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Heme-responsive DNA Binding by the Global Iron Regulator Irr from Rhizobium leguminosarum*

  • Chloe Singleton
    Footnotes
    Affiliations
    Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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  • Gaye F. White
    Footnotes
    Affiliations
    Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich NR4 7TJ, United Kingdom

    School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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  • Jonathan D. Todd
    Affiliations
    School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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  • Sophie J. Marritt
    Affiliations
    Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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  • Myles R. Cheesman
    Affiliations
    Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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  • Andrew W.B. Johnston
    Correspondence
    To whom correspondence may be addressed: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK. Fax: 44-1603-592250
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    School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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  • Nick E. Le Brun
    Correspondence
    To whom correspondence may be addressed: School of Chemistry, University of East Anglia, Norwich NR4 7TJ, UK. Fax: 44-1603-592003
    Affiliations
    Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich NR4 7TJ, United Kingdom
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  • Author Footnotes
    * This work was supported by the UK BBSRC through Grant BB/E003400/1 (to A. W. B. J. and N. E. L. B.), Grants BB/E013252/1 and BB/C007808/1 (to M. R. C.), and the Wellcome Trust through an award from the Joint Infrastructure Fund for equipment.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5 and Table S1.
    1 Both authors contributed equally to this work.
Open AccessPublished:March 16, 2010DOI:https://doi.org/10.1074/jbc.M109.067215
      Heme, a physiologically crucial form of iron, is a cofactor for a very wide range of proteins and enzymes. These include DNA regulatory proteins in which heme is a sensor to which an analyte molecule binds, effecting a change in the DNA binding affinity of the regulator. Given that heme, and more generally iron, must be carefully regulated, it is surprising that there are no examples yet in bacteria in which heme itself is sensed directly by a reversibly binding DNA regulatory protein. Here we show that the Rhizobium leguminosarum global iron regulatory protein Irr, which has many homologues within the α-proteobacteria and is a member of the Fur superfamily, binds heme, resulting in a dramatic decrease in affinity between the protein and its cognate, regulatory DNA operator sequence. Spectroscopic studies of wild-type and mutant Irr showed that the principal (but not only) heme-binding site is at a conserved HXH motif, whose substitution led to loss of DNA binding in vitro and of regulatory function in vivo. The R. leguminosarum Irr behaves very differently to the Irr of Bradyrhizobium japonicum, which is rapidly degraded in vivo by an unknown mechanism in conditions of elevated iron or heme, but whose DNA binding affinity in vitro does not respond to heme.

      Introduction

      Heme is perhaps the most widespread and physiologically important form in which iron is found in life, being involved, for example, in electron transfer, enzyme catalysis, and small molecule metabolism. Heme is also potentially toxic, and in eukaryotic organisms, levels of heme are sensed directly by the transcriptional repressor Bach1, which regulates heme degradation (
      • Ogawa K.
      • Sun J.
      • Taketani S.
      • Nakajima O.
      • Nishitani C.
      • Sassa S.
      • Hayashi N.
      • Yamamoto M.
      • Shibahara S.
      • Fujita H.
      • Igarashi K.
      ), and the transcriptional activator Hap1, a key regulator of the oxidative stress response (
      • Zhang L.
      • Hach A.
      ). Other eukaryotic transcription factors, such as NPAS2 and REV-ERB alpha and beta utilize heme to sense gas molecule analytes (
      • Dioum E.M.
      • Rutter J.
      • Tuckerman J.R.
      • Gonzalez G.
      • Gilles-Gonzalez M.A.
      • McKnight S.L.
      ,
      • Yin L.
      • Wu N.
      • Curtin J.C.
      • Qatanani M.
      • Szwergold N.R.
      • Reid R.A.
      • Waitt G.M.
      • Parks D.J.
      • Pearce K.H.
      • Wisely G.B.
      • Lazar M.A.
      ,
      • Pardee K.I.
      • Xu X.
      • Reinking J.
      • Schuetz A.
      • Dong A.
      • Liu S.
      • Zhang R.
      • Tiefenbach J.
      • Lajoie G.
      • Plotnikov A.N.
      • Botchkarev A.
      • Krause H.M.
      • Edwards A.
      ). In bacteria, only a very few known transcriptional regulators respond to and interact with heme. For example, Rhodospirillum rubrum CooA, a member of the CRP regulatory family, is a CO-sensing regulator of the CO oxidation pathway (
      • Lanzilotta W.N.
      • Schuller D.J.
      • Thorsteinsson M.V.
      • Kerby R.L.
      • Roberts G.P.
      • Poulos T.L.
      ). But, until now, there are no examples of bacterial regulatory proteins that modulate DNA binding affinity directly and solely in response to heme binding.
      Heme and iron metabolism are intimately connected, and, in many organisms, evolution has favored regulatory networks in which iron is sensed directly as Fe2+, giving rise to the iron regulatory paradigms of Fur and DtxR. The very widespread Fur transcriptional regulator contains DNA binding and sensory domains. When bound to Fe2+, the protein binds to Fur box operator sequences 5′ of Fur-regulated genes, thereby (usually) repressing their transcription in iron-sufficient conditions (
      • Hantke K.
      ). When iron is scarce, Fe2+-depleted Fur dissociates from its target operators, switching on genes involved in (e.g.), iron recruitment and uptake. DtxR, a functionally similar but evolutionarily unrelated Fe2+-sensing global iron regulator, occurs in some high GC Gram-positive bacteria (
      • Ding X.
      • Zeng H.
      • Schiering N.
      • Ringe D.
      • Murphy J.R.
      ).
      Recently, it was established that the Fur/DtxR mode of regulation is far from universal in bacteria: a wide range of α-proteobacteria, including some of the most abundant bacteria on Earth, either lack Fur or contain Fur homologues that are relegated to more parochial, or different, regulatory roles including the sensing of manganese, rather than iron, availability (
      • Rudolph G.
      • Hennecke H.
      • Fischer H.M.
      ,
      • Johnston A.W.
      • Todd J.D.
      • Curson A.R.
      • Lei S.
      • Nikolaidou-Katsaridou N.
      • Gelfand M.S.
      • Rodionov D.A.
      ,
      • Rodionov D.A.
      • Gelfand M.S.
      • Todd J.D.
      • Curson A.R.
      • Johnston A.W.
      ). R. leguminosarum, which forms N2-fixing nodules on various legume plants, and its close relatives, the phytopathogens Agrobacterium (
      • Ngok-Ngam P.
      • Ruangkiattikul N.
      • Mahavihakanont A.
      • Virgem S.S.
      • Sukchawalit R.
      • Mongkolsuk S.
      ) and mammalian pathogens Brucella (
      • Martínez M.
      • Ugalde R.A.
      • Almirón M.
      ) and Bartonella (
      • Parrow N.L.
      • Abbott J.
      • Lockwood A.R.
      • Battisti J.M.
      • Minnick M.F.
      ) as well as Sinorhizobium meliloti (
      • Chao T.C.
      • Buhrmester J.
      • Hansmeier N.
      • Pühler A.
      • Weidner S.
      ) and Mesorhizobium loti (
      • Carlton T.M.
      • Sullivan J.T.
      • Stuart G.S.
      • Hutt K.
      • Lamont I.L.
      • Ronson C.W.
      ) (which nodulate alfalfa and Lotus, respectively), contain two novel, wide-ranging iron-responsive transcriptional regulators, RirA and Irr. RirA recognizes cis-acting iron regulatory sequences (RirA-boxes) that precede genes whose function (e.g. iron uptake) are of the type that would be repressed by Fur in bacteria that use Fur as the primary iron-responsive transcriptional regulator (
      • Rodionov D.A.
      • Gelfand M.S.
      • Todd J.D.
      • Curson A.R.
      • Johnston A.W.
      ,
      • Wexler M.
      • Todd J.D.
      • Kolade O.
      • Bellini D.
      • Hemmings A.M.
      • Sawers G.
      • Johnston A.W.B.
      ,
      • Yeoman K.H.
      • Curson A.R.
      • Todd J.D.
      • Sawers G.
      • Johnston A.W.
      ,
      • Todd J.D.
      • Wexler M.
      • Sawers G.
      • Yeoman K.H.
      • Poole P.S.
      • Johnston A.W.B.
      ,
      • Todd J.D.
      • Sawers G.
      • Johnston A.W.
      ). However, RirA is unrelated to Fur, being in a family of regulators that includes IscR and NsrR (
      • Schwartz C.J.
      • Giel J.L.
      • Patschkowski T.
      • Luther C.
      • Ruzicka F.J.
      • Beinert H.
      • Kiley P.J.
      ,
      • Tucker N.P.
      • Hicks M.G.
      • Clarke T.A.
      • Crack J.C.
      • Chandra G.
      • Le Brun N.E.
      • Dixon R.
      • Hutchings M.I.
      ). Like them, RirA is a predicted FeS cluster-binding protein.
      Irr is in the Fur superfamily, but has important differences compared with Fur sensu stricto. Its major role is to repress many genes, such as those involved in heme biosynthesis or bacterioferritin production, under conditions of low iron availability. It does so, mostly, by binding to cis-acting regulatory sequences (iron control element (ICE)
      The abbreviations used are: ICE
      iron control element
      CD
      circular dichroism
      DTT
      dithiothreitol
      EMSA
      electrophoretic mobility shift assay
      EPR
      electron paramagnetic resonance
      HRM
      heme regulatory motif
      ICP-AE
      inductively coupled plasma atomic emission
      NIR-MCD
      near infra-red magnetic circular dichroism.
      boxes) that are 5′ of the target genes, repressing their transcription. This differs from conventional Fur, which represses its target genes under iron-sufficient conditions. Unlike other Fur family members (Fur, Mur, Zur), which require a metal cofactor to act as repressors (
      • An Y.J.
      • Ahn B.E.
      • Han A.R.
      • Kim H.M.
      • Chung K.M.
      • Shin J.H.
      • Cho Y.B.
      • Roe J.H.
      • Cha S.S.
      ,
      • Lee J.W.
      • Helmann J.D.
      ,
      • Lucarelli D.
      • Russo S.
      • Garman E.
      • Milano A.
      • Meyer-Klaucke W.
      • Pohl E.
      ,
      • Díaz-Mireles E.
      • Wexler M.
      • Sawers G.
      • Bellini D.
      • Todd J.D.
      • Johnston A.W.
      ), Irr proteins are repressors in the absence of their analyte molecule. And, rather than sensing iron directly, Irr proteins bind (and, so, sense) heme.
      Thus, R. leguminosarum has two “opposing” regulators, which repress different portfolios of target genes under Fe-sufficient (RirA) or Fe-deficient (IrrRl) levels of iron. Because RirA and Irr respond to the availability of two biologically significant iron-containing molecules, FeS clusters and heme, respectively, their iron regulons may better sense the physiologically relevant status of iron, not just the concentration of the metal per se, as occurs in those organisms that use Fur or DtxR (
      • Johnston A.W.
      • Todd J.D.
      • Curson A.R.
      • Lei S.
      • Nikolaidou-Katsaridou N.
      • Gelfand M.S.
      • Rodionov D.A.
      ). Furthermore, Irr and RirA interact with each other, each affecting the transcription of the other in response to iron availability (
      • Todd J.D.
      • Sawers G.
      • Rodionov D.A.
      • Johnston A.W.B.
      ).
      In contrast, in Bradyrhizobium japonicum, a member of the rhizobia that is related to R. leguminosarum, but which lacks RirA, both the positive and negative regulatory responses to Fe availability can be mediated by its Irr (here termed IrrBj), which has a more wide-ranging set of target genes than does IrrRl in Rhizobium and its close relatives. Indeed, in iron-deficient cells, IrrBj can act as a positive regulator of some promoters, such as those involved in iron uptake although the reasons why some B. japonicum promoters are repressed and others are positively activated by IrrBj are not clear (
      • Small S.K.
      • Puri S.
      • Sangwan I.
      • O'Brian M.R.
      ).
      IrrBj has been studied in some detail (
      • Hamza I.
      • Chauhan S.
      • Hassett R.
      • O'Brian M.R.
      ,
      • Rudolph G.
      • Semini G.
      • Hauser F.
      • Lindemann A.
      • Friberg M.
      • Hennecke H.
      • Fischer H.M.
      ,
      • Sangwan I.
      • Small S.K.
      • O'Brian M.R.
      ,
      • Qi Z.
      • Hamza I.
      • O'Brian M.R.
      ,
      • Yang J.
      • Ishimori K.
      • O'Brian M.R.
      ). Heme binding to IrrBj, which occurs via the enzyme ferrochelatase (
      • Qi Z.
      • O'Brian M.R.
      ), has the remarkable effect of forming a complex that is extremely unstable and is rapidly degraded via an unknown mechanism. Thus, in iron-replete conditions, IrrBj is unavailable to repress or activate its target genes.
      Heme interacts with at least two different sites on IrrBj. One of these, toward the N terminus of IrrBj, is a heme regulatory motif (HRM), which is a widely distributed short sequence motif that is involved in heme-mediated regulation in many different proteins (
      • Zhang L.
      • Guarente L.
      ). The other site is an internal, histidine-rich (HXH) motif (
      • Yang J.
      • Ishimori K.
      • O'Brian M.R.
      ). In addition to functional differences, there are significant differences in the sequences of the IrrBj and the IrrRl polypeptides. Most notably, the latter lacks the important N-terminal HRM motif of IrrBj, and so the mechanism by which it senses heme is unclear. Therefore, we set out to investigate in more detail the functions and the biochemical properties of IrrRl.
      Here, we use in vivo and in vitro studies to show that IrrRl also binds heme, but that this does not affect its stability in vivo. However, it does cause the protein to lose its high affinity DNA binding ability, via a conformational change. This is the first example of a bacterial DNA regulatory protein in which DNA binding affinity is modulated directly by heme.

      Acknowledgments

      We thank Jaeger Hamilton for assistance with in vivo functional studies.

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