The Fur-like protein Mur of Rhizobium leguminosarum is a Mn2+-responsive transcriptional regulator

E. Díaz-Mireles1, M. Wexler1, G. Sawers2, D. Bellini1, J. D. Todd1 and A. W. B. Johnston1

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

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In wild-type Rhizobium leguminosarum, the sitABCD operon specifies a Mn2+ transporter whose expression is severely reduced in cells grown in the presence of this metal. Mutations in the R. leguminosarum gene, mur (manganese uptake regulator), whose product resembles the Fur transcriptional regulator, cause high-level expression of sitABCD in the presence of Mn2+. In gel-shift mobility assays, purified R. leguminosarum Mur protein bound to at least two regions near the sitABCD promoter region, although this DNA has no conventional consensus Fur-binding sequences (fur boxes). Thus, in contrast to {gamma}-proteobacteria, where Fur binds Fe2+, the R. leguminosarum Fur homologue, Mur, act as a Mn2-responsive transcriptional regulator.


Abbreviations: DP, 2,2-dipyridyl; EDDHA, ethylenediamine di(o-hydroxyphenylacetic acid); MRS, Mur-responsive sequence; mur, manganese uptake regulator


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fur is a widely distributed bacterial transcriptional regulator that affects expression of many genes in response to iron availability (see review by Andrews et al., 2003). Fur has been studied in detail in enteric bacteria, Pseudomonas and, to a lesser extent, in other bacteria, including Gram-positives such as Bacillus (Baichoo et al., 2002). A structure of Fur from Pseudomonas aeruginosa was recently presented (Pohl et al., 2003), as was that of a Fur-like protein of Rhizobium leguminosarum (Kolade et al., 2002), the subject of the present study. When complexed with Fe2+ iron, Fur binds to fur boxes, which overlap promoters of the ‘target’ genes, preventing transcription in iron-replete conditions (Andrews et al., 2003). Many Fur-repressed genes have a role in iron uptake, but others are less obviously connected with bacterial ‘iron biology’ (see McHugh et al., 2003). Some operons appear to be activated by Fur, but in at least some cases (e.g. E. coli sdh genes for succinate dehydrogenase), this ‘induction’ is due to Fur-dependent repression of ryhB, which specifies a small RNA that prevents expression of the sdh target gene (Massé & Gottesman, 2002).

The Fur superfamily comprises proteins with similar, but distinct regulatory roles (Escolar et al., 1999). In addition to Fur itself, other members are Zur, which responds to zinc, repressing genes involved in Zn2+ uptake (Gaballa et al., 2002; Hantke, 2001) and PerR, which, in Gram-positives such as Bacillus and Staphylococcus, regulates several genes involved in the oxidative-stress response (Bsat et al., 1998; Horsburgh et al., 2002). Depending on the particular promoter, PerR-dependent repression is mediated by Fe2+ or Mn2+ (Fuangthong et al., 2002).

A fourth member of the Fur family is Irr, found first in Bradyrhizobium japonicum, a symbiotic bacterium that forms N2-fixing nodules on soybeans (Hamza et al., 1998). Irr represses hemB, which is involved in haem biosynthesis, via a complex post-translational mechanism mediated by haem availability (Qi & O'Brian, 2002). Irr also affects expression of hemB in R. leguminosarum, which nodulates peas, clover and beans (Wexler et al., 2003).

R. leguminosarum has a close homologue of Fur which, unusually, does not regulate iron-responsive genes, including those involved in the synthesis and uptake of the siderophore vicibactin and in the uptake of haem (Carter et al., 2002; Wexler et al., 2001, 2003). This Fur-like protein not only has sequence similarity to Fur but partially corrected the regulatory defect of an E. coli fur mutant and can bind to a canonical fur box (Wexler et al., 2003). Similarly, mutations in the fur-like gene (furBj) of B. japonicum did not affect the expression of haem uptake genes in these bacteria (Nienaber et al., 2001), although FurBj regulates irr in response to Fe2+ (Hamza et al., 1999, 2000). Interestingly, FurBj binds near the irr promoter despite the lack of fur boxes in this region (Friedman & O'Brian, 2003). The R. leguminosarum genes involved in iron acquisition were regulated, not by this Fur-like protein, but by a very different regulator, termed RirA, close homologues of which occur only in R. leguminosarum and its near relatives (Todd et al., 2002). Thus, the iron regulon in rhizobia differs markedly from that in many other bacteria (Johnston et al., 2001).

The fur-like gene of the closely related Sinorhizobium meliloti adjoins an operon termed sitABCD (Platero et al., 2003; see genome sequence at http://sequence.toulouse.inra.fr/meliloti.html). It was so named because it was first thought to specify an ABC-type iron transporter in Salmonella (Zhou et al., 1999), but was later found to have a higher affinity for Mn2+ transport (Kehres et al., 2002). In Sinorhizobium, too, mutations in sitABCD severely reduce growth in medium low in Mn2+ (Platero et al., 2003). Here, we show that the Fur-like protein of R. leguminosarum represses transcription of sitABCD in response to Mn2+ and so is more appropriately termed ‘Mur’ (manganese uptake regulator). Independently, similar observations were made on sitABCD of S. meliloti (S. Weidner, unpublished observations).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Strains, plasmids, media and growth conditions.
Bacterial strains and plasmids are listed in Table 1. R. leguminosarum and Escherichia coli were grown routinely as described by Beringer (1974). In metal-depleted media, the chelators 2,2-dipyridyl (DP) (30 µM) or ethylenediamine di(o-hydroxyphenylacetic acid) (EDDHA) (200 µM) were present; MnCl2 or FeCl3 was added (at 20 or 50 µM as appropriate). Peas were grown, inoculated and examined for nodulation as described by Wexler et al. (2001). {beta}-Galactosidase and {beta}-glucuronidase assays were done essentially as described by Miller (1972) and Yeoman et al. (2000) respectively, rhizobia being grown in liquid, minimal (Y) or complete (TY) medium with different concentrations of MnCl2, FeCl3 or DP.


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Table 1. Strains and plasmids used in this study

 
In vivo and in vitro genetic manipulations.
Plasmids were transferred by conjugation, mutagenized with Tn5lac and mutant alleles introduced into the R. leguminosarum genome as described by Wexler et al. (2001). Routine methods were used for cloning, hybridizations and plasmid isolations. Primers to amplify the sitABCD promoter region from R. leguminosarum genomic DNA and to determine the sitABCD transcriptional start site by primer extensions (Sawers & Böck, 1989) are listed in Table 2. They were based on the genome sequence of R. leguminosarum strain 3841 (http://www.sanger.ac.uk/Projects/R_leguminosarum/).


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Table 2. Sequences of oligonucleotide primers used in this study

Relevant restriction sites are underlined.

 
Mobility shift assays.
DNA mobility shift assays, using four different DNA fragments, were performed as described by Ochsner et al. (1995), with minor modifications. The fragments used were PCR products as follows: (i) a 352 bp fragment that contains the Fur-regulated pvdS promoter of P. aeruginosa (Wexler et al., 2003), (ii) a 344 bp fragment, containing the R. leguminosarum irr promoter (Wexler et al., 2003), (iii) a 504 bp fragment (termed 1/504) spanning the R. leguminosarum MRS1 and MRS2 (MRS, Mur-responsive sequence), (iv) a 222 bp fragment (1/222) spanning MRS1 and (v) a 304 bp fragment (200/504) spanning MRS2. The DNA fragments were 3'-end labelled with DIG-11-ddUTP, using the DIG Gel Shift Kit 2nd Generation (Roche). Non-denaturing gels contained 0·1 mM Mn2+. Mur protein was overexpressed and purified as described by Wexler et al. (2003). Two different concentrations (0·36 µM and 3·6 µM) of purified Mur were added to the DIG-labelled DNA, in the presence of unlabelled, non-competitor DNA [1 µg µl–1 poly(dI-dC), sodium salt]. Following electrophoresis, DNA–protein complexes were blotted and visualized by enzyme immunoassay, using anti-Digoxigenin-AP, Fab-fragments and the colorimetric substrate NBT/BCIP (DIG High Prime DNA Labelling and Detection Starter Kit I) as described by the manufacturer.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Identification and cloning of a homologue of the sitABCD operon in R. leguminosarum
The genome sequence of R. leguminosarum strain 3841 has a close homologue of the sitABCD operon of S. meliloti (Platero et al., 2003), the Sit products being very similar (SitA 80 % identical, SitB 74 %, SitC 74 %, SitD 72 %) in these two rhizobial species. Indeed, these SitABCD proteins are very similar to those of several distantly related bacteria, such as Salmonella and the pathogenic strain of E. coli CFT073 (but not strain K-12) (see Table 3); however, there are no close homologues in near-relatives of R. leguminosarum, such as Brucella and Mesorhizobium. This suggests that they had been acquired by R. leguminosarum relatively recently by lateral gene transfer. The operator–proximal gene, sitA, encodes the periplasmic binding protein, followed by sitB, which encodes an ATPase. Finally, sitC and sitD encode the membrane-located transporters. In S. meliloti, sitABCD is next to a gene whose product is 42 % identical to E. coli Fur (http://sequence.toulouse.inra.fr/meliloti.html). In contrast, R. leguminosarum sitABCD is between two ORFs that specify a possible adenine deaminase (adm) and a putative amidohydrolase (aho) (Fig. 1), and is very distant from the fur-like gene on the R. leguminosarum chromosome (http://www.sanger.ac.uk/Projects/R_leguminosarum/).


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Table 3. Similarities of R. leguminosarum SitA, B, C and D proteins to Sit proteins of other bacteria

The deduced amino acid sequences of the R. leguminosarum SitA, SitB, SitC and SitD proteins were compared with those of a selection of Sit-like proteins in other bacterial genera. Figures show percentage identities over the entire length of the polypeptides.

 


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Fig. 1. Representation of the regulatory region upstream of R. leguminosarum sitA. (a) Locations of the sitABCD genes, between an ORF encoding a putative adenine deaminase (adm) and a putative amidohydrolase (aho) are shown. (b) Expanded version of the intergenic space between the stop codon of adm (set at position 1) and the sitA translational start (at position 308 bp). The two putative sitA transcriptional starts, TS1 and TS2 are shown. The dimensions of fragments (1/222 and 200/504) used in gel shifts are delineated. The two conserved MRS motifs are indicated by the boxes, striped for MRS1 and checked for MRS2. (c) Comparison of the MRS1 and MRS2 sequences with a region upstream of sitA of S. meliloti (Smel). The transcriptional starts, TS1 and TS2, are shown. Solid arrows above MRS1 show the dimensions of the 7-mer inverted repeat, the corresponding bases being in lower case. Sequences spanning MRS1 and MRS2 are superimposed; nucleotides in MRS2 identical to those in MRS1 are underlined and in bold. Note the gap ‘X’ in MRS1. In the Smel DNA sequence, the dotted arrow and lower case bases indicate an inverted repeat in that species and the underlined, bold bases correspond to those that are identical to ones in R. leguminosarum MRS1.

 
To obtain the intact sitABCD operon, colonies of E. coli harbouring a gene library of R. leguminosarum strain 3841 DNA cloned in the wide-host-range cosmid pLAFR1, were probed with a labelled PCR-generated fragment that spanned R. leguminosarum sitA. Two hybridizing plasmids, termed pBIO1457 and pBIO1458, were confirmed to contain sitABCD, plus flanking DNA.

The sitABCD operon of R. leguminosarum is involved in manganese uptake
Given the similarity of sitABCD of S. meliloti and R. leguminosarum, we tested if the cloned sitABCD genes of the latter could correct a sit mutant of S. meliloti for its poor growth on low-Mn2+ medium. The R. leguminosarum sitABCD-containing plasmid pBIO1457 was mobilized into the S. meliloti sitB mutant H36 and the transconjugants were grown on TY medium whose divalent cations had been depleted by adding the chelator EDDHA and in EDDHA-treated medium that had been replenished by adding FeCl3, MnCl2 or both. As expected (Platero et al., 2003), H36 grew very poorly on EDDHA-containing medium, unless MnCl2 was added. However, H36 containing pBIO1457 grew nearly as well as did wild-type S. meliloti on low-Mn2+ medium, showing that the R. leguminosarum sit genes corrected the defect in Mn2+ uptake of this S. meliloti sitB mutant (Fig. 2).



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Fig. 2. Effect of cloned R. leguminosarum sitABCD genes on growth of S. meliloti sitB mutant in manganese-deficient medium. Cells were diluted into complete (TY) medium containing 200 µM chelator EDDHA plus either 50 µM MnCl2 (a) or 50 µM FeCl2 (b) and growth at 28 °C was monitored for 100 h by measuring OD620. Strains: wild-type S. meliloti 242 ({blacklozenge}), S. meliloti sitB mutant H36 ({blacksquare}) and H36 containing the R. leguminosarum sitABCD operon cloned in pBIO1457 ({blacktriangleup}).

 
To isolate Sit mutants in R. leguminosarum, pBIO1457 and pBIO1458 were mutagenized with Tn5lac, which specifies kanamycin resistance and has a promoterless lacZ gene at one end, generating transcriptional lacZ fusions. The exact locations and orientations of two Tn5lac insertions in the sitABCD operon were identified by restriction mapping and verified by sequencing, with mutant plasmids as template and an oligonucleotide at the 5' end of the lacZ in Tn5lac as sequencing primer. In one of these, pBIO1459, Tn5lac is in sitD, with lacZ and sitD in the same orientation. In the other, pBIO1460, Tn5lac is in sitB, with lacZ and sitB being in opposite orientations.

The sitB and sitD mutations were transferred by marker exchange from their plasmid-borne locations to the genome of wild-type R. leguminosarum strain 3841, to form strains J400 and J401, respectively. These were examined for their growth in media that varied in metal availability. Unlike S. meliloti, the R. leguminosarum Sit mutants were indistinguishable from wild-type in their growth rates on Mn2+-depleted medium, suggesting that R. leguminosarum might have another functional Mn2+ transporter. The R. leguminosarum and S. meliloti genomes both contain ORFs whose deduced products have significant homology to MntH, a different type of Mn2+ transporter in the Nramp family which is widespread in bacteria (Cellier et al., 2001), and which, in E. coli, is regulated by Fur (Patzer & Hantke, 2001). It is unclear why the phenotypes of Sit mutants differ in the two rhizobial species and it remains to be established if the mntH-like genes are functional Mn2+ transporters in R. leguminosarum and/or S. meliloti.

The two R. leguminosarum Sit mutants were each inoculated onto peas. The numbers, morphologies and times of appearance of the nodules were indistinguishable from those induced by wild-type J251.

sitABCD expression is repressed by Mn2+, derepressed in the absence of Mur but is unaffected by the nod gene inducer naringenin
To examine expression of sitABCD, the sitD–lacZ fusion plasmid pBIO1459 was mobilized into wild-type R. leguminosarum 3841. Cells were grown in minimal medium and in medium containing the chelator DP, to which MnCl2 or FeCl3, or neither had been added. As seen in Table 4, sitABCD expression was markedly enhanced in the metal-depleted compared to the metal-replete medium. However, addition of MnCl2 greatly reduced, but did not abolish, expression of the fusion. In contrast, adding FeCl3 had no repressive effect; indeed, the iron-supplemented cells had slightly higher {beta}-galactosidase activity, perhaps due to non-specific enhancement of their metabolic activity.


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Table 4. Expression of R. leguminosarum sit–lacZ and S. meliloti sit–gus fusions in wild-type and mur mutant in media varying in the concentration of Mn2+ and Fe2+

{beta}-Galactosidase or {beta}-glucuronidase activities, as appropriate, were measured in triplicate for wild-type R. leguminosarum J251 or the mur mutant J325 and for derivatives of both strains containing the R. leguminosarum sit–lacZ fusion plasmid pBIO1459 or the S. meliloti sitA–gus fusion plasmid pTCC1. Cells were grown in media depleted for metals by adding the chelator DP and in media supplemented with 10 µM FeCl3 or 10 µM MnCl2 as indicated. Figures are activities in Miller units with standard errors in parentheses.

 
Given that sitABCD and fur are adjacent in S. meliloti and that the fur-like gene of R. leguminosarum had no known function, we determined if it was involved in controlling sitABCD expression. When pBIO1459 was mobilized into the R. leguminosarum Fur strain J325, expression of sit–lacZ was extremely high, even in medium with added Mn2+ (Table 4). Thus, Mn2+-dependent repression of sitABCD is the first function so far ascribed to the fur-like gene of R. leguminosarum. Based on these findings, we propose to name this gene mur (manganese uptake regulator).

To distinguish further the genes involved in Mn2+- and Fe2+-dependent gene regulation, pBIO1459 was conjugated into J397, a RirA mutant of R. leguminosarum. Expression of sitA–lacZ was the same as in the wild-type J251, in high and low levels of MnCl2 and/or FeCl3 (not shown).

Given that the sequences 5' of sitA in R. leguminosarum and S. meliloti had similarities, but also some differences (see below), it was of interest to see if the Mn2+-regulated promoter of S. meliloti sitABCD responded to Mn2+ and/or to the Mur of R. leguminosarum. Therefore, plasmid pTCC1, which contains a sit–gus fusion of S. meliloti was conjugated into wild-type R. leguminosarum and into the mur mutant J325 and the strains were grown and assayed for {beta}-glucuronidase. The results resembled those obtained with the sit–lacZ fusion of R. leguminosarum itself; Mn2+ repressed S. meliloti sit–gus expression in the wild-type but not in the mur mutant (Table 4).

In a transcriptomic survey of S. meliloti, Ampe et al. (2003) showed that expression of sitABCD was enhanced (aprox. 10-fold) in cells grown in the presence of luteolin, a flavonoid inducer of nodulation (nod) genes of that species (Peters et al., 1986). Therefore, wild-type R. leguminosarum containing the sitD–lacZ fusion plasmid pBIO1459 was grown in the presence of naringenin, a potent inducer of nod gene expression in R. leguminosarum (Firmin et al., 1986). However, sitABCD expression was unaffected by naringenin, irrespective of the presence or absence of added manganese (data not shown). It is unclear if this reflects a real difference in the regulatory properties of the two species or whether, for unknown reasons, the data obtained from microarray experiments differ from those using lac fusions.

Effects of Mn2+ and Fe2+ on the regulation of a classical fur box by R. leguminosarum Mur protein
The E. coli bfd gene is normally repressed in iron-replete conditions by the binding of Fur to a canonical fur box near the bfd promoter. Wexler et al. (2003) showed that the cloned R. leguminosarum mur gene, when overexpressed in iron-replete E. coli cells, partially corrected the constitutive expression of a bfd–lac fusion in a fur mutant of E. coli (JRG2653). In R. leguminosarum, the Mur protein responds to Mn2+, rather than Fe2+, so we re-examined the ability of cloned R. leguminosarum Mur to act on a conventional fur box, but this time in response to Mn2+. To do this, the fur mutant E. coli JRG2653 containing pBIO1153 (R. leguminosarum mur cloned in pUC18) was grown in minimal medium to which additional FeCl3 or MnCl2 had been added or in which both had been removed by DP. It was confirmed that the bfd–lac fusion was repressed by R. leguminosarum Mur, in iron-replete medium, but not in the Mn2+-replete medium (Table 5). Thus, the ability of R. leguminosarum Mur to bind to a canonical fur box and to repress bfd transcription is dependent on iron, not manganese.


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Table 5. Effect of cloned R. leguminosarum mur gene on expression of bfd–lacZ fusion in E. coli

{beta}-Galactosidase assays were done in triplicate on E. coli JRG2653, which is Fur and contains a chromosomal bfd–lacZ fusion. Assays were also done on a derivative of JRG2653 containing the cloned R. leguminosarum mur gene in pBIO1153. Cells were grown in media depleted for metals by adding DP and in media supplemented with 10 µM FeCl3, 10 µM MnCl2 or both. Figures are activities in Miller units with standard errors in parentheses.

 
The effects of E. coli Fur on expression of R. leguminosarum sitABCD were also determined. To do this, the sitD–lacZ fusion plasmid pBIO1459 was mobilized into wild-type E. coli strain MC4100 and the cells were grown in metal-depleted medium and in medium supplemented with Mn2+ or with Fe2+. Neither metal affected expression of the fusion. Furthermore, when pBIO1459 was mobilized into E. coli fur mutant strain H1941, there was no enhancement in its expression (data not shown). Thus E. coli Fur does not alter the expression of the R. leguminosarum sitABCD genes, in response to either Mn2+ or Fe2+, at least when present in the heterologous E. coli host.

The fact that sitABCD was expressed in E. coli, irrespective of the metal status of the medium, was somewhat surprising, since many R. leguminosarum ‘housekeeping’ genes are poorly expressed in this host (see Johnston et al., 1978). This may reflect the recent acquisition (see above) of the sitABCD operon, perhaps together with a promoter that is recognized by enteric bacteria.

Transcription of sitABCD
To locate the sitABCD transcriptional start sites, primer extension experiments were done, in which RNA was harvested from wild-type and from mur mutant J325, grown in media containing DP, with or without added Mn2+. Two 5' ends (termed TS1 and TS2) were seen, which were located, respectively, 102 and 43 bp upstream of the sitA translational start codon (Fig. 3). The TS2 revealed two adjacent 5' ends; TS1 initiated at an adenosine residue. The intensities of both these transcripts were markedly reduced (approx. sixfold) in J251 (wild-type) grown in Mn2+-replete medium compared to when this strain was depleted of Mn2+ by the presence of DP (Fig. 3). In contrast, with RNA harvested from the mur mutant J325, there was high-level expression of both transcripts, even in cells grown with Mn2+. It is unknown if the smaller mRNA is derived from the larger one or if the two RNAs are the products of two separate promoters, both of which are repressed in response to Mn2+ in Mur+ R. leguminosarum.



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Fig. 3. Primer extensions. RNA was harvested from wild-type (J251) and mur mutant (J325) R. leguminosarum after growth on medium containing the chelator DP (D) or with 20 µM MnCl2 (M). Primer extensions used primer sit-PE, which was also used for DNA sequencing, with the corresponding cloned region as template. Locations of the TS1 and TS2 transcriptional starts are indicated (see also Fig. 1).

 
Inspection of the DNA in the sitABCD promoter region revealed some interesting features. Most prominent were two closely related sequences (23 of 27 bp identical) that each lie 5' of the two putative sitABCD transcript start sites TS1 and TS2 (Fig. 1). For convenience, we respectively refer to these as MRS1 and MRS2 (Mur-responsive sequence). The 27 bp MRS1, which extends 14–40 bp 5' of TS1 is shorter by 1 bp than MRS2, which is located 19–46 bp 5' of TS2 (see Fig. 1). Within MRS1 is an inverted 7 bp repeat, separated by 7 bp, a motif that is very similar to one that occurs upstream of the sitA gene of S. meliloti (Fig. 1). In addition, there are several shorter direct and inverted repeats in the MRS1 of R. leguminosarum.

Binding of R. leguminosarum Mur to two regions near the sitABCD promoter(s)
We saw no similarity in the sequences near either of the putative sitABCD promoters to canonical fur boxes as reinterpreted by Baichoo & Helmann (2002) and by Lavrrar & McIntosh (2003), nor was there detectable similarity to the unusual Fur-binding sequence near the B. japonicum irr promoter (Friedman & O'Brian, 2003).

To establish if there was a direct interaction between R. leguminosarum Mur and cis-acting regulatory sequences upstream of sitA, we did gel-shift experiments, using purified Mur protein and three different fragments that spanned the sitABCD promoter(s). One fragment (1/504) spanned the entire region between sitA and adn; the other two spanned MRS1 (fragment 1/222) or MRS2 (fragment 200/504) individually (see Fig. 1). For comparison, two previously described fragments (Wexler et al., 2003) were also used. One was a 352 bp fragment spanning a conventional fur box of the P. aeruginosa pvdS gene (Ochsner et al., 1995), which can bind to R. leguminosarum Mur. A negative control was the region 5' of R. leguminosarum irr, which did not bind Mur (Wexler et al., 2003). The two fragments that contained each of the individual MRSs were retarded at similar concentrations of added Mur protein (Fig. 4), as was the fragment that contained both of these motifs (not shown). Thus, at least two separate regions upstream of sitABCD can bind to Mur in vitro. As expected, the pvdS promoter fragment, with its conventional fur box, was retarded but the negative irr control fragment was not (Fig. 4).



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Fig. 4. Gel retardation assays of R. leguminosarum Mur protein with DNA fragments spanning the sitA regulatory region. R. leguminosarum Mur protein (lanes 2, 0·36 µM Mur; lanes 3, 3·6 µM Mur; lanes 1, no added Mur, as control) was added to four different DIG-labelled fragments. A, fragment spanning irr promoter region; B, spanning P. aeruginosa pvdS promoter; C, fragment 200/504 spanning MRS2 motif 5' of R. leguminosarum sitA; D, fragment 1/222 spanning MRS1 motif upstream of R. leguminosarum sitA (see Fig. 1).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The few studies on iron-regulated gene expression in the {alpha}-proteobacteria indicate that these may have different mechanisms from those found in other bacterial groups. Thus, in R. leguminosarum, for example, the regulation of many genes involved in iron acquisition is mediated by RirA, a protein with no detectable sequence similarity to Fur (Todd et al., 2002).

Although many {alpha}-proteobacteria have a protein whose sequence resembles Fur of, for example, E. coli, it does not appear to play a pivotal role in ‘global’ iron-responsive gene regulation, at least in the two rhizobial species R. leguminosarum and B. japonicum, since mutations in their fur-like genes do not affect the expression of genes involved in iron uptake (Wexler et al., 2003; Nienaber et al., 2001). Consistent with this, no known iron-regulated promoters of R. leguminosarum have fur boxes (Wexler et al., 2003). In iron-replete media, FurBj can repress transcription of the irr gene, which regulates a gene involved in haem synthesis and which is itself in the fur superfamily. However, even here, although FurBj binds to classical E. coli fur boxes, its binding site at the irr promoter has no resemblance to a fur box (Friedman & O'Brian, 2003). Thus, FurBj has features of a classical Fur, but also some atypical characteristics.

Like that of B. japonicum, the Fur-like protein of R. leguminosarum can also bind to classical fur boxes that are experimentally provided in vitro, and cloned R. leguminosarum mur partially corrects the regulatory defect of a fur mutant of E. coli (Wexler et al., 2003). However, in R. leguminosarum itself, Mur has a different role and responds to a different metal, repressing transcription of the sitABCD Mn2+ transport operon in response to manganese. It is striking that the genome of Mesorhizobium loti (http://www.kazusa.or.jp/rhizobase/Mesorhizobium/index.html), which nodulates Lotus, has no fur homologue, pointing to the redundancy of this gene for the rhizobia. It may be significant that M. loti also has no sitABCD operon; however, unlike Rhizobium, it does have a homologue of MntR (see below).

The sitABCD operon was first located in a pathogenicity island in Salmonella typhimurium (Zhou et al., 1999), the cloned sitABCD genes correcting the growth defect on low-iron medium of an E. coli mutant that was defective in siderophore synthesis. Thus, it was originally thought that SitABCD was, primarily, a transporter of iron. However, it also imports Mn2+, particularly in alkaline conditions (Boyer et al., 2002; Kehres et al., 2002), and this is important for pathogenesis. Not only is SitABCD a dual function transporter for Mn2+ and Fe2+, it is regulated by both metals, but in different ways. Transcription of Salmonella sitABCD is repressed either by Fe2+ or by Mn2+; significantly, the Fe2+-dependent repression requires Fur but Mn2+-dependent repression does not, pointing to a different Mn2+-dependent regulator, possibly MntR (Runyen-Janecky et al., 2003).

Close homologues (>60 % identical) of the Sit proteins occur, sporadically, in a wide range of eubacteria, but the relatedness of the sequences is not congruent with bacterial taxonomy. Among {alpha}-proteobacteria, Rhizobium, Agrobacterium, Sinorhizobium, Rhodobacter and Rhodospirillum all have very close Sit homologues but Mesorhizobium, Caulobacter and Brucella do not. This strongly suggests that these genes are particularly susceptible to lateral gene transfer and that the R. leguminosarum sitABCD operon was acquired relatively recently from another bacterial taxon.

Regulation of sitABCD in R. leguminosarum shares some features with those in Salmonella. Thus, it is repressed in response to added Mn2+ and mutations in a member of the fur superfamily dramatically affect its transcription. However, R. leguminosarum sitABCD is not repressed by FeCl3 and, almost paradoxically, the Mn2+-dependent repression requires Mur whereas in Salmonella, a different regulator (see above) mediates Mn2+-dependent control. The ability of a member of the Fur superfamily to respond to Mn2+ is not, however, unique to R. leguminosarum. PerR of Bacillus represses several promoters of genes in response to Mn2+. Of particular relevance here, PerR represses genes involved in protection from oxidative stress in response to either Mn2+ or to Fe2+ but PerR-mediated repression of itself and of fur of Bacillus is responsive only to Mn2+ (Fuangthong et al., 2002). This is akin to our observations that R. leguminosarum Mur corrects a conventionally acting fur mutant of E. coli in response to Fe2+ but not Mn2+, whereas the reverse is true for its repression of sitABCD in R. leguminosarum itself. Given that the putative Mur-binding MRS motifs in the promoter region of sitABCD in R. leguminosarum have no sequence similarity to the fur box at the pvdS promoter, this points to a subtle interaction between the Mur protein and the specific metal, which determines the sequences to which the Mur can bind. Likewise, in the DtxR family of regulators, DtxR itself is primarily responsive to Fe2+ whereas the MntR of Bacillus is a Mn2+-sensing regulator, but substitution of just two amino acids in the latter conferred responsiveness to the former metal (Guedon & Helmann, 2003).

The concept of Fur-mediated regulation being effected through an interaction of a canonical fur box with the repressor protein, bound to its cognate metal co-repressor, may be an oversimple model. Even with a conventional fur box, there is still a debate as to the nature of the core elements that determine its function (Baichoo & Helmann, 2002). Moreover, it is becoming increasingly clear that Fur proteins can bind to cis-acting regulatory sequences that do not resemble classical fur boxes. Baichoo et al. (2002) identified several such ‘exceptions to the rule’ among several B. subtilis genes that were regulated by Fur in response to Fe2+ in these bacteria. Our observations here, together with those of Friedman & O'Brian (2003), show that in the rhizobia, too, Fur-like proteins can recognize very different sequences, further prompting a re-examination of what exactly defines a fur box.

Given that some Fur-like proteins can distinguish Mn2+ and Fe2+, we note that most studies involving in vitro gel shifts with ‘genuine’ Fur and cognate fur boxes utilize Mn2+ as the co-repressor, rather than the authentic ligand Fe2+. Although these studies have yielded coherent observations, the findings described here show that there is a risk of artifactual outcomes if in vitro binding experiments are the only source of information used to identify the metallic co-repressor.

R. leguminosarum Mur can bind to DNA containing either of two conserved sequences, MRSs, in the sitABCD promoter region, 5' of two likely promoters for this operon. DNase protection experiments with purified Mur protein will be required to show that these two MRSs are binding sites and, if so, where the DNA–protein contact points are located. It also remains to be established if the two distinct RNA products of the primer extensions represent two different promoters, each of which responds to the binding of Mur to its cognate MRS.

Mur can recognize two very different sets of DNA sequences, namely a canonical fur box and the MRS regions in the R. leguminosarum sitABCD promoter region, in response to two different metals. It will be of interest to determine the allosteric effects of Fe2+ and Mn2+ on Mur and, ideally, to examine the structure of the Mur protein, in combination with these two co-repressor metals on its different target sequences.

In conclusion, the results presented here reaffirm that regulation of iron-responsive genes in R. leguminosarum is not mediated by Fur. It is now clear that Mur, its Fur-like protein, is something of a specialized ‘outrider’ perhaps with a minor role, dedicated to regulating an operon that was acquired relatively recently. Transcriptomic analyses may be needed to elucidate if Mur regulates other R. leguminosarum genes. Given that some other {alpha}-proteobacteria have fur-like genes, it will be interesting to discern their roles and to establish directly if Fur or other, unknown, genes affect iron-responsive regulation in well-studied genera such as Caulobacter or Rhodobacter.


   ACKNOWLEDGEMENTS
 
The work was funded by the UK BBSRC and a CONACYT (México) Scholarship to E. D-M. We are grateful to Kay Yeoman for technical advice, to Simon Andrews, Phil Poole and Elena Fabiano for generous provision of strains, and to Stefan Weidner for useful discussions and for S. meliloti sit–lac fusion plasmids.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 3 December 2003; revised 19 January 2004; accepted 21 January 2004.



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