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
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ABSTRACT |
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INTRODUCTION |
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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).
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METHODS |
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RESULTS |
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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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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 sitDlacZ 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
-galactosidase activity, perhaps due to non-specific enhancement of their metabolic activity.
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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 sitAlacZ 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 sitgus 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 -glucuronidase. The results resembled those obtained with the sitlacZ fusion of R. leguminosarum itself; Mn2+ repressed S. meliloti sitgus 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 sitDlacZ 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 bfdlac 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 bfdlac 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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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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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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DISCUSSION |
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Although many -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 -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 DNAprotein 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 -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.
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ACKNOWLEDGEMENTS |
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Received 3 December 2003;
revised 19 January 2004;
accepted 21 January 2004.
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