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. OBrian. Tel: +1 716 829 3200. Fax: +1 716 829 2725. e-mail: mrobrian{at}buffalo.edu
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ABSTRACT |
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Keywords: Bradyrhizobium japonicum, iron metabolism, Fur, Irr, haem metabolism
Abbreviations: ALA, -aminolevulinic acid
a Present address: Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA.
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INTRODUCTION |
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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 ).
-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.
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METHODS |
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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
, XL-1 Blue or TB1 were used for propagation of plasmids. E. coli strains were grown at 37 °C on LuriaBertani 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
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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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, 1116 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 23 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 SmaIHpaI 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 [
-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 & OBrian, 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 manufacturers 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 & OBrian, 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.
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RESULTS |
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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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DISCUSSION |
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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 & OBrian, 1993
; Sangwan & OBrian, 1991
); thus the need for the two enzymes may be different in that context.
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ACKNOWLEDGEMENTS |
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Received 24 August 1999;
revised 12 November 1999;
accepted 18 November 1999.