMSAs).
These were performed as described by de Lorenzo et al. (1988) . Two oligonucleotide primers were synthesized to amplify the fhu promoter from the genomic DNA of S. aureus. In the forward primer, an XbaI site was incorporated (5'-CGCGTCTAGAAATTTCCGTACT-3') and a KpnI site was incorporated in the reverse primer (5'-CGTCAGGTACCATGGTATGAAG-3') (restriction enzyme sites are underlined). The sir promoter was also amplified from the genomic DNA of S. aureus using two primers (5'-TGGACGGCATACTAAATCGTGA-3' and 5'-GCTAGTCTAGAGTACCCATTGCATGTT-3'). The XbaI restriction enzyme site in the reverse primer is underlined. The amplified fhu and sir promoter DNAs were digested with XbaI and then end labelled with [
-32P]dCTP by the fill-in reaction using the Klenow fragment of DNA polymerase I. The standard binding assays contained (in a total volume of 20 µl) 1 µg BSA, 1 µg sheared calf thymus DNA and various concentrations of Fur protein in 1xbinding buffer (100 µM MnCl2, 1 mM MgCl2 and 25 mM KCl in 10 mM Tris-borate, pH 7·5). The reaction mixture was incubated at 25 °C for 15 min. The diluted end-labelled DNA was added to the reaction mixture and incubated for an additional 20 min at 25 °C. Three microlitres of loading buffer (0·1% cyanol blue and 40% sucrose in DNA-binding buffer) was added and 10 µl of the mixture was loaded onto a 5% nondenaturing PAGE. Electrophoresis was carried out at 90 V for 2·5 h using 10 mM Tris-borate, pH 7·5, containing 100 µM MnCl2 as electrode buffer. The gels were dried and the DNAprotein complexes were visualized by autoradiography.
DNase I-protection assay.
A DNA fragment containing the fhu promoter region was amplified as above and the amplified DNA fragment was cloned into pCR2.1, resulting in the plasmid pCR2.1-pfhu. A 500 ml culture of E. coli JM109 containing the above construct was grown and plasmid DNA was isolated using Wizard Plus Maxi Prep Columns (Promega). The isolated DNA (200 µg) was digested with EcoRI and subsequently with XbaI. The fragment representing the fhu promoter was gel purified and the XbaI overhang was end-labelled and used for the DNase I-protection assays essentially as described by Leblanc & Moss (1994) with a slight modification described by Singh et al. (1999)
. First, the binding reaction was performed in a total volume of 50 µl of the 1xbinding buffer. The binding assay contained 1 µg BSA, 20 ng end-labelled DNA and varying concentrations of Fur. After 20 min incubation, 50 µl cofactor solution (10 mM MgCl2, 5 mM CaCl2) and 0·4 units of DNase I (Sigma) were added and incubated for an additional 2 min at room temperature. DNase I activity was terminated by the addition of 100 µl stop solution (1% SDS, 200 mM NaCl, 20 mM EDTA, pH 8·0). The reaction products were extracted with phenol/chloroform, precipitated with 2·5 vols 100% ethanol and air dried. The DNase I-digested fragments were then analysed on a 5·0% polyacrylamide sequencing gel. To determine the exact area protected, manual nucleotide sequencing reactions of the fhu promoter fragment were carried out in parallel to the DNase I-footprint fragments, using the primer 5'-CTAGAAATTTCCCTACTTTC-3'.
Primer extension.
Total RNA from S. aureus RN450 grown in iron-limited minimal media was isolated with the TRI reagent kit (Molecular Research Center). The primer-extension assay was performed as described previously (Xiong & Jayaswal, 1998 ), using an oligonucleotide primer (5'-TTCATAATTTCCCTACTTTC-3') specific to the fhuC coding region. A sequencing ladder generated by using the same primer on pCR2.1-pfhu was coelectrophoresed to determine the position of the transcription-start site.
Complementation of the fur- mutation in B. subtilis.
A ~1·9 kb fragment containing the entire fur gene was amplified using S. aureus RN450 genomic DNA as the template. The primers used were 5'-GGTGCAGTTGCTGTTTGTGC-3' and 5'-AGTGGCGTAACGTATGTGGC-3'. The fur gene was cloned into pCR2.1 and then subcloned into the EcoRI site of the shuttle vector pCU1. To test whether the S. aureus Fur complemented the fur- mutants, the resulting plasmid pCU1-fur was transferred by electroporation into the fur- strain of B. subtilis HB6543. The complementation test in B. subtilis HB6543 was performed by measuring the siderophore production (OD510) against the culture density (OD600), as described by Chen et al. (1993) and Bsat et al. (1998)
.
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RESULTS AND DISCUSSION |
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The hydropathy profile revealed S. aureus Fur to be a hydrophilic protein, suggesting a cytoplasmic location. The analysis of the C terminus of Fur indicated the presence of a conserved metal binding domain (HXHHH) and motifs (CXXCG and CXXC) that coordinate the binding of metal ions. The N terminus contains an HTH DNA-binding motif common among most of the metalloregulatory proteins (Lam et al., 1994 ; Cook et al., 1998
; Pohl et al., 1999
). These secondary-structure-associated features suggest that S. aureus Fur is a metal-responsive regulatory protein.
Cloning of a putative ferrichrome uptake (fhu) operon from S. aureus
The ferrichrome-uptake system (fhu) in B. subtilis has been reported to be regulated by Fur (Bsat et al., 1998 ; Mademidis & Koster, 1998
). To determine whether Fur regulated such a ferrichrome-uptake system in S. aureus as well, we cloned a 4 kb fragment containing the fhu operon from the cosmid library into pTZ18R. Nucleotide sequence analysis of this fragment revealed three ORFs designated fhuC, fhuB and fhuD (Fig. 2a
). fhuC showed significant sequence homology with fhuC of B. subtilis, encoding an ATP-binding protein. fhuB and fhuD showed significant homologies with many bacterial ferrichrome-transport permeases. In fhuC, the ribosome-binding sequence (AGTAGG), was found 6 nt upstream of the proposed translation-initiation codon ATG (Fig. 2a
). Possible -35 (TAGTCA) and -10 (TATAAT) sequences were also identified. Three imperfect repetitions of the sequence GATAAT (analogous to the so-called Fur box) were found upstream of fhuC. This sequence is identical to the consensus E. coli Fur box sequence and to the Fur box of the dhb operon of B. subtilis except for 2 bp (Fig. 2b
) (Rowland et al., 1996
; Escolar et al., 1998
). However, it differed at five nucleotide positions from the recently identified Fur box upstream of sirA in S. aureus (Fig. 2b
) (Heinrich et al., 1999
).
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In this study, we observed that Co2+, Cu2+, Mn2+ and Ni2+ metal ions also interacted with Fur in vitro, similar to other systems (Bagg & Neilands, 1987 ). This suggests that Fur may be regulating not only iron but the intracellular levels of other metal ions as well. However, the interaction of Fur with these cognate metal ions remains controversial. Bsat & Helmann (1999)
have recently reported that B. subtilis Fur interacted very tightly both in vitro and in vivo, with the operator region of the dhb operon. Interestingly, the affinity of the Fur protein with the dhb operon was iron independent. Recently, Althaus et al. (1999)
have reported that Fur purified from E. coli contained two molecules of zinc per Fur molecule. While the first zinc molecule could be easily detached from native Fur, the other could be detached only under denaturing conditions. While the two metalated forms of Fur were able to interact with the operatorpromoter regions, the apoprotein did not bind specifically to these regions.
In conclusion, S. aureus Fur is likely to function as an iron-responsive metalloregulatory protein that interacts with at least two operons, fhu and sir, in S. aureus. Currently we are constructing mutations in the fur gene, and also in the individual ORFs of the fhu operon to determine their biological functions. An understanding of these systems may provide potential targets to control the proliferation of this pathogenic bacterium.
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ACKNOWLEDGEMENTS |
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Received 1 September 1999;
revised 7 December 1999;
accepted 9 December 1999.