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Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331-2902, USA
Correspondence
Daniel J. Arp
arpd{at}science.oregonstate.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Ammonia catabolism requires many Fe-containing proteins (Hooper et al., 1972
). Iron acquisition by N. europaea is supported by approximately 90 genes that have high similarity to Fe uptake genes in other organisms; these genes include approximately 29 genes with similarity to TonB-dependent outer-membrane (OM) siderophore transporter genes (Chain et al., 2003; Wei et al., 2006). Genomics analysis suggests that N. europaea has the potential to take up a broad spectrum of siderophores (Wei et al., 2006). However, with the exception of genes for citrate synthesis, the N. europaea genome has no predicted genes for siderophore biosynthesis (Chain et al., 2003). Although N. europaea has a greater Fe requirement than Escherichia coli and Pseudomonas species, under Fe-limiting conditions without added siderophores, N. europaea grows moderately well by assimilating most of the Fe present in the culture medium (Wei et al., 2006). However, the cells grown under Fe-limited conditions have lower haem and cellular Fe contents and fewer membrane layers than cells grown under Fe-replete conditions, and they upregulate the production of several Fe siderophore receptors and diffusion proteins (Wei et al., 2006).
There are several processes for Fe acquisition that are tightly regulated (reviewed by Braun & Killmann, 1999; Clarke et al., 2001; Faraldo-Gomez & Sansom, 2003). Under Fe-replete conditions, the ferric uptake regulator (Fur, with Fe2+ as a co-factor) represses the expression of Fe uptake systems, while Fe limitation leads to derepression (Braun et al., 2003; Escolar et al., 1999; Visca et al., 2002). There are additional levels of regulation, such as those through FecI–FecR–FecA (ECF 
-factor–anti--factor–OM-transducer) (Mahren et al., 2005). One main avenue for Fe acquisition is via siderophore uptake systems. Siderophores are low-molecular-mass, Fe-chelating compounds that micro-organisms produce and secrete in low-Fe environments to sequester Fe. The Fe-loaded siderophores are then recaptured by the micro-organisms via OM siderophore transporters (Braun & Killmann, 1999; Neilands, 1995). In some cases, siderophores produced by one micro-organism can be taken up by another. For instance, Arthrobacter flavescens is able to take up siderophores made by soil fungi and bacteria (Winkelmann, 1991), and the non-siderophore-producing Pseudomonas fragi uses exogenous siderophores, such as ferrioxamine, enterobactin and some pyoverdines, for its Fe nutrition (Champomier-Verges et al., 1996). Exogenous siderophores, such as cepabactin and coprogen, promote Fe uptake by Pseudomonas aeruginosa PAO1, while others, such as ferrichrome A, have no effect on its Fe nutrition, indicating the requirement for specific siderophore transporters (Meyer, 1992). The hydroxamate siderophore ferrioxamine [also known as desferrioxamine (DFX) for the Fe-free form] is of particular interest because it is produced by many soil bacteria, such as Erwinia, Nocardia, Streptomyces, Arthrobacter, Chromobacterium and Pseudomonas species (Berner et al., 1988; Gunter et al., 1993; Meyer & Abdallah, 1980; Muller & Raymond, 1984; and references therein), and it is used by many other bacteria for Fe uptake (Deiss et al., 1998; Kingsley et al., 1999). In its predominant habitats (soils and wastewaters), with low available Fe levels, N. europaea is likely to encounter and use ferrioxamine for its Fe requirements.
A major challenge to the characterization of the function and regulation of the OM siderophore transporters in N. europaea is the large number of such genes, and their deduced functional redundancy (Chain et al., 2003; Wei et al., 2006). For example, multiple genes are predicted to encode OM ferrichrome (hydroxamate-type) transporters, catechol-type siderophore transporters, and OM transporters for other siderophores. However, of the hydroxamate-type siderophore transporters in N. europaea, only NE1097 and NE1088 putatively code for OM ferrioxamine transporters. These two genes are located in one of the two islands on the N. europaea chromosome where Fe-transport-related genes are concentrated (Chain et al., 2003; Wei et al., 2006). NE1097 is more similar than NE1088 to the well-characterized foxA genes of other bacteria. Thus, in this study, NE1097 is designated foxA1, and NE1088 as foxA2 (and the corresponding proteins are designated FoxA1 and FoxA2). In other bacteria, ferrioxamine transporters and ferrichrome transporters cannot substitute for each other (Gaspar et al., 1999; Killmann & Braun, 1998), thereby allowing biochemical and genetic characterization of one type of hydroxamate-type siderophore transporter, without interference from another.
This study characterized the role of OM Fe-ferrioxamine transporters in Fe acquisition in N. europaea through both physiological and genetic approaches. Mutants of N. europaea lacking functional ferrioxamine transporter genes lost the ability to grow in media containing excess DFX (all Fe being chelated in ferrioxamine). This study provides evidence for the functionality of two of the many predicted Fe-acquisition genes in N. europaea, and support for the view that this bacterium can acquire Fe siderophores secreted by other microbes in natural environments.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Stein & Arp, 1998). The standard (Fe-replete) medium contained 10 µM Fe3+ (FeCl3) complexed with EDTA to prevent Fe from precipitation. Fe-limited medium was made from reagent-grade chemicals, without addition of any Fe salt, and contained 0.2 µM Fe (Wei et al., 2006). All media, buffers and other reagents were made in double-deionized water. All glassware was soaked in 1 % HNO3 overnight, and then rinsed thoroughly with double-deionized water. N. europaea cultures were grown at 30 °C on a rotary shaker, and late-exponential-phase cells were collected by centrifugation and thorough washes for the analyses. 
concentrations in N. europaea cultures were determined colorimetrically by the Griess reagent (Hageman & Hucklesby, 1971), and were used as a second indicator for the growth rates and growth phases, since the accumulation of 
is consistently proportional to the increase in cell mass during growth. Citric acid and Fe-free ferrichrome (desferrichrome, DFC) and Desferal (deferoxamine/DFX mesylate) were purchased from Sigma. No siderophores pre-loaded with Fe were used in this study. Siderophores were dissolved in double-deionized water, filter-sterilized, and added to Fe-limited medium, or to media with specified Fe concentrations, in the siderophore feeding experiments. The generic siderophore name ferrioxamine is also used to describe a medium or a culture containing both Fe and desferrioxamine (often in excess), and Fe-ferrioxamine (Fe-DFX) is used to refer to the Fe-loaded form, when necessary. In this study 10 µM DFX was used to ensure the complete chelation of Fe (0.2 µM) in the Fe-limited medium. In the media that contained other metals, and whose pH changed at different culture phases as a result of 
accumulation, complete chelation of Fe appeared to require at least fivefold DFX (1.0 µM) relative to the Fe level (0.2 µM) in the regular Fe-limited medium, as indicated by the complete inhibition of the growth of the double ferrioxamine transporter mutant (see below).
Determination of Fe and haem contents.
Total Fe contents in thoroughly washed N. europaea cells were determined by the ferrozine assay following HNO3 (5 %) digestion of cells at 100 °C (Carter, 1971). Fe concentrations below 1 µM were determined by inductively coupled plasma mass spectrometry (ICP-MS), as described (Houk, 1994). Preparation of a cell-soluble fraction, and determination of haem contents following extraction with pyridine, were done as described (Berry & Trumpower, 1987; Wei et al., 2006).
59Fe siderophore complex uptake experiments.
Isotopic iron (59Fe) in ferric chloride form [specific activity: >5 Ci g–1 (185 GBq g–1)] was purchased from Perkins-Elmer Life and Analytical Sciences. 59Fe was chelated to DFX by mixing it with DFX in double-deionized water (adjusted to pH 6.0). Cultures (OD600 approx. 0.02) of the wild-type (in Fe-limited medium, with and without DFX) and the double mutant (in Fe-limited medium) were divided into treatment and control, and tested for the uptake of 59Fe-DFX. To the control culture (for non-specific binding of 59Fe), 200 µM allylthiourea (an inhibitor of ammonia monooxygenase activity) and 3 mM NaN3 were added before the addition of 59Fe-DFX (concentrations specified in Results). Fractions of the cultures were taken over a time course, and filtered through a 0.45 µm type HA filter (Millipore) to separate cells from unincorporated 59Fe-DFX. The cells retained on the filters were washed with 5 ml 10 mM sodium citrate solution (pH 7), followed by 20 ml double-deionized water. The radioactivity in the cells attached to the filters was measured by liquid scintillation counting.
RNA extraction and real-time RT-PCR.
RNA extraction was conducted as described (Wei et al., 2004). RNA samples were treated with DNase (Ambion), and then quantified by A260 measurements using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). The levels of gene expression (mRNA) in late-exponential-phase cells were determined by real-time (quantitative) RT-PCR (qRT-PCR) in a MyiQ real-time PCR system, with a SYBR-Green-I-based detection kit (Bio-Rad), as described by the manufacturer; the relative expression levels for the genes tested were calculated according to the 
CT method (Livak & Schmittgen, 2001; Pfaffl, 2001), using the N. europaea 16S rRNA as a reference. All primers (Table 1) for qRT-PCR were designed to produce PCR products of approximately 150 bp, with an annealing temperature at 59 °C.
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). The ferrioxamine transporter genes foxA1 and foxA2 (Fig. 1a) were amplified by PCR using Taq DNA polymerase (Promega) on an iCycler Thermal Cycler (Bio-Rad), as described by the manufacturers (see Table 1
for primers). The resulting DNA fragments were cloned into the pGEM-T Easy vector (Promega) to form two clones. A gentamicin-resistance cassette (from pUCGM; Schweizer, 1993) was inserted into the HindIII site of the ORF of foxA1 (Fig. 1b). For foxA2, a 540 bp EcoRV segment was deleted from the ORF. A kanamycin-resistance cassette (Kovach et al., 1995) was end-filled, and then ligated into the foxA2 ORF (Fig. 1b). The plasmid constructs with the insertions were introduced back into the N. europaea wild-type cells by electroporation on the ElectroPorator (Invitrogen) at 1300 V, with a capacitance at 50 µF, and a load resistance at 500 
. Successful transformants were selected in liquid medium using gentamicin sulfate (15 µg ml–1) or kanamycin sulfate (20 µg ml–1). Aliquots from these cultures were streaked onto Nylon disk membranes, which were placed on semi-solid plates, to isolate clonal mutant strains, as described (Hommes et al., 1996). The double-knockout mutant was obtained similarly by using the single mutant strains as recipient cells for electroporation, selected in the presence of both kanamycin and gentamicin. All mutants were verified by Southern analysis (Fig. 1c, and Results). Southern blotting, labelling of DNA probes, hybridization and imaging were done as described previously (Wei et al., 2004).
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). Briefly, cells were broken by ultrasonication, the sonicated material was centrifuged at 1500 g for 1 min to pellet unlysed cells, and the top phase (cell lysate) was transferred to ultracentrifuge tubes. Crude total membranes were collected by ultracentrifugation of the cell lysates, and washed thoroughly by homogenization in Tris buffer (0.1 M, pH 7.8) containing 1 M KCl. Total membranes were collected again by ultracentrifugation, and resuspended in Tris buffer (50 mM, pH 7.8). Protein contents in whole cells and cell fractions were estimated by using the Micro BCA Protein Assay kit (Pierce), and BSA was used as a protein standard. The peptide composition of cell membranes was analysed using SDS-PAGE [with 12 % (w/v) acrylamide in resolving gels], as described previously (Hyman & Arp, 1993). HPLC tandem MS (HPLC/MS/MS) analyses and identification of proteins were performed as previously described (Wei et al., 2006). |
RESULTS |
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), suggesting that the ferrioxamine uptake system in N. europaea requires the presence of the siderophore for its induction. We tested whether the lag phase would be shortened by using cells grown in the presence of ferrioxamine as the inoculum (0.2 µM Fe, 10 µM DFX). The lag phase was shorter when the inoculum used was grown in medium containing ferrioxamine than when the inoculum was grown in Fe-limited medium in the absence of ferrioxamine (Fig. 2). The lag phase of the culture inoculated with ferrioxamine-exposed cells was comparable to that of the ferrioxamine-free Fe-limited culture (Wei et al., 2006).
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The total cellular Fe content [14.7±0.68 nmol (mg protein)–1] in cells grown in DFX-containing Fe-limited medium was similar to that in cells grown in DFX-free Fe-limited medium [13.6±2.2 nmol (mg protein)–1; Wei et al., 2006]. The haem content in the soluble fraction of the N. europaea cells cultured in Fe-limited DFX-containing medium was also similar to that in DFX-free Fe-limited medium [3.04±0.03 versus 2.87±0.71 nmol (mg protein)–1; Wei et al., 2006]. These results indicate that N. europaea can take up and process ferrioxamine-chelated Fe. Furthermore, in the presence of DFX, cells took up about 90 % of the Fe in the Fe-limited medium, compared with 70 % in the absence of DFX (Wei et al., 2006).
Ferrioxamine induction of foxA1 and foxA2 expression, and of FoxA protein production
Induction of the ferrioxamine uptake system was further confirmed by the elevated mRNA levels of foxA1 and foxA2 in cells grown in ferrioxamine-containing medium (Table 2). The mRNA levels of foxA1 and foxA2 increased about twofold by Fe limitation alone, but the inclusion of ferrioxamine resulted in additional eight- and sixfold increases, respectively, in mRNA level. The expression levels of foxA1 were higher than those of foxA2 under all three growth conditions (Table 2). Clearly, the expression of foxA1 and foxA2 was highly induced by the presence of ferrioxamine.
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) were uniquely observed in the cells cultured in ferrioxamine-containing medium, compared with cells grown in Fe-limited medium (lane 2, Fig. 3) and those grown in ferrichrome-containing medium (lane 4, Fig. 3). The identities of the membrane proteins differentially expressed under Fe-limited ferrioxamine-supplemented conditions were determined by HPLC/MS/MS analyses. Given that the predicted molecular masses of the two mature proteins (minus signal peptides) FoxA1 and FoxA2 are 87.4 and 86.3 kDa, band 4 in lane 3 (Fig. 3) was a good candidate to contain these proteins. Indeed, band 4 contained two proteins identified as the products of foxA1 (NE1097) and foxA2 (NE1088) that are homologous to the foxA of other bacteria (Bell et al., 2004; Killmann & Braun, 1998). The Mascot (www.matrixscience.com) scores for FoxA1 and FoxA2 obtained from this protein sample were 3310 and 670 respectively, both being greatly above the peptide Mascot cutoff value of 30. FoxA1 and FoxA2 were highly produced in cells grown in the ferrioxamine-containing Fe-limited medium, and not in the cells grown in Fe-limited medium containing ferrichrome (Fig. 3, lane 4) or citrate (not shown). These results indicate that production of OM ferrioxamine transducers requires ferrioxamine, consistent with the elevated foxA1 and foxA2 mRNA levels detected in cells grown in ferrioxamine-containing medium (Table 2), and suggest a role for FoxA1 and FoxA2 in ferrioxamine uptake. Ferrioxamine-exposed cells also shared two major protein bands with cells grown in siderophore-free and ferrichrome-containing media (bands 5 and 6, Fig. 3). These two bands contained the same siderophore transporters (Wei et al., 2006), except that an additional OM siderophore transducer encoded by NE2433 was detected in the ferrioxamine-exposed cells (data not shown).
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; Kingsley et al., 1999), while FoxA2 has 32 % aa identity (51 % similarity) to the S. enterica FoxA. FoxA1 and FoxA2 have, respectively, 33 and 31 % aa identity to the Yersinia enterocolitica FoxA (Baumler & Hantke, 1992), which is highly similar to another characterized FoxA in Erwinia herbicola (Berner & Winkelmann, 1990; Deiss et al., 1998). FoxA1 and FoxA2 share a 46 % aa identity (61 % similarity) with each other. Interestingly, the primary sequences of FoxA1 and FoxA2 are typical of OM transducers (with an extended N terminus). Thus, FoxA1 and FoxA2 are about 100 aa longer than the OM ferrioxamine receptors in the enteric species mentioned above. Both FoxA1 and FoxA2 have a 36 % aa identity to a transducer-type FoxA recently reported in P. aeruginosa (Llamas et al., 2006).
Constructs of foxA1 and foxA2 with antibiotic-resistance cassette inserts (Fig. 1b) were electroporated into the N. europaea wild-type cells. Both single and double insertional mutants were obtained through homologous recombination, and were verified by Southern hybridizations (Fig. 1c). The gentamicin-cassette probe detected the band corresponding to the 3.66 kb (the calculated size based on the DNA sequences) BglI–EcoRI fragment in the single mutant foxA1 : : Gm (NE1097 : : Gm) and in the double mutant (foxA1 : : Gm/foxA2 : : Km), as detected with the foxA1 probe (not shown). The kanamycin-cassette probe detected the same 3.31 kb (the calculated size based on the DNA sequences) EcoRI–ApaI fragment in single mutant foxA2 : : Km (NE1088 : : Km) and in the double mutant, as detected with the foxA2 probe (not shown). These results confirm that a single copy of antibiotic-resistance cassette was correctly inserted in each targeted gene in the whole genome. In addition, weak hybridization bands were observed because foxA1 and foxA2 have regions of high nucleotide sequence homology (approx. 550 bp at the 5' end having approx. 88 % nucleotide identity to each other). These bands (Fig. 1c, upper low-intensity bands) with correct predicted fragment sizes (refer to Fig. 1b, c, and legend) further confirmed that only the targeted gene had an insert in the two single mutants, while both genes had an insert in the double mutant, and no undesired recombination occurred between the two genes.
Characterization of these ferrioxamine transporter mutants showed that, as predicted, the double mutant could not grow in Fe-limited (0.2 µM) medium containing DFX at 10 µM, nor could it grow in DFX-containing medium with increased Fe (1.2 µM) (Fe-sequestered). Surprisingly, the single mutant with a disrupted foxA1 had the same phenotype as the double mutant (Fig. 4), suggesting that this transporter (FoxA1) has an essential role in the Fe-ferrioxamine-acquisition system. Growth of the single mutant foxA2 : : Km was tested in 10 µM DFX-containing medium with 0.2, 0.4 and 1.2 µM Fe. Little growth was observed after 8 days' incubation with Fe at 0.2 µM (Fig. 4). When Fe was at approximately 0.4 µM, the growth rate and cell mass after 10 days' incubation (data not shown) were less than half of those of the wild-type grown in Fe-limited (0.2 µM) medium with 10 µM DFX. Interestingly, when the Fe (Fe-ferrioxamine) concentration was raised to 1.2 µM, the growth rate and final cell mass (Fig. 4) were comparable to those for the wild-type grown in medium with 0.2 µM Fe and 10 µM DFX (Fig. 2).
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Uptake of 59Fe-ferrioxamine by N. europaea wild-type and mutants
Late-exponential-phase cultures of N. europaea wild-type grown in Fe-limited DFX-containing medium were fed with 59Fe (0.1 µM) and DFX (10 µM). During a 4 h incubation, DFX-induced N. europaea wild-type cells could take up 59Fe-DFX, and they accumulated 28 pmol 59Fe (ml cells)–1 or 28 % of the total added 59Fe. Cells cultured in DFX-free Fe-limited medium (i.e. not induced with DFX) did not take up 59Fe-DFX during a 21 h incubation, but did take up approximately 50 % of the free 59Fe (0.1 µM, without DFX). These results are consistent with the growth patterns and the requirement of a 3-day induction for non-DFX-induced cells to grow on DFX-chelated Fe (Fig. 2). As expected, the double ferrioxamine transporter mutant failed to accumulate 59Fe in a 3-day incubation after cells were transferred from Fe-limited medium to the same medium amended with 2 µM 59Fe and 10 µM DFX.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Second, the proteins FoxA1 and FoxA2 were produced only in the cells grown in ferrioxamine-containing medium (Fig. 3). Third, inactivation of foxA1 and foxA2 abolished the uptake of Fe-DFX for cell growth (59Fe feeding, and Fig. 4). This study is the first to confirm the functionality of two of the many siderophore transporters in N. europaea.
Addition of the siderophore ferrioxamine or ferrichrome to Fe-limited medium led to increased N. europaea growth (total cell mass), indicating that both siderophores can be used for efficient Fe uptake (Wei et al., 2006). However, these two siderophores reveal two different regulatory responses used by N. europaea cultured under low Fe and in the presence of these siderophores. N. europaea cultured in ferrioxamine-containing medium showed a prolonged lag phase, indicating that cells initially could not take up Fe-ferrioxamine. The significant reduction of the lag phase by exposure of the inoculum to ferrioxamine (Fig. 2) indicated an induction mechanism. In contrast to the ferrioxamine, addition of excess ferrichrome to Fe-limited medium did not prolong the lag phase (Wei et al., 2006), which is consistent with the observation that one ferrichrome transporter (a receptor, encoded by NE1089) is already highly expressed in cells grown in ferrichrome-free Fe-limited medium (Wei et al., 2006).
Our results also reveal the specificity of the siderophores towards their transporters. Both ferrioxamine and ferrichrome are hydroxamate-type siderophores, and ferrioxamine transporter genes and ferrichrome transporter genes of N. europaea are phylogenetically close (data not shown). Nonetheless ferrichrome did not induce the expression of OM ferrioxamine transporters in N. europaea (Fig. 3), and this indicates a high specificity of each transporter to its corresponding siderophore, and hence the specificity for the regulation pathways involving particular and anti- factors. The failure of the double mutant to grow in media where all Fe was chelated by DFX also confirmed that none of the other predicted OM siderophore transporter genes, including transducer gene NE2433 that was uniquely expressed in ferrioxamine-grown cells, could compensate for the inactivated foxA1 and foxA2.
Induction (or autoregulation) of the expression of OM siderophore transporters by their binding of specific siderophores has been observed in other bacteria (e.g. Braun, 1997; Kim et al., 1997; Llamas et al., 2006), and typically involves a regulatory pathway with and anti- factors. OM transducers are those transporters that have N-terminal extensions that interact with anti- factor (FecR-type) that activates the factor (FecI-type), which turns on the transcription of the OM transducer genes (Braun et al., 2003; Schalk et al., 2004). Both N. europaea FoxA1 and FoxA2 have an N-terminal extension typical of OM transducers. Therefore, the N. europaea ferrioxamine uptake system is likely to be subjected to dual regulations: global regulation by available Fe levels through Fur, and specific regulation by available ferrioxamine through /anti- factors; our results (Fig. 3, Table 2) indicate that the latter plays a major role. However, only foxA1 has cognate and anti- factor genes (Fig. 1a), similar to the fecI–fecR–fecA gene cluster in E. coli (Mahren & Braun, 2003; Visca et al., 2002).
The phenotypes of the two single mutants described above (Fig. 4) point to possible different roles of FoxA1 and FoxA2 in the transport of ferrioxamine across the OM, and possibly in signal transduction. First, one may conclude that FoxA2 is simply nonfunctional, since the single mutant foxA1 : : Gm (with a functional foxA2) failed to grow when all the Fe was chelated in ferrioxamine (Fig. 4). However, the other single mutant foxA2 : : Km (functional foxA1) could not grow at the same rate as the wild-type in ferrioxamine-containing Fe-limited medium, indicating a role of FoxA2 in Fe-ferrioxamine uptake. An alternative explanation could be that FoxA2 is functional for transporting Fe-ferrioxamine, but that it cannot activate the signal transduction pathway for its own expression when foxA1 is nonfunctional (see below). Second, qRT-PCR results (Table 2) suggest that foxA1 is expressed constitutively at low levels under Fe limitation in the absence of ferrioxamine, probably for the initial sensing of the siderophore. Therefore, both FoxA1 and FoxA2 could be functional for transporting Fe-ferrioxamine in the wild-type cells, and FoxA2 might be even more efficient given that FoxA1 could not fully compensate for the loss of FoxA2 at low Fe-ferrioxamine concentrations (Fig. 4).
A number of possible mechanisms can be explored to explain the roles and regulation of the N. europaea FoxA1 and FoxA2 by using the extensive studies done in model species such as E. coli. Our results show that FoxA1 is essential for the ferrioxamine uptake, but it may be an inefficient transporter since the foxA2-disrupted mutant could barely grow in medium with 10 µM DFX and 0.2 µM Fe (Fig. 4). One possible reason for the lower efficiency of FoxA1 in Fe-ferrioxamine uptake could be different specificities of FoxA1 and FoxA2 to different types of ferrioxamine. Several types of ferrioxamine such as B, E and G have been described, and different subspecies of S. enterica use different types of ferrioxamine (Kingsley et al., 1999). Another possibility for the inefficiency of FoxA1 for transporting Fe-ferrioxamine may be that it binds DFX (the unloaded form) more tightly than FoxA2 does, and thus requires a higher ratio of Fe-loaded ferrioxamine to the total added DFX than FoxA2 for effective transport of Fe-loaded ferrioxamine (Fig. 4). Binding of Fe-free siderophores by OM receptors has been observed in E. coli and P. aeruginosa (Hoegy et al., 2005; Stintzi et al., 2000; Yue et al., 2003). The presence of excess Fe-free siderophores might interfere with the transport of Fe-loaded siderophores (Schalk et al., 2001), and this may be the case with N. europaea FoxA1. Comparison of the rates and efficiency of Fe-loaded ferrioxamine uptake cannot be made between the two single mutants because the foxA1-disrupted mutant cannot grow in medium in which all Fe is sequestered by ferrioxamine.
The mRNA level of foxA2 under the Fe-limited DFX-free condition was similar to that under Fe-replete condition (Table 2), i.e. foxA2 was not induced by Fe limitation alone, suggesting that its expression requires a specific factor. It is also possible that FoxA2 is not produced at initial growth stages, which means that foxA2 alone cannot initiate the uptake of Fe-DFX complex, and thus cell growth. But RT-PCR detected foxA2 mRNA in wild-type cells at early growth phase (OD600 0.009) (data not shown). Thus, the results described above suggest that only FoxA1 could turn on the expression of both foxA1 and foxA2, probably through the interaction of its N-terminal extension with an anti- factor that in turn activates a particular factor. This hypothesis is supported by protein sequence analysis. Alignment of the N-terminal extensions shows that out of the 14 conserved amino acid residues that are believed to be involved in the interaction with anti- factors (Schalk et al., 2004), four are different in FoxA2, compared with 13/14 that are conserved in FoxA1 (Fig. 5). It is worth noting that these 14 AA residues are well conserved in eight different OM transducers across different species (Schalk et al., 2004), and in N. europaea FoxA1, but not as well conserved in FoxA2. This difference might explain why FoxA2 could not initiate the expression of the Fe-ferrioxamine uptake system even though it is required for the efficient uptake of Fe-ferrioxamine. The nucleotide sequences of the putative promoter regions of foxA1 and foxA2 (predicted with the program at http://www.fruitfly.org/seq_tools/promoter.html) are 84 % identical, suggesting the possibility of interaction with the same factor (the one activated by FoxA1). Future studies using techniques such as site-directed mutagenesis and the bacterial two-hybrid system could help to dissect the specific interactions among components of the ferrioxamine and other siderophore uptake systems in N. europaea.
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ACKNOWLEDGEMENTS |
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Edited by: R. van Spanning
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Received 13 June 2007;
revised 15 August 2007;
accepted 16 August 2007.
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, inoculated with cells not exposed to ferrioxamine; 
, inoculated with cells previously exposed to ferrioxamine. The two inoculum cultures were at a similar growth stage, and equal inoculum amounts were used (normalized by OD600). Data shown are means of triplicates, with variation less than 10 % among the triplicates. The experiment was repeated several times, and produced similar results or the same trend.
, single mutant NE1097 : : Gm in medium with 1.2 µM Fe and 10 µM DFX; 