Fur-independent regulation of iron metabolism by Irr in Bradyrhizobium japonicum

Iqbal Hamzaa,1, Zhenhao Qi1, Natalie D. King1 and Mark R. O’Brian1

Department of Biochemistry and Center for Microbial Pathogenesis, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214, USA1

Author for correspondence: Mark R. O’Brian. Tel: +1 716 829 3200. Fax: +1 716 829 2725. e-mail: mrobrian{at}buffalo.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bradyrhizobium japonicum expresses both Fur and Irr, proteins that mediate iron-dependent regulation of gene expression. Control of irr mRNA accumulation by iron was aberrant in a fur mutant strain, and Fur repressed an irr::lacZ promoter fusion in the presence of iron. Furthermore, metal-dependent binding of Fur to an irr gene promoter was demonstrated in a region with no significant similarity to the Fur-binding consensus DNA element. These data suggest that the modest control of irr transcription by iron is mediated by Fur. However, Irr protein levels were regulated normally by iron in the fur strain, indicating that Fur is not required for post-transcriptional control of the irr gene. Accordingly, regulation of hemB, a haem biosynthesis gene regulated by Irr, was controlled normally by iron in a fur strain. In addition, the hemA gene was shown to be controlled by Fur, but not by Irr. It was concluded that Fur cannot be the only protein by which B. japonicum cells sense and respond to iron, and that Irr may be involved in Fur-independent signal transduction. Furthermore, iron-dependent regulation of haem biosynthesis involves both Irr and Fur.

Keywords: Bradyrhizobium japonicum, iron metabolism, Fur, Irr, haem metabolism

Abbreviations: ALA, {delta}-aminolevulinic acid

a Present address: Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A considerable body of work on the regulation of iron homeostasis in bacteria has focused on Fur, a transcriptional regulator that controls genes in an iron-dependent manner. In the classic view, ferrous iron binds directly to Fur to confer binding to a defined cis-acting DNA element for transcriptional repression. Thus, Fur is both an iron sensor and a mediator of iron-dependent regulation. However, this model may not be universally applicable; Fur can bind DNA in the absence of metal (Althaus et al., 1999 ; Bsat & Helmann, 1999 ) and evidence for Fur as a positive effector or having activity in the absence of metal has been reported (Foster & Hall, 1992 ; Litwin & Calderwood, 1994 ).

Very recently, several Fur-like proteins have been identified that are not functional Fur homologues, but instead they are involved in maintenance of zinc homeostasis (Gaballa & Helmann, 1998 ; Patzer & Hantke, 1998 ), manganese-dependent response to oxidative stress (Bsat et al., 1998 ) or iron-dependent regulation of haem biosynthesis (Hamza et al., 1998 ). Additional fur-like genes have been identified from genome sequencing and from screens for genes involved in pathogenesis (Camilli & Mekalanos, 1995 ; Wang et al., 1996 ). There now appears to be a family of Fur proteins that are functionally diverse, but are all involved in metal-dependent regulation.

Irr from Bradyrhizobium japonicum may be the most divergent of the Fur-like proteins described thus far in that it is active only under metal limitation and contains a single cysteine residue rather than the multiple cysteines found in the other proteins. Moreover, irr gene expression is strongly regulated by iron whereas fur is essentially constitutive. Iron represses the irr gene moderately at the transcriptional level and strongly at protein turnover (Hamza et al., 1998 ; Qi et al., 1999 ). The latter mechanism involves iron-dependent binding of haem to a haem regulatory motif of the Irr protein which is necessary for its degradation (Qi et al., 1999 ). As a result, irr mRNA is diminished but detectable under high iron conditions, whereas protein levels are undetectable.

Haem is iron-protoporphyrin and Irr mediates iron control of the haem biosynthetic pathway (Hamza et al., 1998 ). {delta}-Aminolevulinic acid (ALA) synthase and ALA dehydratase catalyse the first two steps of haem biosynthesis and are encoded by hemA and hemB respectively. Under iron limitation, Irr negatively regulates haem biosynthesis at hemB, and an irr strain shows constitutively high levels of hemB mRNA and protein. The hemA gene is also controlled by iron (Page et al., 1994 ), but a regulator for it has not been defined and is addressed herein. B. japonicum is the only organism described thus far that contains a Fur-like protein in addition to bona fide Fur that is involved in iron metabolism (Hamza et al., 1999 ). This led us to ask what the relationship between Fur and Irr may be, and whether Fur is involved in haem biosynthesis. Here we report that Fur is involved in control of irr transcription, but that post-transcriptional control by iron is Fur-independent and therefore B. japonicum must have at least two iron-dependent regulatory systems. We propose that these sensory mechanisms allow for differential control of the hemA and hemB genes.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Chemicals and reagents.
All chemicals were reagent grade and were purchased from Sigma or from J. T. Baker, NJ. Purified noble agar and yeast extract were obtained from Difco. 59FeCl3, [{alpha}-32P]dNTPs [3000 Ci mmol-1 (111 GBq mmol-1)] and [{gamma}-32P]ATP [3000 Ci mmol-1 (111 GBq mmol-1)] were obtained from Dupont-NEN Life Science Products. [{alpha}-32P]UTP [800 Ci mmol-1 (29·6 GBq mmol-1)] used for RNase protection assays was obtained from ICN Biomedicals.

Bacterial strains, plasmids, media and growth.
Bacterial strains used in this study are listed in Table 1. B. japonicum strain I110 was the parent strain used; strains LODTM5 and GEM4 are irr and fur mutants, respectively, and were described previously (Hamza et al., 1998 , 1999 ) B. japonicum strains were routinely grown at 28 °C in GSY media as described previously (Frustaci et al., 1991 ). Cultures for growth of strain GEM4 were supplemented with 100 µg ml-1 each of spectinomycin and streptomycin, and LODTM5 was grown in the presence of 100 µg ml-1 each of kanamycin and streptomycin. The medium used for culturing cells under iron limitation was a modified GSY medium (0·5xGSY) in which 0·5 g yeast extract l-1 was used instead of 1 g l-1 and no exogenous iron source was added. The actual iron concentration of the media was 0·23 µM as determined with a Perkin Elmer model 1100B atomic absorption spectrometer. High-iron media contained 6 µM added FeCl3. Glassware was rinsed extensively with 6 M HCl and then washed with distilled water (Milli-Q PF plus), followed by rinsing with metal-free water (Milli-Q UV plus). Escherichia coli strains DH5{alpha}, XL-1 Blue or TB1 were used for propagation of plasmids. E. coli strains were grown at 37 °C on Luria–Bertani broth or 2xyeast-tryptone medium with appropriate antibiotics. pMH15fur was provided by K. Hantke, University of Tübingen, Germany, and contains the E. coli fur gene cloned into pACYC184. pGDIrr-fuse contains a 608 bp SmaI/BamHI fragment of the irr gene that includes 175 bp upstream of the transcription start site cloned into pGD499 (Ditta et al., 1985 ), resulting in an irr::lacZ transcriptional fusion. pSKD{Delta}lac contains a deletion from the HindIII site in the multiple cloning site of pBluescript SK to an unidentified region upstream of the multiple cloning site created by digestion with T4 DNA polymerase. The plasmid does not encode ß-galactosidase activity in E. coli strains harbouring the omega complementation fragment of ß-galactosidase.


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Table 1. Bacterial strains used in this study

 
ß-Galactosidase assay of E. coli liquid cultures.
Analysis of iron-dependent repression of an irr::lacZ fusion by BjFur and EcFur in E. coli was carried out by measuring ß-galactosidase activity in cells grown under high- or low-iron conditions. Cultures of E. coli strain DH5{alpha} harbouring pGDIrr-fuse and either pSKBJF800 (BjFur), pMH15fur (EcFur) or pSKD{Delta}lac (control) were grown in LB media containing either 100 µM FeCl3 or 200 µM {alpha},{alpha}-dipyridyl for high- and low-iron conditions, respectively. ß-Galactosidase activity was measured in cells as described by Miller (1972) . Cells were grown aerobically at 37  °C to late-exponential phase. Cells were spun down and resuspended in 800 µl Z buffer (60 mM Na2HPO4 . 7H2O, 40 mM NaH2PO4 . H2O, 10 mM KCl, 1 mM MgSO4 . 7H2O and 50 mM ß-mercaptoethanol, pH 7·0). One hundred microlitres of suspension, corresponding to 1x108 cells, was used per reaction. The data are represented in Miller units and each value is a mean of triplicate samples corrected for background. Absorbance of o-nitrophenol formed from ONPG by ß-galactosidase was recorded at 420 nm and normalized for cell density at OD550.

Gel mobility shift assay.
E. coli strain H1780 with plasmids pMH15fur, pSKBJF800 and pSKSBIrr, was inoculated from an overnight culture into a fresh 250 ml 2xYT liquid culture with antibiotics, 100 µM FeCl3 and 1 mM IPTG. Cells were harvested at mid-exponential phase, washed twice and resuspended in TNG buffer [50 mM Tris, 50 mM NaCl and 5% (v/v) glycerol, pH 7·4] and 1 mM PMSF. Supernatants were obtained by passage of the cells twice through a French pressure cell at 900 p.s.i. and clarification at 14000 g. Protein concentration was estimated by the Bradford (1976) protein assay. On average, 11–16 mg total protein ml-1 was obtained per preparation. For gel mobility shift assays (modified from de Lorenzo et al., 1988 ), 15 µg crude extract, 2·5 µg poly(dI-dC).poly(dI-dC) (Pharmacia Biotech) and 1x105 c.p.m. labelled DNA probe were mixed in 1xbinding buffer [10 mM bis-Tris borate, pH 7·5, 1 mM MgCl2, 40 mM KCl, 5% glycerol, 0·1% (v/v) Nonidet P-40 and 1 mM DTT] and incubated on ice for 5 min in a 20 µl reaction volume. A 5% nondenaturing polyacrylamide gel in electrophoresis buffer (20 mM bis-Tris borate, pH 7·5) was prerun for 15 min at 200 V constant voltage and loaded with 20 µl of the binding reaction mixture without dye. After electrophoresis at 4 °C for 2–3 h at 200 V, the gel was dried and autoradiographed. For assays in the presence of metal, 100 µM MnCl2 was added to the assay mix, electrophoresis buffer and the polyacrylamide gel. Plasmid pSKES3, containing a 300 bp SmaI–HpaI fragment (175 bp of upstream sequence from the irr transcriptional start site), was used to isolate various deletions of the irr upstream sequence for mobility shift experiments. The restriction-enzyme-digested DNA probes were either purified from 4% GTG Nusieve agarose (FMC BioProducts) or 15% acrylamide gels and radiolabelled at 30 °C for 1 h using [{alpha}-32P]dNTP and the Klenow fragment of DNA polymerase I.

Analysis of ALA dehydratase and Irr protein.
The presence of ALA dehydratase or Irr in whole cells or cell extracts was detected by immunoblot analysis of 10 or 15% SDS-PAGE gels using antibodies raised against the respective protein. Anti-ALA dehydratase and anti-Irr antibodies were prepared previously (Chauhan & O’Brian, 1995 ; Hamza et al., 1998 ) Cross-reactive material that bound to the membrane was analysed with peroxidase-conjugated goat anti-rabbit IgG and visualized by chemiluminescence by using the Renaissance kit (DuPont-NEN) according to the manufacturer’s instructions. Autoradiograms were quantified using a Bio-Rad model GS-700 imaging densitometer in the transmittance mode and the Molecular Analyst software package, version 1.5. Several exposures of a single blot were analysed to be certain that the data were examined within the linear range of the densitometer.

Isolation of RNA and mRNA analysis.
Total RNA was prepared and analysed as described previously (Chauhan & O’Brian, 1997 ) and quantified by measuring absorption at 260 and 280 nm. Cultured cells were grown to mid-exponential phase and steady-state levels of irr, hemA, hemB or hemH mRNA were analysed by the Ribonuclease Protection Assay kit (HybSpeed RPA; Ambion). Antisense RNA probes to the respective genes were synthesized and gel-purified using the T7 MAXIScript In Vitro Transcription kit (Ambion) as recommended by the manufacturer. The bands on autoradiograms of RNA gels were quantified using a Bio-Rad model GS-700 imaging densitometer in the transmittance mode as described above. The transcription start site of the irr gene was determined by primer extension analysis as described by Ausubel et al. (1994) using RNA isolated from B. japonicum cells grown in iron-limited media.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Fur regulates irr at the mRNA level
We showed previously that irr mRNA levels are iron-regulated (Hamza et al., 1998 ) and that B. japonicum Fur has metal-dependent DNA-binding activity (Hamza et al., 1999 ). Therefore, the effects of iron on irr mRNA expression were examined in the parent strain I110 and the fur strain GEM4. As demonstrated previously (Hamza et al., 1998 ), irr mRNA was approximately fivefold greater in iron-limited cells of the parent strain I110 compared with those grown in iron-rich media (Fig. 1). However, irr mRNA was not down-regulated in iron-replete cells of fur strain GEM4 compared with those grown under iron limitation, indicating that normal control of irr mRNA by iron was lost in the fur strain. By contrast, mRNA levels of hemH, a gene unresponsive to iron (Chauhan et al., 1997 ; Hamza et al., 1998 ), were not altered in the fur strain (Fig. 1). These data indicate that Fur is involved in iron-dependent control of irr mRNA accumulation. irr mRNA levels were lower in iron-limited cells of the mutant than those of the parent strain, which may indicate an activity for Fur when iron availability is low. If so, the activity would likely be indirect because data below suggest that Fur binds to the irr promoter in the presence of metal.



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Fig. 1. Effects of a fur gene mutation on iron-dependent accumulation of irr mRNA. Cells from parent strain I110 or fur strain GEM4 were grown in media containing either no (-) or 6 µM (+) added FeCl3. Cells were analysed for irr or hemH mRNA by RNase protection analysis. Two micrograms of total RNA was analysed per reaction. hemH is a control for a gene not regulated by iron.

 
Evidence that Fur directly regulates the irr gene
We addressed the effects of Fur on irr promoter activity in E. coli cells using an irr::lacZ fusion (Fig. 2) along with plasmid-borne fur genes of B. japonicum or E. coli. The irr promoter was active in E. coli as discerned by ß-galactosidase activity, which was only slightly iron-responsive in control cells lacking a plasmid-borne fur gene. However, introduction of the B. japonicum fur gene resulted in over a threefold repression of activity in the presence of iron compared to cells grown under iron deficiency, indicating that a B. japonicum Fur (BjFur)-responsive element was present in the reporter gene fusion. Surprisingly, reporter activity was not significantly repressed by E. coli Fur (EcFur) compared to the control, indicating an activity for BjFur that is absent in EcFur. We observe that lacZ reporter fusions are not sensitive tools in B. japonicum when mRNA synthesis levels are easily discernible, even in the repressed or uninduced state (S. Chauhan & M. R. O’Brian, unpublished observations). Consistent with this, ß-galactosidase activity was repressed only 30–50% by iron in B. japonicum harbouring pGDIrr-fuse (data not shown) even though iron affects irr mRNA synthesis and steady-state levels three to fivefold (Hamza et al., 1998 ; Fig. 1). Thus, we did not use the reporter for further experimentation in B. japonicum cells.



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Fig. 2. Dependence of an irr::lacZ reporter on Fur and iron in E. coli. Strain DH5{alpha} cells bearing pGDIrr-fuse and either pSKBJF800 (BjFur), pMH15fur (EcFur) or pSK{Delta}lac (Control) were grown in LB media containing either 200 µM of the iron chelator {alpha},{alpha}-dipyridyl (-) or 100 µM FeCl3 (+). ß-Galactosidase activity was measured and expressed in Miller units as described in the text. The data are means of triplicate samples±SD.

 
The effects of Fur on irr gene expression were assessed further by gel mobility shift assays using irr gene upstream DNA and overexpressed Fur proteins. In addition, the assays were carried out in the presence or absence of Mn2+, which has been shown to substitute for Fe2+ as a cofactor of Fur in vitro (de Lorenzo et al., 1988 ). Mn2+ is used because Fe2+ is readily oxidized to Fe3+ in air, which is not functional as a Fur cofactor. A 191 bp SmaI–MluI fragment corresponding to positions -175 to +16 with respect to the irr transcription start site was bound by BjFur in the presence of Mn2+ but not in its absence (Fig. 3). Collectively, the data in Figs 1–3 indicate that BjFur controls the irr gene directly and is a negative regulator in the presence of metal. Recombinant Irr did not bind to its promoter region in either the absence or presence of metal, suggesting that the irr gene does not directly regulate its own expression (I. Hamza & M. R. O’Brian, unpublished data). Interestingly, EcFur did not form a complex with the irr upstream region in a gel mobility shift assay (Fig. 3), which is consistent with the irr::lacZ reporter gene data (Fig. 2). These findings indicate that BjFur has a DNA-binding activity not present in EcFur.



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Fig. 3. Metal-dependent binding of BjFur to the irr gene upstream region. Gel mobility shift assays were carried out in the presence (a) or absence (b) of 100 µM MnCl2 using a 32P-labelled 191 bp SmaI–MluI fragment including nucleotides -175 to +16 of the irr upstream region (see Fig. 5). The protein samples used were from E. coli fur strain H1780 containing plasmids pSKBluescript(+) (pSK), pMH15fur (EcFur) or pSKBJF800 (BjFur). The DNA was also run in the absence of protein (Free).

 


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Fig. 5. Localization of the BjFur-binding region upstream of the irr gene. (a) Gel mobility shift assays were carried out using 32P-labelled subclones of the irr upstream region. ‘+’ denotes that BjFur bound to the DNA element in the presence of metal as indicated by a mobility shift. ‘-’ denotes no mobility shift. The bent arrow denotes the transcription start site of irr. ‘ATG’ denotes the translation initiation codon. (b) Mobility shift assays using the 63 bp BanII–SfaNI DNA fragment containing nucleotides -82 to -20 and extracts containing BjFur or EcFur, and controls containing either extracts with no Fur protein (pSK) or no protein (Free). (c) Nucleotide sequence of the 63 bp BanII–SfaNI fragment that binds to BjFur (underlined) and adjacent DNA (GenBank accession number AF052295). The bent arrow denotes the transcription start site. The boxed DNA shows sequences with positional and sequence similarity to the -35 and -10 promoter region genes from B. japonicum and other bacteria. The lower case ‘atg’ denotes the translation initiation codon.

 
BjFur binds to a unique region in the irr upstream region
Both BjFur and EcFur bind the Fur box consensus sequence (Hamza et al., 1999 ) and therefore the inability of EcFur to bind to the irr upstream region suggested a BjFur-binding site that is dissimilar from the Fur box consensus. Indeed, examination of the irr gene upstream region revealed no element with significant similarity to the Fur box consensus sequence. To further localize the BjFur-binding region, the transcription start site of irr was determined by primer extension analysis (Fig. 4) and restriction fragment subclones of the 191 bp SmaI–MluI fragment upstream region were analysed by a gel mobility shift assay (Fig. 5). The smallest subclone tested that bound BjFur was a 63 bp BanII–SfaNI DNA fragment corresponding to nucleotides -82 to -20 of the irr promoter region (Fig. 5c). This fragment contains no substantial similarity to the Fur consensus sequence (best alignment is 7 of 19 nucleotides) and was not bound by EcFur (Fig. 5b). Both the BjFur and EcFur protein preparations used in the analysis bound to the Fur box consensus DNA (data not shown). We suggest that BjFur has a unique DNA-binding activity not found in EcFur in addition to its Fur-box-binding activity.



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Fig. 4. Primer extension mapping of the 5' end of irr mRNA. Total RNA was isolated from strain I110 cells grown under iron limitation. The oligonucleotide primer used is complementary to nucleotides between +29 and +46 of the irr gene. DNA sequencing reactions were carried out with the same oligonucleotide. The arrow indicates the 5' end of the irr transcript.

 
Control of Irr protein level by iron is independent of Fur. Irr protein turns over rapidly in the presence of iron and is undetectable in cells grown in iron-replete media. (Hamza et al., 1998 ; Qi et al., 1999 ). To further address the post-transcriptional control of the irr gene, we analysed the effects of iron on Irr protein accumulation by immunoblot analysis in parent strain I110 and fur mutant strain GEM4 (Fig. 6) Interestingly, the fur strain showed an iron-dependent Irr expression pattern similar to the wild-type (Fig. 6) despite the aberration in message accumulation (Fig. 1). These findings confirm the conclusion that post-transcriptional control is the predominant influence on expression of the irr gene. Furthermore, the normal iron-dependent accumulation of Irr in the fur strain indicates that post-transcriptional control of irr by iron is not Fur-dependent, and therefore B. japonicum must have another mechanism for sensing and responding to iron.



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Fig. 6. Iron-dependent accumulation of Irr and ALA dehydratase (ALAD) in wild-type parent strain I110 and fur strain GEM4. Cells were grown in media containing either no (-) or 6 µM (+) added FeCl3. Irr and ALA dehydratase proteins were detected in whole cell extracts by Western blot analysis using anti-Irr or anti-ALA dehydratase antibodies. Twenty-five micrograms of protein extract was loaded per lane.

 
Fur does not directly control expression of the haem biosynthesis enzyme ALA dehydratase
The haem biosynthesis enzyme ALA dehydratase, encoded by hemB, is iron-regulated in B. japonicum, and Irr mediates negative control of that gene in response to iron limitation (Chauhan et al., 1997 ; Hamza et al., 1998 ). Consequently, hemB mRNA and protein levels are repressed over 100-fold in iron-limited cells of the wild-type, but remain high in an irr mutant strain. To determine whether Fur was involved in iron-dependent ALA dehydratase expression, immunoblot analysis was carried out using total protein from cells of parent strain I110 and fur strain GEM4 grown in low- or high-iron media (Fig. 6). ALA dehydratase accumulated to high levels in cells of both the wild-type and mutant grown in iron-replete media and was very low in iron-limited cells; thus regulation was essentially normal in the fur strain. These findings indicate that Fur is not required for full expression of hemB. The normal regulated expression of ALA dehydratase in the fur strain was likely due to the fact that Irr protein, which controls ALA dehydratase, remained iron-dependent in the mutant (Fig. 6).

Fur and Irr mediate iron-dependent control of hemA and hemB, respectively
The B. japonicum hemA and hemB genes are both regulated by iron (Hamza et al., 1998 ; Page et al., 1994 ), but only a regulator of hemB has been described previously (Hamza et al., 1998 ). The current work indicates that Fur and Irr can mediate cellular responses to iron independently, and a region with weak similarity to the Fur box sequence was found in the hemA promoter by Page et al. (1994) . Therefore, regulation of hemA and hemB transcripts by iron was examined in irr and fur strains by RNase protection analysis (Fig. 7). Transcripts of hemA and hemB were low in cells grown under iron deprivation and high under iron-replete conditions in parent strain I110. As shown previously (Hamza et al., 1998 ), control of hemB by iron was lost in the irr strain, resulting in elevated transcript even under iron deprivation. hemB mRNA levels were controlled normally in fur strain GEM4, which is in agreement with the observed protein levels (Fig. 6) and shows that hemB is not regulated by Fur. By contrast, hemA mRNA levels were unresponsive to iron in the fur strain, with the maintenance of high levels under both high- and low-iron conditions (Fig. 7), and thus Fur is involved in iron-dependent expression of hemA. However, hemA was regulated normally in strain LODTM5, indicating that Irr does not control that gene. These data strongly indicate different mechanisms for the regulation of hemA and hemB by iron, and show that Irr and Fur can act independently of each other.



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Fig. 7. Effects of a fur or irr gene mutation on iron-dependent accumulation of hemA and hemB mRNA. Cells from wild-type (WT) parent strain I110, irr strain LODTM5 or fur strain GEM4 were grown in media containing either no (-) or 6 µM (+) added FeCl3. Cells were analysed for hemA or hemB mRNA by RNase protection analysis. Two micrograms of total RNA was analysed per reaction.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the present study, we demonstrate that Fur controls the irr gene at the mRNA level in a metal-dependent manner and that it binds to the irr promoter in a region dissimilar to the Fur box consensus. Screens for DNA elements from Salmonella typhimurium (Tsolis et al., 1995 ) or Pseudomonas aeruginosa (Ochsner & Vasil, 1996 ) that bind to Fur by an in vitro selection procedure or an in vivo titration assay, respectively, did not reveal elements with significant deviation from the iron box consensus sequence. However, fur genes have been identified in numerous bacteria in which the respective proteins have not been extensively characterized, and thus new properties for Fur may be found in those organisms. The fur gene of Rhizobium leguminosarum has been cloned (De Luca et al., 1998 ) and it would be interesting to assess the DNA-binding properties of that protein. The significance of the transcriptional control of irr by iron is not obvious since the accumulation of Irr was essentially normal in the fur strain under the conditions examined. It is possible that an increase in the irr mRNA level upon a decrease in the cellular iron concentration would facilitate Irr synthesis, but the steady-state level of protein is more greatly affected by post-transcriptional control.

The iron responsiveness of Irr accumulation in the fur strain shows that B. japonicum must have a mechanism for sensing and responding to the cellular iron level in addition to Fur. Haem mediates iron-dependent degradation of Irr (Qi et al., 1999 ) and thus haem may be the form of iron to which Irr responds. Also, iron may in some way activate the protein(s) that degrades Irr. In either event, the findings indicate that aspects of iron metabolism need not be under the control of the Fur regulon in B. japonicum. Furthermore, Irr is a regulatory protein and therefore ALA dehydratase may be one of numerous iron-dependent cellular processes that do not require Fur. Two-dimensional PAGE analysis of proteins from S. typhimurium and Vibrio cholerae wild-type and fur strains revealed proteins regulated by iron, but which are Fur-independent (Foster & Hall, 1992 ; Litwin & Calderwood, 1994 ). Thus, alternative mechanisms of regulating iron-dependent processes may be generally applicable in bacteria that express Fur. The need for multiple iron regulatory proteins will likely be clearer when the form of iron to which Irr responds is known, and when the respective regulons are more completely characterized.

The presence of two systems for mediating iron control of gene expression was underscored further by examination of the haem biosynthesis genes hemA and hemB. Both genes are regulated by iron, but analysis of mutants shows that hemA is only affected by Fur whereas hemB is controlled only by Irr under the conditions examined. Furthermore, the aberration in hemA expression in the fur strain was found under low iron, indicating a direct or indirect role for Fur under those conditions. Although many studies, including this one, demonstrate activity in the presence of metal, other studies show that Fur can bind to DNA in the absence of metal (Althaus et al., 1999 ; Bsat & Helmann, 1999 ), and that it can have a physiological function under iron limitation (Foster & Hall, 1992 ). Finally, ALA synthase and ALA dehydratase, the respective hemA and hemB products, are part of a pathway committed to the same products; thus it is intriguing that they are regulated by iron via different systems. A role for ALA dehydratase in addition to haem synthesis has been reported in animals (Guo et al., 1994 ), but no similar function has been described in bacteria. The substrates for ALA synthase, glycine and succinyl coenzyme A, are involved in numerous cellular processes whereas ALA metabolized by ALA dehydratase is a committed intermediate. It is possible that ALA synthase and ALA dehydratase need to be coordinated with different enzymes even though they are part of the same pathway. Furthermore, B. japonicum can acquire ALA from its soybean host in symbiosis (Chauhan & O’Brian, 1993 ; Sangwan & O’Brian, 1991 ); thus the need for the two enzymes may be different in that context.


   ACKNOWLEDGEMENTS
 
The authors thank Yali Friedman for determining the transcription start site of irr. This work was supported by a grant from the National Science Foundation to M.R.O’B.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 24 August 1999; revised 12 November 1999; accepted 18 November 1999.