Fur is not the global regulator of iron uptake genes in Rhizobium leguminosarum

M. Wexler1, J. D. Todd1, O. Kolade1, D. Bellini1, A. M. Hemmings1, G. Sawers2 and A. W. B. Johnston1

1 School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK
2 John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK

Correspondence
A. W. B. Johnston
a.johnston{at}uea.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Rhizobium leguminosarum fur mutants were unaffected in Fe-dependent regulation of several operons that specify different Fe uptake systems, yet cloned R. leguminosarum fur partially corrected an Escherichia coli fur mutant and R. leguminosarum Fur protein bound to canonical fur boxes. The lack of a phenotype in fur mutants is not due to functional redundancy with Irr, another member of the Fur superfamily found in the rhizobia, since irr fur double mutants are also unaffected in Fe-responsive regulation of several operons involved in Fe uptake. Neither Irr nor Fur is needed for symbiotic N2 fixation on peas. As in Bradyrhizobium japonicum, irr mutants accumulated protoporphyrin IX. R. leguminosarum irr is not regulated by Fur and its Irr protein lacks the motif needed for haem-dependent post-translational modification that occurs in B. japonicum Irr. The similarities and differences in the Fur superfamily in the rhizobia and other Gram-negative bacteria are discussed.


Abbreviations: CAS, chrome azurol sulphonate; GFP, green fluorescent protein; PPIX, protoporphyrin IX; VB, vicibactin


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The regulation of genes involved in iron acquisition is highly conserved both within and between many bacterial taxa. Acquisition of Fe poses special challenges: it is essential, being used in many co-enzymes; very insoluble in its usual, ferric, form; and cytotoxic, catalysing the production of free radicals. Therefore genes involved in Fe acquisition or metabolism are tightly regulated in response to external Fe availability (Crosa, 1997).

Since its discovery (Hantke, 1981), one Fe-dependent regulator has attracted most attention, at least in Gram-negative bacteria. This protein, Fur (ferric uptake regulator), is a transcriptional repressor of more than 90 different genes in for example Escherichia coli and Pseudomonas aeruginosa (Hantke, 2001a). Many of these genes, such as those involved in synthesis or uptake of siderophores, are directly involved in Fe nutrition. Other genes, including those for production of exotoxin by pathogens, encode adaptive responses to Fe-deficient conditions. In its repressive mode, Fur protein attached to Fe2+ binds to motifs (fur boxes) that abut target promoters, preventing transcription. Fur also appears to positively regulate some genes (Hantke, 2001a). This, too, may be an indirect repressive effect in which E. coli Fur represses transcription of ryhB, which encodes a small RNA molecule that in turn inhibits genes whose expression is enhanced in Fe-rich conditions (Masse & Gottesman, 2002). A molecular structure for Fur of the N2-fixing bacterium Rhizobium leguminosarum biovar viciae, the microsymbiont of peas and vetches, and the subject of the present study, has recently been presented (Kolade et al., 2002).

In some Gram-positive bacteria, Fe-responsive gene regulation is mediated by members of the DtxR family, identified in Corynebacterium diphtheriae as a regulator of Fe-dependent diphtheria toxin (Boyd et al., 1990). Although Fur and DtxR have no sequence homology, their structures may have some similarities (de Peredo et al., 2001). In addition to Fur itself, there are at least three other subgroups in the Fur superfamily. PerR is an oxidative-stress-response regulator (Bsat et al., 1998) and Zur regulates genes involved in Zn uptake (Hantke, 2001b).

The fourth member of the Fur family is Irr, which occurs in bacteria known collectively as the rhizobia. These induce N2-fixing nodules on the roots of legumes and in this symbiotic state, have a very high demand for Fe, since several polypeptides made specifically in the nodule (e.g. nitrogenase and leghaemoglobin) are Fe-proteins (see Johnston et al., 2001). Irr was identified in Bradyrhizobium japonicum (which nodulates soybeans) as a transcriptional repressor of hemB, which specifies {delta}-aminolaevulinic acid dehydratase, in the haem biosynthetic pathway (Hamza et al., 1998). Irr is subject to complex post-translational modification such that it is degraded when bound to haem, which is delivered by the enzyme ferrochelatase (Qi et al., 1999; Qi & O'Brian, 2002). Irr is restricted to a few {alpha}-proteobacteria including rhizobia, Agrobacterium, the animal pathogen Brucella and Rhodopseudomonas palustris, a photosynthetic bacterium very closely related to B. japonicum. Transcription of irr is moderately repressed in Fe-replete conditions, this being dependent on a protein (FurBj) that is homologous to Fur (Hamza et al., 1999), even though the irr promoter region has no sequence similarity to canonical fur boxes (Hamza et al., 2000). Interestingly, FurBj also positively regulates hemA, which specifies {delta}-aminolaevulinic acid synthase, the first step in haem biosynthesis (Hamza et al., 2000).

These observations suggested that rhizobial Fur differs from that of other bacteria, including E. coli, in which Fur has been studied in detail. A further difference occurs in the regulation of the rhizobial hmu genes, which specify haem transporters in B. japonicum and R. leguminosarum. As in other bacteria that use haem as an Fe source, rhizobial hmu genes are repressed in Fe-replete conditions (Nienaber et al., 2001; Wexler et al., 2001). In many other bacteria, this is mediated by Fur (e.g. Ochsner et al., 2000), but fur mutants of B. japonicum and R. leguminosarum express their hmu genes normally. Also, transcription of R. leguminosarum tonB, which is required for haem utilization, was Fe-regulated but again this was unaffected by a fur mutation (Wexler et al., 2001).

The ability to use haem is unusual, being normally a property of pathogens. Therefore, the failure of rhizobial hmu genes to be Fur-regulated may not be typical of Fe uptake systems. Most strains of R. leguminosarum make vicibactin (VB), a trihydroxamate siderophore (Dilworth et al., 1998). Genes for VB synthesis and uptake were identified, as follows: fhuAF and fhuCDB specify the receptor and ABC uptake transporter for VB; the vbsC, vbsGSO and vbsADL operons specify enzymes for VB synthesis; and rpoI encodes an RNA polymerase {sigma} factor that initiates transcription of vbsGSO and vbsADL. All these operons are expressed at high levels only in Fe-depleted conditions (Carter et al., 2002; Stevens et al., 1999; Yeoman et al., 1999, 2000).

Interestingly, each of the above operons is deregulated in R. leguminosarum with mutations in rirA, a newly found gene whose product has no sequence similarity to Fur but which has close homologues in other rhizobia, Agrobacterium and Brucella (Todd et al., 2002). This further suggests novelty in Fe-responsive regulation in rhizobia. Here, we establish if Fur of R. leguminosarum (FurRl) has any properties of ‘classical’ Fur proteins and test if there is functional redundancy between Fur and Irr.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Strains, plasmids, media and growth conditions.
Bacterial strains and plasmids are listed in Table 1. Strains were grown routinely as in Beringer (1974). In high-Fe medium, FeCl3 (20 µM in liquid, 200 µM in agar) was added; low-Fe medium had no added Fe, but had 2,2'-dipyridyl (30 µM in liquid, 150 µM in agar). Other Fe sources were 10 µM haemin or 20 µM ferric citrate. Peas were assayed for N2 fixation as in Beynon et al. (1980). Chrome azurol sulphonate (CAS) plate tests for siderophores were as in Yeoman et al. (1997).


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Table 1. Strains and plasmids used in this study

 
{beta}-Galactosidase assays, expression of gfp promoter clones and protoporphyrin IX (PPIX) accumulation.
{beta}-Galactosidase assays were done essentially as in Miller (1972). Colonies were screened for green fluorescent protein (GFP) by plating on high- and low-Fe agar medium, then examining them under 365 nm UV light. Colonies were screened for PPIX by their fluorescence under 365 nm UV light on high- and low-Fe agar medium. PPIX was identified using spectrofluorimetry (Yeoman et al., 1997).

In vivo and in vitro genetic manipulations.
Plasmids were conjugated from E. coli to R. leguminosarum by triparental mating. Tnlac insertions in irr were obtained and introduced into the genome of R. leguminosarum as in Wexler et al. (2001). Sequencing used the dideoxy chain-termination method. Primers to amplify the hemA promoter region from R. leguminosarum genomic DNA and to determine the irr mRNA starts by primer extensions (Sawers & Böck, 1989) are listed in Table 2.


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Table 2. DNA primers used in this study

 
Expression and purification of Fur protein.
Cosmid pBIO929 (de Luca et al., 1998) was used as template to amplify R. leguminosarum fur, with primers shown in Table 2. The PCR fragment was cloned into the pET-21a overexpression vector to form pBIO1151, the insert being verified by DNA sequencing. This plasmid was transformed into E. coli BL21(DE3)(plysS) and the cells were grown at 37 °C to OD600 0·6–0·8, followed by induction of Fur expression at 30 °C for 3 h with 0·4 mM IPTG. Cell-free extracts were applied to an IDA metal-chelate column (Novagen) charged with 50 mM NiSO4. Binding of the protein resulted from the intrinsic metal-binding capacity of Fur. Protein, eluted with imidazole, was applied to a Superdex 75 HiLoad 16/60 HR gel filtration column (Amersham Pharmacia Biotech). The pooled, dimeric FurRl peaks were dialysed against 20 mM Tris/HCl pH 8·0, 10 mM DTT and stored at -20 °C.

Mobility shift assays.
DNA mobility shift assays, using three different DNA fragments, were performed as described by Ochsner et al. (1995), with minor modifications. One of these fragments, synthesized by MWG Biotech, was a 25 bp synthetic DNA fragment containing an E. coli fur box, labelled with fluorescein at the 5' terminus of each single-stranded oligonucleotide. The oligonucleotides were based on a 19 bp fur box sequence (Escolar et al., 1999) extended by three randomly chosen nucleotides at the 5' and 3' termini (5'-att gat aat gat aat cat tat cga c-3' and 5'-GTC GAT AAT GAT TAT CAT TAT CAA T-3'). Double-stranded DNA was prepared by mixing equal concentrations of the single-stranded oligonucleotides and heating at 90 °C for 10 min followed by cooling to room temperature over 1 h. The other two DNA fragments were (i) a 352 bp EcoRI–HindIII fragment from pVDS, which contains the Fur-regulated pvdS promoter of P. aeruginosa (Ochsner et al., 1995) and (ii) a 344 bp EcoRI–HindIII fragment containing the R. leguminosarum irr promoter, end-labelled with [35S]dATP{alpha}S (Table 2). Non-denaturing gels contained 0·2 mM Mn2+. Various concentrations (0–50 µM) of purified Fur protein were added to the fluorescein-labelled DNA (2 µM), in the presence of unlabelled, non-competitor DNA [200 µM poly(deoxyinosinic-deoxycytidylic) acid, sodium salt]. For radiolabelled fragments purified Fur (0–200 nM) was added to approximately 100 ng DNA. Following PAGE, dried gels were exposed to a Molecular Dynamics phosphor-imager screen, digitally quantified with a Storm 840 laser scanner (Molecular Dynamics) and visualized with ImageQuant software.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Fur does not regulate R. leguminosarum genes involved in iron acquisition
Iron-responsive transcription of two divergently transcribed operons, hmuPSTUV and orf1–tonB, had previously been found to be independent of the R. leguminosarum fur gene (Wexler et al., 2001). We extended the range of Fe-responsive genes studied for their response to FurRl. These genes were involved in VB uptake (fhuAF and fhuCDB), VB synthesis (vbsGSO, vbsADL and vbsC) and an ECF {sigma} factor (rpoI). Previously isolated plasmids carrying lacZ transcriptional fusions to each of these operons were mobilized into wild-type R. leguminosarum J251 and the fur mutant J325. Transconjugants, grown in high and low levels of FeCl3, were assayed for {beta}-galactosidase. In the wild-type, as expected, expression of these promoters was greater in low- than high-Fe medium; however, this was unaffected by the fur mutation, in either medium (Table 3).


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Table 3. Effect of R. leguminosarum fur mutation on expression of Fe-responsive promoters

Wild-type strain J251 and fur mutant J325 were used as recipients for various Fe-responsive lac fusion plasmids and the strains were grown in Fe-replete or Fe-deficient minimal media before assaying {beta}-galactosidase (given as Miller units, with standard errors in parentheses).

 
Transcription of rirA, which regulates many Fe-responsive genes in R. leguminosarum, is itself responsive to Fe availability, its expression being about twofold higher in Fe-replete cells than when grown in low-Fe medium (Todd et al., 2002). The rirA–lacZ fusion plasmid pBIO1439 was mobilized into J325 and the transconjugant assayed for {beta}-galactosidase. However, the fur mutation did not affect expression of rirA–lacZ in either high- or low-Fe media (Table 3). Nor did FurRl affect its own expression; a fur–lacZ fusion plasmid, pBIO939, displayed somewhat elevated {beta}-galactosidase expression in Fe-replete medium, but to the same degree in J251 and J325 (Table 3).

We had also identified an operon, sfuABC (Todd et al., 2002), which is also likely involved in Fe uptake. Its products closely resemble SfuA, SfuB and SfuC of Serratia and YfuABC of Yersinia, two similar ABC iron importers of a type normally found only in pathogens and whose expression is Fur-regulated (Angerer et al., 1992). The promoter of R. leguminosarum sfuABC was cloned in the gfp reporter plasmid pOT2, forming pBIO1378. When transferred to wild-type J251 and to J325, pBIO1378 conferred high-level expression (green UV fluorescence) in both backgrounds on -Fe but not +Fe agar (Fig. 1). Thus, sfuABC is Fe-regulated, but not via Fur.



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Fig. 1. Effect of fur and irr mutations on Fe-responsive expression of sfu–gfp and PPIX accumulation. Derivatives of wild-type (J251), fur mutant (J325) and irr fur mutant (J387) each containing sfu–gfp fusion plasmid pBIO1378 on Fe-replete (left) and Fe-depleted medium (right). Note pink UV fluorescence in J387 in Fe-replete medium due to PPIX and the green fluorescence in J251 and J325 in low-Fe medium due to GFP expression. The yellow colour in J387 in low-Fe medium is due to PPIX plus GFP.

 
Consistent with the lack of Fur-dependent regulation of all these R. leguminosarum genes, we noted that none of their promoters contained a sequence that resembled a canonical fur box.

Effects of R. leguminosarum Fur on hemA expression
In B. japonicum, transcription of the hemA gene is higher in Fe-replete than in Fe-depleted media, this regulation depending on Fur of this species (Hamza et al., 2000). We investigated the effects of FurRl on the expression of R. leguminosarum hemA, which we identified from its genome sequence (http://www.sanger.ac.uk/Projects/R_leguminosarum/). A 405 bp fragment 5' of hemA, which spanned its promoter(s), was amplified from this sequenced strain's genomic DNA using hemRlS and hemRlE primers (Table 2). The PCR product was cloned into the reporter plasmid pMP220 to form pBIO1437, which was mobilized into J251 and into J325. For both of the resultant transconjugants, hemA–lacZ expression was enhanced, about threefold, in high-Fe conditions (Table 3). Thus, expression of hemA responds to Fe, but this does not involve Fur.

R. leguminosarum Fur functions heterologously in E. coli as a ferric uptake regulator
Given its lack of effect on Fe-responsive genes, we tested if FurRl has at least some properties of Fur from {gamma}-proteobacteria. This was done both genetically and biochemically. First, its ability to correct a fur mutant of E. coli was examined. R. leguminosarum fur was cloned in pUC18, oriented to be under the control of the lac promoter (pBIO1153) or lacking a functional promoter (pBIO1154). Both plasmids were transferred into two E. coli strains containing a bfd–lacZ fusion (bfd specifies bacterioferritin-associated ferredoxin and is Fur-regulated). One strain (JRG2652) is Fur+; the other (JRG2653) has a fur deletion. {beta}-Galactosidase was assayed for each strain, grown in high- and low-Fe media with IPTG. As expected, {beta}-galactosidase expression was Fe-regulated in JRG2652, but in the fur mutant, JRG2653, Fe regulation of bfd–lacZ was absent (Table 4). However, when pBIO1153 (but not pBIO1154) was present in JRG2653, Fe-dependent regulation was partially restored. The expressed FurRl antagonized E. coli Fur in wild-type JGR2652, perhaps by forming a heterodimer with E. coli Fur, the hybrid being less effective than native E. coli Fur.


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Table 4. Effect of cloned R. leguminosarum fur gene on expression of bfd–lacZ fusion in E. coli

Derivatives of Fur+ (JRG2652) and Fur- (JRG2653) E. coli containing a plasmid in which R. leguminosarum fur was (pBIO1153) or was not (pBIO1154) expressed were grown in Fe-replete or Fe-depleted media. After growth, {beta}-galactosidase activities were determined (given as Miller units, with standard errors in parentheses).

 
R. leguminosarum Fur binds to fur boxes
We also examined the ability of FurRl to bind in vitro to a synthetic, 25 bp oligonucleotide that contained a canonical fur box (see Methods). As shown in Fig. 2, addition of FurRl protein caused a gel shift of this fragment, even in the presence of excess non-specific competitor DNA. In a similar experiment, purified FurRl retarded a radiolabelled 352 bp fragment spanning the promoter of the pvdS gene of P. aeruginosa (not shown). This gene encodes an Fe-responsive {sigma} factor whose expression is regulated via Fur, due to the presence of a fur box (Ochsner et al., 1995).



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Fig. 2. R. leguminosarum Fur binds to the E. coli fur box in vitro. Gel retardation assay using a 25 bp fluorescein-labelled DNA fragment based on a canonical E. coli fur box. Lanes 1–6 contain 0, 2, 8, 16, 25 and 50 µM of purified dimeric R. leguminosarum Fur protein.

 
Other characteristics of fur mutants
Fur- mutants of some bacteria show enhanced Mn2+ resistance (Hantke, 1987). Strains J325 and J251 were therefore grown on agar with MnCl2 (5, 10, 15, 20 mM); no difference was seen in their sensitivities. Consistent with this, mutations in R. leguminosarum which conferred resistance to Mn2+ were not in the fur gene (de Luca et al., 1998). E. coli fur mutants fail to grow on succinate as sole C source (Hantke, 1987) due to the Fur-dependent expression of the corresponding genes (see Masse & Gottesman, 2002). However, the fur mutant J325 grew normally with succinate as sole C source. Siderophore-excreting colonies exhibit orange haloes on CAS agar. The CAS halo of J325 was wild-type in appearance, consistent with its apparently normal expression of genes involved in Fe uptake and siderophore synthesis. Indeed, the fur mutation had no effect on the growth rates in liquid minimal medium containing various Fe sources (ferric citrate, haemin or low-level iron). The fur mutant nodulated and fixed N2 in pea nodules in a manner indistinguishable from the wild-type J251. Thus, FurRl is not needed for nodule development or N2 fixation, under the conditions used here.

Analysis of R. leguminosarum irr and of irr fur mutants
The lack of effect of FurRl on the expression of Fe-responsive genes in R. leguminosarum, coupled to the fact that Irr is confined to the rhizobia, raised the possibility that there may be functional redundancy between Irr and Fur, even though these proteins have non-identical functions in B. japonicum. We therefore identified irr of R. leguminosarum, with a view to obtaining a double mutant strain, defective in both Irr and Fur.

The R. leguminosarum genome sequence has one gene whose product is very similar (73 % identical) to B. japonicum Irr. A probe internal to R. leguminosarum irr was used to probe E. coli colonies harbouring a pLAFR1-based cosmid gene bank of R. leguminosarum genomic DNA (de Luca et al., 1998). A 4·2 kb fragment that included irr was subcloned from one hybridizing cosmid into pUC18 to form pBIO1340. The insert was sequenced to confirm the presence of intact irr. Interestingly, R. leguminosarum Irr has no cysteines and lacks the haem-binding pocket that is needed for degradation of Irr of B. japonicum (Qi et al., 1999). We noted that Irr of Agrobacterium, Brucella, Sinorhizobium, Mesorhizobium and one of the two Irr proteins of Rps. palustris also lacked this motif, suggesting that B. japonicum Irr may be unusual in its haem-dependent instability.

To mutate R. leguminosarum irr, the 4·2 kb EcoRI fragment from pBIO1340 was cloned into pLAFR1, to form pBIO1374, which was mutagenized with Tnlac. A mutant plasmid (pBIO1375) with an insertion 36 bp 3' of the irr translational start was isolated, with irr and lacZ of Tnlac in the same orientation. This mutation was marker-exchanged into the genome of wild-type J251 and into the fur mutant J325 to form strains J386 and J387, respectively. Having ratified (by Southern blots) the two homogenotes, we examined several of their phenotypes.

As in B. japonicum (Hamza et al., 2000), the R. leguminosarum irr mutants, J386 and J387, showed strong pink UV fluorescence (Fig. 1), consistent with accumulation of PPIX and the loss of repression of hemB. Fluorescence spectra (excited at 405 nm and emission measured at 633 nm) of J386 extracts identified the pink fluorescent compound as PPIX (not shown). Although growth of these mutants in liquid minimal media with ferric citrate, haemin or low Fe as sole Fe sources was slightly reduced compared to those of J251, on CAS agar the haloes of both mutants were indistinguishable from that of the wild-type. Strains J386 and J387 were also inoculated onto peas; both mutants nodulated and fixed N2 in a similar manner to wild-type strain J251.

Since J386 and J387 both contain an irrlacZ fusion, to assay the effects of Irr on expression of Fe-responsive genes, we used a reporter gene other than lacZ for these fusions. Accordingly, several plasmids were made, based on the wide-host-range vector pOT2, in which different Fe-responsive promoters control the gfp reporter (Table 1). These were pBIO1378 (sfu–gfp), pBIO1364 (vbsA–gfp), pBIO1369 (rpoI–gfp), pBIO1371 (tonB–gfp), pBIO1370 (hmu–gfp), pBIO1363 (vbsS–gfp) and pBIO1365 (fhuA–gfp). They were transferred into J251 (wild-type), J325 (fur) and into derivatives of strains J386 (irr) and J387 (fur irr), cured of pPH1JI (used for the marker-exchange and which confers resistance to gentamicin, as does pOT2). Transconjugants were plated on high- and low-Fe agar medium and examined under UV light. The results are illustrated in Fig. 1 with the sfu–gfp fusion plasmid pBIO1378 (note the yellow colour, due to combined fluorescence from GFP and PPIX). Fluorescence was seen only in Fe-deficient agar, confirming the Fe-mediated repression of sfu. The differential expression in high and low Fe was unaffected by mutations in fur (J325) or in irr (J386 and J387). Similar results occurred with all the other gfp fusion plasmids (not shown).

Transcription of R. leguminosarum irr
Expression of a B. japonicum irr–lacZ transcriptional fusion is less (by about 30–50 %) in cells grown in high-Fe medium (Hamza et al., 1998, 2000). To examine R. leguminosarum irr expression, we transferred pBIO1373, which contains irr–lacZ, into wild-type J251 and into J325 and assayed {beta}-galactosidase activity after growth in different Fe conditions (Table 3). In both recipients, expression of irr–lacZ was enhanced, by about 20 %, under low-Fe conditions. Essentially the same results were obtained when the irr–lacZ fusion was present in the fur mutant, J325. Thus, in R. leguminosarum, unlike B. japonicum, the modest, Fe-dependent effect on irr expression is not mediated via Fur. Consistent with this, purified FurRl did not bind in vitro to a 344 bp PCR fragment spanning the irr promoter region, which had been mapped by primer extension (not shown). Also, there is no sequence near the R. leguminosarum irr promoter region that resembles the B. japonicum Fur-binding site (Hamza et al., 2000).

Genomic analyses of the Fur superfamily in relation to rhizobial homologues
Given the unusual nature of the rhizobial Fur regulator, we determined the sequence relatedness (using CLUSTAL W) among members of Fur superfamily (Fur, Irr, Zur and PerR) in the rhizobia and their very close relatives Brucella and Agrobacterium. These sequences were compared with a selection of members of the Fur superfamily in other bacterial taxa. The deduced proteomes of Agrobacterium, Bradyrhizobium, Brucella, Rhizobium and Sinorhizobium all contained one protein whose sequence clearly placed them in the Fur (in sensu stricto) clade of the Fur superfamily. The Fur-like proteins of these genera were closely related to each other and were all within the branch that included the ratified Fur proteins of (e.g.) E. coli and Pseudomonas. However, the deduced proteome of Mesorhizobium loti has no protein whose sequence places it within this Fur clade, indicating that it can survive with no Fur-like protein whatsoever.

The analysis confirmed that proteins most closely related to the canonical Irr of B. japonicum were confined to the rhizobia and their very close relatives, though it was noted that both Brucella and Rps. palustris (a very near neighbour of Bradyrhizobium) both have two Irr-like proteins in their proteome. All the rhizobia also contain a single protein that is closely related to Zur, the Zn-responsive regulator of E. coli, but none has a PerR-like protein.

Conclusions
The results presented here and in studies on Fur of B. japonicum (Nienaber et al., 2001) show that the regulation of many Fe-responsive genes in the rhizobia is not mediated by Fur. Further, none of the transcripts examined here contained sequences that resembled fur boxes, nor did we find such cis-acting sequences in the expected regions (i.e. 5' of putative operons) anywhere in the genome of R. leguminosarum. It may be that FurRl recognizes different regulatory DNA motifs, as appears to be the case in B. japonicum (Hamza et al., 1999).

Fur proteins of rhizobia have some properties attributed to ‘classical’ Fur proteins. FurBj (Hamza et al., 1999) and FurRl (this work) both bind to canonical Fur boxes in a metal-dependent way and, as shown here, a fur mutant of E. coli was partially corrected by the cloned fur of R. leguminosarum. The lack of an expected phenotype in fur mutants is not due to functional redundancy between Fur and Irr, judged from the phenotype of irr fur double mutants.

It seems that, instead of Fur, Rhizobium uses RirA as a regulator of Fe-responsive operons. Excepting hemA, all the genes described here are deregulated in rirA strains of R. leguminosarum but the mechanisms involved are unknown (Todd et al., 2002). RirA has not been studied in bacteria other than R. leguminosarum, but close homologues occur in the proteomes only of its very close relatives (Sinorhizobium, Mesorhizobium, Agrobacterium, Brucella), suggesting that its role as a regulator may be confined to a very small group of bacteria.

Future studies, perhaps using transcriptomics or proteomics, may be needed to determine if R. leguminosarum Fur regulates any genes in this species and if so, whether iron, or some other molecule, is the co-repressor molecule.


   ACKNOWLEDGEMENTS
 
The work was funded by the UK BBSRC. We thank P. Doughty and S. Andrews for strains, C. Punginelli for technical assistance, and B. Emerson for phylogenetic analyses.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 18 November 2002; revised 21 January 2003; accepted 28 January 2003.