1 Mikrobiologie/Membranphysiologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany
2 Mikrobiologie/Biotechnologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany
Correspondence
Klaus Hantke
hantke{at}uni-tuebingen.de
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ABSTRACT |
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Protein purification and cleavage data are available as supplementary material with the online version of this paper.
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INTRODUCTION |
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METHODS |
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Isolation of salmochelin and enterobactin.
M63 medium (50 ml) was inoculated with 0·5 ml cells, freshly grown in TY medium, and incubated for 18 h with shaking at 37 °C. After centrifugation, 2 mM FeCl3 was added to the supernatant. The precipitate formed was removed by centrifugation; the clear supernatant was loaded onto a small column containing 0·5 g DE 52 cellulose suspended in water. After washing with five column volumes of water, siderophores were eluted with 2 M ammonium chloride; the coloured fractions were combined and analysed by HPLC.
Salmochelins were prepared as described by Bister et al. (2004). Enterobactin and its degradation products were prepared according to Young & Gibson (1979)
, and further purified by preparative HPLC as described by Bister et al. (2004)
.
Construction of plasmids.
Plasmid pKHI18 was digested with BlnI and XbaI, and ligated to generate plasmid pKHI20. This treatment removed a 5·3 kb fragment encoding iroN and the C-terminal end of iroE. To generate plasmid pKHI21, plasmid pKHI18 was digested with BstBI and ligated; this removed a 2·9 kb fragment encoding the N-terminal half of iroN. Plasmid pKHI23 was generated by HpaI digestion of pKHI18 and ligation of the two fragments, the first fragment encoding the vector with the promoter region and the first 75 bp of the iroB gene, and the second fragment (7·4 kb) encoding the C-terminal end of iroC (234 bp) and iroDEN.
The primers iroDNdeI (5'-GGATGCCATATGCTGAACATGCAACAACATCC-3') and iroXhoI (5'-GGACCTCGAGTCAACTCAACCCTGTAGTAAACC-3') were used to amplify iroD from pKHI18. The 1·2 kb PCR fragment and the vector pET19b were digested with NdeI/XhoI, ligated, and introduced into E. coli DH5 by transformation. E. coli BL21(DE3) was always transformed freshly with the resulting plasmid pMZ2038 for isolation of the His-tagged IroD protein (NHis-IroD).
Primers IroEEcoR (5'-GACGAATTCAAGCCGGATATG-3') and IroEHind (5'-TGCCAAGCTTACTATGCCGGAGTTAC-3') were used to amplify the region of iroE encoding residues 33 to the C-terminus in pKHI18. The 0·9 kb fragment and the vector pMAL-p2X were digested with EcoRI/HindIII and ligated. The resulting plasmid, pKHI22, encoded the MalEIroE fusion protein with a signal sequence that allowed export of the fusion protein into the periplasm.
The iroE mutant plasmid pSP211 iroBCDN was obtained by religation of pKHI18 after BlnI/SfiI digestion, and treatment with the Klenow fragment to obtain blunt-ended DNA.
Primers PSMalr1 (5'-CAGAATTCCAACCGGACCCCGAGGCG-3') and PSMalr2 (5'-ACAAGCTTCAGCGCTGGCGTTCCACC-3') were used to amplify the pfeE region from Pseudomonas aeruginosa. The 0·9 kb fragment and the vector pMAL-p2X were digested with EcoRI /XhoI, and ligated to generate plasmid pMZ243.
Plasmid pMZ2108 was obtained by amplifying the McmK coding region from E. coli CA46 using primers MCMKxhoII (5'-GCTGTCCTCGAGTATGATACCTATGAAAAA-3') and MCMKpstI (5'-ATGCTGCTGCAGTGCTTACACAAAGTTATT-3'), digesting the 1·2 kb fragment obtained and the vector pBAD/HisB with PstI /XhoI, and ligating them.
Isolation of fusion proteins.
E. coli BL21(DE3)(pMZ2038) was grown in TY medium containing 50 µg ampicillin ml1 to a density of 5x108 cells ml1. IPTG (1 mM) was then added, and the incubation was continued for 3 h. Cells were harvested, washed in 50 mM Tris/HCl pH 8·0, 2 mM EDTA, and stored as a frozen sediment at 70 °C. Cells from 50 ml cultures were suspended in 40 ml binding buffer (5 mM imidazole, 0·5 M NaCl, 20 mM Tris/HCl, pH 7·9). After sonification, inclusion bodies and cellular debris were collected by centrifugation at 20 000 g for 15 min, and washed once in 20 ml binding buffer. The sediment was solubilized on ice in 5 ml binding buffer containing 6 M urea. After 1 h, the insoluble material was removed by centrifugation at 39 000 g for 20 min. A Ni-chelate column was loaded with the extract, and washed with 10 column volumes of binding buffer. After washing the column with six column volumes of binding buffer containing 20 mM imidazole, the protein was eluted with binding buffer containing 300 mM imidazole. Fractions were analysed by SDS-PAGE (see the supplementary figure Fig. S1 with the online journal). The purified NHis-IroD protein was dialysed against 50 mM Tris/HCl pH 7·5, 1 mM MgSO4, 10 mM DTT for 18 h to renature the protein.
Plasmids pKHI22 and pMZ243 were separately introduced into E. coli MC4100 by transformation. The transformants were grown in TY medium with 0·2 % glucose and 50 µg ampicillin ml1 to a density of about 3x108 cells ml1. IPTG (0·3 mM) was added to induce expression of the gene encoding the fusion protein. After 3 h, the cells were harvested, and the fusion protein was enriched from osmotic shock fluid, which mainly contains periplasmic proteins. The fusion protein was bound to amylose-agarose beads (New England Biolabs) and eluted with maltose as described by the manufacturer.
Determination of IroD, IroE and PfeE activities.
Freshly dialysed NHis-IroD protein was used since the protein lost its enzymic activity within 48 h. The reaction mixture contained, if not indicated otherwise, 5 µl NHis-IroD (6·5 mg ml1 in renaturation buffer), 2 µl buffer (0·2 M sodium phosphate, pH 8·0), 8 µl salmochelin (5 mg ml1, final concentration 2 mM), 5 µl water. The mixture was incubated at room temperature. After 5, 10 and 30 min, 5 µl aliquots were withdrawn, and 55 µl stop buffer (6 % acetonitrile/0·1 % trifluoroacetic acid) was added. The sample was stored on ice until a 20 µl aliquot was analysed by HPLC.
The assay mixture for IroE or PfeE activity determination contained, if not indicated otherwise, 5 µl MalEIroE (10 mg ml1) or MalEPfeE (10 mg ml1), 2 µl buffer (0·2 M sodium phosphate buffer, pH 8·0), 8 µl salmochelin (5 mg ml1), 5 µl water. The assay mixture was incubated under the same conditions as above.
For Km determinations, reaction mixtures contained salmochelin S4 (0·125, 0·25, 0·5, 1, 2 and 4 mM), 0·8 µl buffer (200 mM phosphate buffer, pH 8·0), 2 µl MalEIroE (10 mg ml1) or 2 µl IroD (6·5 mg ml1), and water up to 8 µl final volume. The mixture was incubated at room temperature for 1 min, a sample of 5 µl was withdrawn, and 55 µl stop buffer (6 % acetonitrile/0·1 % trifluoroacetic acid) was added. The samples were stored on ice until a 20 µl aliquot was analysed by HPLC.
HPLC analysis.
Samples were analysed by HPLC (Shimadzu; LC10 pumps) on a reversed-phase column (Nucleosil C18, 5 µm, 4x250 mm) using a gradient increasing from 6 to 40 % acetonitrile in water (both with 0·1 % trifluoroacetic acid) with detection at 220 nm. The substrates were purified by preparative HPLC (Shimadzu; LC8 pumps) on a reversed-phase Nucleosil 100 C18 column (20x250 mm, 7 µm; Grom) using a gradient of 6 to 40 % acetonitrile in water, both containing 0·1 % trifluoroacetic acid, over 20 min (flow rate 5 ml min1, detection at 220 nm).
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RESULTS |
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From the culture supernatants of salmochelin S4-producing strains, a compound, called S0, was isolated. S0 did not stimulate iron-dependent growth. However, biologically active salmochelin S4 was obtained from HPLC-pure salmochelin S0 by concentrating the HPLC fraction by evaporation. S0 was not cleaved by IroD (Supplementary Fig. S3), which indicated that salmochelin S0 has a structure that does not allow cleavage.
Enterobactin was also recognized by NHis-IroD and cleaved to linear oligomers of DHBS (see the supplementary figure Fig. S5 with the online journal). Fe-enterobactin was not cleaved by NHis-IroD (Supplementary Fig. S3).
IroD complements a fes mutant
Since IroD was able to cleave enterobactin, the iroD gene was tested for the ability to complement a fes mutant. E. coli AN273 fes was transformed with the plasmid pMZ2038 iroD. Colonies of E. coli AN273 fes were pink on TY plates, characteristic of enterobactin production, whereas colonies of E. coli AN273 fes(pMZ2038 iroD) were colourless, as typical of E. coli on these plates. On iron-limiting NTADE plates, enterobactin and (DHBS)3 had a strong growth-stimulating effect on E. coli AN273 fes(pMZ2038 iroD), whereas the fes mutant grew only weakly (Table 2). Salmochelin S4 caused only a very faint growth response of E. coli AN273 fes(pMZ2038 iroD), which indicated that the enterobactin transport system does not efficiently transport salmochelin S4.
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Salmochelin S4 was used as a substrate to determine the function of IroE. Analysis of the reaction mixture after 5 and 30 min by HPLC revealed two peaks in the position of salmochelins S2 and S3 (Fig. 3). The major peak was analysed by mass spectrometry and was confirmed to be salmochelin S2 (data not shown). S3 had been shown to be an oxidation product without siderophore activity (Hantke et al., 2003
). No further degradation of salmochelin S2 by IroE was observed. In addition, IroE cleaved Fe3+-salmochelin S4 and the iron-free form of salmochelin S4 with nearly the same efficiency (Fig. 3
). The Km of MalEIroE with deferri-salmochelin S4 as a substrate was approximately 2·6±0·5 mM.
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Function of IroE and IroD in iron-salmochelin uptake
S. enterica serotype Typhimurium ATCC 14028 (Bäumler et al., 1998) produces mainly the degradation products of salmochelin S4, namely salmochelins S2, S1, SX (pacifarinic acid) and DHBS. The mutant S. enterica H5547 iroD : : kan produces mainly salmochelin S4 and DHBS (Hantke et al., 2003
). This is in accordance with the observation that IroD degraded salmochelin S4 in vitro to the linear trimers, dimers and monomers. However, considering the operon organization and the phenotype of S. enterica H5547 iroD : : kan, it is highly probable that this mutation has a polar effect on iroE; this could lead to salmochelin S4 not being cleaved in the periplasm, which possibly is a prerequisite for uptake. To test these possibilities, various mutants derived from plasmid pKHI18 iroBCDEN were constructed.
In the mutant plasmid pSP211 iroBCDN, approximately 700 bp were removed from the iroE gene. E. coli K-12 MG1655 transformed with pSP211 iroBCDN grew slightly slower in iron-poor M63 minimal medium (growth yield after 24 h 0·88 mg cell dry wt ml1) than E. coli MG1655 transformed with pKHI18 iroBCDEN (growth yield after 24 h 1·13 mg cell dry wt ml1). This might indicate that the uptake of Fe3+-salmochelin S4 via IroN without degradation to Fe3+-salmochelin S2 is not very efficient. Furthermore, E. coli MG1655(pSP211 iroBCDN) produced more salmochelins S0 and S4 than E. coli MG1655(pKHI18 iroBCDEN), which produced mainly salmochelins S1 and S2 (Table 3).
The periplasmic enterobactin-binding protein FepB was tested to see whether it was necessary for salmochelin S4 uptake. E. coli AN311 fepB was transformed with the plasmids pKHI18 iroBCDEN and pSP211 iroBCDN. In minimal medium M63, strain AN311 produces mainly enterobactin and some trimeric 2,3-DHBS, which might result from enterobactin hydrolysis in the medium (Table 3). E. coli AN311 fepB(pKHI18 iroBCDEN) produced mainly the linear salmochelin S1, and the monomers salmochelin SX and DHBS, which indicated that salmochelin S4 uptake was independent of FepB. In addition, salmochelin S5, a dimer of salmochelin SX, was found (Fig. 1
), which had not been observed before in wild-type strains. Since the salmochelins were degraded to salmochelin SX and DHBS, they must have been taken up into the cytoplasm, where IroD is localized, since IroE in the periplasm degrades salmochelin only to the linear trimer salmochelin S2. Very similar results were obtained with fepC and fepD mutants (data not shown); therefore, uptake through the cytoplasmic membrane might be accomplished by IroC. In contrast, E. coli AN311 fepB(pKHI20 iroBCD) produced more undegraded salmochelins S4 and S0 than AN311 fepB(pKHI18 iroBCDEN) (Table 3
), which indicated that cleavage of salmochelin S4 to salmochelin S2 is necessary for efficient uptake.
Function of IroC in iron-salmochelin uptake
IroC had been assumed to be an exporter for salmochelin S4 because of its similarities to eukaryotic multidrug resistance (MDR) proteins (Hantke et al., 2003). The results presented above indicated that IroC might be responsible for the uptake of iron-salmochelin. A test with E. coli AN311 fepB(pKHI18 iroBCDEN) showed that salmochelin S4 as well as salmochelin S2 stimulated growth on iron-limiting medium (Table 4
). Since bacterial binding-protein-dependent ABC transporters are not functional without their cognate binding protein, it was presumed that the Fep-ABC-transporter would be inactive without FepB. Therefore, IroC was assumed to be the transporter of the salmochelins. Application of enterobactin to filter paper discs on agar seeded with E. coli H1882(pKHI18 iroBCDEN) produced a weak growth zone, which indicated that IroC allows not only the uptake of Fe3+-salmochelin S2, but also the uptake of Fe3+-(DHBS)3, the linear form of Fe3+-enterobactin (Table 5
). Strain E. coli H1882(pSP211 iroBCDN) was not able to grow with enterobactin, possibly because without IroE there is no degraded Fe3+-enterobactin in the periplasm.
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For reasons that are not understood, strains unable to produce enterobactin (e.g. those with mutations in entC, aroB, aroA) transformed with iro genes grew only poorly with various salmochelins. In addition, the strong growth response of E. coli H5687 entC (Table 5
) showed that the Fep uptake system is more efficient than the Iro proteins in the uptake of enterobactin.
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DISCUSSION |
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A third member of the Fes protein family, McmK, encoded in microcin biosynthesis operons (Patzer et al., 2003), is also able to complement Fes activity. Microcin E492 is modified by a linear enterobactin. One glucose residue bridges the C-terminal serine of the microcin peptide with the trimer of DHBS (Thomas et al., 2004
). In the maturation process of microcins E492, M and H47, an IroB-like protein (MceC or McmK) might C-glycosylate enterobactin with a glucosyl residue, which is then linked to the C-terminal serine residue of the microcin peptide (Thomas et al., 2004
). The Fes-like esterases MceD or McmK might cleave the cyclic triester of enterobactin, when bound via one glucose residue to the respective microcin peptide part. Cleavage of the cyclic enterobactin might be necessary for the export of the microcins via the TolC-coupled ABC exporter (MchEF or MceGH).
The IroE protein, isolated as a MalEIroE fusion protein from the periplasm, had a different esterase specificity. It only cleaved the cyclic seryl-triester, and did not cleave the linear forms of salmochelin. Although it also cleaved the Fe3+-chelates, it did not cleave the Ga3+-salmochelin S4, which indicated that not only the ester ring is recognized.
A BLAST search revealed several IroE-related proteins in other bacteria. A dendrogram of some selected proteins is shown in Fig. 5. The PfeE protein, with 37 % identity to IroE, is encoded downstream of the enterobactin receptor gene cluster pfeRSA in P. aeruginosa and has functions similar to those of IroE. The cloned protein cleaved salmochelin S4, enterobactin and the iron-containing salmochelin S4. Similarly, a periplasmic protein with 32 % identity to IroE is predicted downstream of bfeA, the enterobactin receptor of Bordetella parapertussis. Even in the Gram-positive bacterium Bacillus subtilis, two homologues are found. ybbA (25 % identity) is downstream of feuABC, which encodes an iron-Fur-regulated ABC transporter with unknown substrate specificity, and yuiI (28 % identity) is downstream of the entA (2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase) homologue. It is tempting to speculate that all these proteins hydrolyse cyclic, ester-linked catecholate siderophores, such as salmochelin, enterobactin and corynebactin (the cognate siderophore of B. subtilis, which also has a cyclic ester structure (Budzikiewicz et al., 1997
; May et al., 2001
)).
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However, during these studies, we observed that strains with the complete set of iro genes, which were unable to produce enterobactin owing to mutations in aroA, aroB or entC, showed no growth response or only a faint response with salmochelin S4. This might indicate that in addition to iron and Fur (Bäumler et al., 1996), there is another, unknown, level of regulation.
Based on the observations discussed above, we propose the following model of salmochelin-dependent iron uptake in E. coli (Fig. 6). Salmochelin S4 is synthesized from enterobactin by IroB, and excreted by an unknown mechanism into the medium, where it complexes Fe3+. The complex is taken up preferentially by IroN in a TonB-dependent manner (Bister et al., 2004
; Hantke et al., 2003
). In the periplasm, Fe3+-salmochelin S4 is cleaved by IroE to form the linear trimer Fe3+-salmochelin S2, which is taken up via IroC. In the cell, Fe3+-salmochelin S2 is bound by IroD, the iron is reduced and removed by an unknown mechanism, and salmochelin S2 is cleaved to the dimer salmochelin S1, the monomer salmochelin SX (pacifarinic acid) and DHBS. The degradation products either are secreted by an unknown mechanism out of the cell and might act as low-affinity siderophores for further iron uptake, or are degraded by the periplasmic copper oxidase CueO (Grass et al., 2004
), since salmochelin SX is often observed in the culture supernatant in amounts lower than expected.
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ACKNOWLEDGEMENTS |
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Received 12 January 2005;
revised 10 March 2005;
accepted 21 April 2005.
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