Interaction of HapX with the CCAAT-binding complex—a novel mechanism of gene regulation by iron
2007; Springer Nature; Volume: 26; Issue: 13 Linguagem: Inglês
10.1038/sj.emboj.7601752
ISSN1460-2075
AutoresPeter Hortschansky, Martin Eisendle, Qusai Al Abdallah, André D. Schmidt, Sebastian Bergmann, Marcel Thön, Olaf Kniemeyer, Beate Abt, Birgit Seeber, Ernst R. Werner, Masashi Kato, Axel A. Brakhage, Hubertus Haas,
Tópico(s)Insect Resistance and Genetics
ResumoArticle14 June 2007free access Interaction of HapX with the CCAAT-binding complex—a novel mechanism of gene regulation by iron Peter Hortschansky Peter Hortschansky Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Martin Eisendle Martin Eisendle Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Qusai Al-Abdallah Qusai Al-Abdallah Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author André D Schmidt André D Schmidt Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Sebastian Bergmann Sebastian Bergmann Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Marcel Thön Marcel Thön Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Olaf Kniemeyer Olaf Kniemeyer Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Beate Abt Beate Abt Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Birgit Seeber Birgit Seeber Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Ernst R Werner Ernst R Werner Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Masashi Kato Masashi Kato Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Axel A Brakhage Corresponding Author Axel A Brakhage Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Hubertus Haas Corresponding Author Hubertus Haas Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Peter Hortschansky Peter Hortschansky Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Martin Eisendle Martin Eisendle Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Qusai Al-Abdallah Qusai Al-Abdallah Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author André D Schmidt André D Schmidt Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Sebastian Bergmann Sebastian Bergmann Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Marcel Thön Marcel Thön Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Olaf Kniemeyer Olaf Kniemeyer Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Beate Abt Beate Abt Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Birgit Seeber Birgit Seeber Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Ernst R Werner Ernst R Werner Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Masashi Kato Masashi Kato Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan Search for more papers by this author Axel A Brakhage Corresponding Author Axel A Brakhage Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany Search for more papers by this author Hubertus Haas Corresponding Author Hubertus Haas Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria Search for more papers by this author Author Information Peter Hortschansky1,‡, Martin Eisendle2,‡, Qusai Al-Abdallah1, André D Schmidt1, Sebastian Bergmann1, Marcel Thön1, Olaf Kniemeyer1, Beate Abt2, Birgit Seeber2, Ernst R Werner3, Masashi Kato4, Axel A Brakhage 1 and Hubertus Haas 2 1Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Jena, Germany 2Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria 3Division of Biological Chemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria 4Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan ‡These authors contributed equally to this work *Corresponding authors: Division of Molecular Biology, Biocenter, Innsbruck Medical University, Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria. Tel.: +43 512 9003 70205; Fax: +43 512 9003 73100; E-mail: [email protected] Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology (HKI), and Friedrich-Schiller-University Jena, Beutenbergstrasse 11a, 07745 Jena, Germany. Tel.: +49 3641 656601; Fax: +49 3641 656603; E-mail: [email protected] The EMBO Journal (2007)26:3157-3168https://doi.org/10.1038/sj.emboj.7601752 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Iron homeostasis requires subtle control systems, as iron is both essential and toxic. In Aspergillus nidulans, iron represses iron acquisition via the GATA factor SreA, and induces iron-dependent pathways at the transcriptional level, by a so far unknown mechanism. Here, we demonstrate that iron-dependent pathways (e.g., heme biosynthesis) are repressed during iron-depleted conditions by physical interaction of HapX with the CCAAT-binding core complex (CBC). Proteome analysis identified putative HapX targets. Mutual transcriptional control between hapX and sreA and synthetic lethality resulting from deletion of both regulatory genes indicate a tight interplay of these control systems. Expression of genes encoding CBC subunits was not influenced by iron availability, and their deletion was deleterious during iron-depleted and iron-replete conditions. Expression of hapX was repressed by iron and its deletion was deleterious during iron-depleted conditions only. These data indicate that the CBC has a general role and that HapX function is confined to iron-depleted conditions. Remarkably, CBC-mediated regulation has an inverse impact on the expression of the same gene set in A. nidulans, compared with Saccharomyces cerevisae. Introduction The cis-acting sequence CCAAT is present in approximately 30% of eukaryotic promoters (Bucher, 1990). An evolutionarily conserved protein complex able to bind to this motif has been found in all eukaryotes analyzed so far, ranging from yeast to mammals. It has been designated Hap complex in Saccharomyces cerevisiae (Pinkham and Guarente, 1985; McNabb et al, 1995), Kluyveromyces lactis (Mulder et al, 1994), and Arabidopsis thaliana (Edwards et al, 1998), Php in Schizosaccharomyces pombe (McNabb et al, 1997), AnCF in Aspergillus species (reviewed in Brakhage et al, 1999), CBF in Xenopus laevis (Li et al, 1998), and NF-Y in mammals (Hooft van Huijsduijnen et al, 1990; Maity et al, 1990), respectively. The S. cerevisiae Hap complex was the first CCAAT-binding complex to be identified. It comprises four subunits, Hap2p, Hap3p, Hap4p and Hap5p. Hap2/3/5p form the core CCAAT-binding complex (here termed CBC), which is responsible for DNA binding, while Hap4p is involved in transcriptional activation (McNabb et al, 1995). Orthologs of Hap2/3/5p are present in all eukaryotes. Moreover, S. cerevisiae Hap2p, A. nidulans HapB and human NF-YA are functionally interchangeable (Becker et al, 1991; Tuncher et al, 2005), which demonstrates high evolutionary conservation of the CBC. However, with the exception of the yeast species K. lactis and Hansenula polymorpha, clear evidence for a Hap4p ortholog in other organisms is inconclusive (Bourgarel et al, 1999; Sybirna et al, 2005). In S. cerevisiae, the Hap4p/CBC complex acts as an activator of genes involved in oxidative phosphorylation in response to growth on non-fermentable carbon sources (Pinkham and Guarente, 1985). The A. nidulans CBC, consisting of the Hap2/3/5p orthologs HapB/C/E, modulates the expression of numerous genes, including the anabolic penicillin biosynthesis genes acvA, ipnA and aatA (Litzka et al, 1996; Then Bergh et al, 1996) and the catabolic acetamidase encoding amdS (Littlejohn and Hynes, 1992). In this respect it has also been shown that the activation of gene expression by pathway-specific regulators can depend on the presence of a functional CBC (Steidl et al, 1999). At the same time, evidence for CBC-mediated repression of gene expression was found in A. nidulans for homoaconitase-encoding lysF and in the auto-regulation of hapB expression (Steidl et al, 2001; Weidner et al, 2001). Recently, the CBC was also found to act as a repressor of mitochondrial electron transport components in Candida albicans (Johnson et al, 2005). However, a clear picture of the CBC regulon in fungi is missing so far. A yeast-two-hybrid screen suggested physical interaction of A. nidulans HapB with a protein of a yet unknown function, termed HapX (Tanaka et al, 2002). HapX displays no similarity to S. cerevisiae Hap4p, apart from an N-terminal 17 amino-acid-motif, which has been shown to be essential for interaction of Hap4p with the S. cerevisiae CBC (McNabb and Pinto, 2005). Deletion of hapX in A. nidulans did not result in a slow-growth and weak-conidiation phenotype, as caused by a deletion of any of the three CBC subunit-encoding genes (Steidl et al, 1999). Furthermore, expression of hapX in S. cerevisiae did not complement the deletion of Hap4. Therefore, a function of HapX in CBC-mediated gene expression appeared unlikely at that time. We resumed the functional analysis of hapX, as we found that its expression is repressed by iron via the GATA factor SreA. As a cofactor for numerous enzymes and in electron transport systems, iron is indispensable for all eukaryotes. However, an excess of iron is toxic, due to its capacity to catalyze the production of cell damaging reactive oxygen species (Halliwell and Gutteridge, 1984). Therefore, subtle control systems are required to maintain iron homeostasis. In A. nidulans, iron represses siderophore-mediated iron uptake, which is the major iron acquisition system and is essential for fungal virulence (Schrettl et al, 2004; Oide et al, 2006), and induces iron-dependent pathways (Haas et al, 1999; Oberegger et al, 2001, 2002b). The repression of the siderophore system under iron-replete (+Fe) conditions is mediated, at least in part, by SreA. Here, we demonstrate that HapX mediates repression of iron-dependent pathways under iron-depleted (−Fe) conditions via interaction with the CBC. HapX target genes were identified by a proteomic approach. The importance of a tight interplay between HapX and SreA is demonstrated by mutual transcriptional control between hapX and sreA and synthetic lethality resulting from deletion of both regulatory genes. Results Deletion of hapX or of genes encoding CBC subunits impedes growth during −Fe conditions A differential mRNA display screen for SreA target genes (Oberegger et al, 2001) suggested that the steady-state level of hapX mRNA is repressed by iron in the wild type and partially derepressed in an sreA deletion (Δ) strain (data not shown). This expression pattern was confirmed using Northern analysis (Figure 1A). Notably, deletion of sreA results in about 30% derepression, which is typical for SreA target genes, for example, siderophore transporter-encoding mirB (Haas et al, 2003). Figure 1.Iron-regulated gene expression in A. nidulans wild-type, ΔsreA, ΔhapX and ΔhapC strains. For Northern analysis, the total RNA was isolated from A. nidulans strains grown for 24 h under +Fe and −Fe conditions. As a control for loading and RNA quality, blots were hybridized with the γ-actin encoding acnA gene. (A) Expression of hapX but not hapC is partially controlled by SreA-mediated iron regulation. (B) Deletion of hapX or hapC causes derepression of sreA, during −Fe conditions but not the SreA regulon, during +Fe conditions. (C) Deletion of hapX or of hapC causes derepression of iron-dependent pathways during iron starvation. (D) A. nidulans siderophores. (E) A. nidulans siderophore metabolism. Known genes involved in siderophore biosynthesis and uptake are shaded in gray. Download figure Download PowerPoint We compared the phenotypic consequences of hapX deletion during +Fe and −Fe conditions, because of the iron-dependent expression of hapX. Consistent with the previous analysis (Tanaka et al, 2002), during +Fe conditions, ΔhapX displayed no significant difference to a wild-type strain with respect to conidiation and growth rate on a solid medium (data not shown) and in a liquid medium (Table I). During −Fe conditions, however, the growth rate was significantly reduced in submerged cultures (Table I), and, on a solid medium, the mycelium was less dense and conidiation was severely impaired (data not shown). Ectopic integration of a functional hapX gene (strain hapXR) cured these and all other defects of ΔhapX (Table I; Figure 2), which demonstrates that the ΔhapX phenotype is a direct result of the loss of HapX activity. Figure 2.Deletion of hapX or genes encoding CBC subunits leads to cellular accumulation of PpIX, decreased TAFC synthesis and increased FC production, during −Fe conditions. (A) Mycelia of A. nidulans strains after growth for 24 h during +Fe and –Fe conditions. (B) Characteristic red auto-fluorescence caused by PpIX accumulation during −Fe conditions. During +Fe conditions, no auto-fluorescence was detectable in any strain (data not shown). (C) Quantification of PpIX accumulation. (D) Representative chromatograms of porphyrin analysis of wild-type, ΔhapX and ΔhapC strains after growth under −Fe conditions. C8, C7, C6, C5, C4 and C2 denote uroporphyrin, heptacarboxylporphyrin, hexacarboxylporphyrin, pentacarboxylporphyrin, coproporphyrin and protoporphyrin, respectively. (E) Quantification of siderophore production during −Fe conditions normalized to that of the wild type. The data represent the mean±s.d. of three simultaneously harvested flasks. Download figure Download PowerPoint Table 1. Deletion of hapX, hapB, hapC or hapE causes a reduced growth rate during −Fe conditions Strain Growth condition Ratio +Fe −Fe −Fe/+Fe Wild type 100.0 66.9±8.4 0.67 ΔhapX 91.6±7.0 49.5±5.3 0.54 hapXR 114.3±5.8 74.2±6.4 0.65 ΔhapB 46.3±5.9 23.7±3.1 0.51 ΔhapC 61.6±7.6 28.3±4.2 0.46 ΔhapE 54.6±6.3 25.5±2.7 0.47 Strains were grown for 24 h in –Fe and +Fe conditions. Dry weights were normalized to that of the wild type grown during iron-replete conditions, which was 0.58±0.04 g. HapX was initially found to interact with the CBC subunit HapB. To ascertain a possible metabolic connection, we also compared the growth rates of strains lacking individual CBC subunits during +Fe and –Fe conditions. Deletion of hapB, hapC or hapE impairs growth during +Fe, as shown previously (Steidl et al, 1999), but in particular during −Fe conditions (Table I), which indicates a special function of the CBC during −Fe conditions consistent with a possible HapX/CBC interaction. Deletion of hapX or of genes encoding CBC subunits causes accumulation of the iron-free heme precursor protoporphyrin IX (PpIX) during −Fe conditions In contrast to the wild type, ΔhapX, ΔhapB, ΔhapC and ΔhapE mycelia displayed a red pigmentation concomitant with red auto-fluorescence during −Fe but not +Fe conditions (Figure 2A and B), which is typical for PpIX accumulation. Determination of the PpIX content revealed no significant difference between −Fe and +Fe conditions in the wild type (Figure 2C). In contrast, the PpIX content of ΔhapX increased slightly during +Fe conditions, and increased about 80-fold during −Fe conditions (Figure 2C and D). Moreover, the content of the PpIX precursors uroporphyrin, heptacarboxylporphyrin, hexacarboxylporphyrin, pentacarboxylporphyrin and coproporphyrin was significantly increased in ΔhapX during −Fe conditions (Figure 2D). Similarly, deletion of any of the CBC subunit-encoding genes caused a 20- to 50-fold increase in PpIX accumulation (Figure 2C). Deletion of hapX or of genes encoding CBC subunits causes decreased extracellular and increased intracellular siderophore production A. nidulans produces two major siderophores (Figure 1D), which are essential for growth (Eisendle et al, 2003); triacetylfusarinine C (TAFC) mobilizes extracellular iron and ferricrocin (FC) stores iron intracellularly (Eisendle et al, 2006). Because of the observed growth defect, we analyzed siderophore production in strains lacking HapX or CBC subunits (Figure 2E). During +Fe conditions, production of TAFC and FC was low and resembled the wild type in all strains (data not shown). During −Fe conditions, however, deletion of hapX or of any CBC subunit-encoding gene caused significantly decreased TAFC and increased FC production. Notably, the decrease in TAFC production was more pronounced in CBC subunit deletion mutants and FC accumulation was higher in a ΔhapX mutant. Supplementation with iron-free TAFC to a final concentration of 20 μM only partially cured the growth defect of ΔhapX during −Fe conditions, whereas PpIX accumulation remained unaffected (data not shown). These data suggest that reduced TAFC production does not account for the full extent of growth reduction and PpIX accumulation resulting from hapX deletion. HapX represses sreA expression without a general effect on the SreA regulon To further investigate the altered siderophore production of ΔhapX and ΔhapC strains, we performed a Northern analysis of the known genes involved in siderophore metabolism (Haas et al, 2003): sreA (repressor of siderophore metabolism), mirB (transporter for uptake of ferric TAFC), sidA (ornithine monooxygenase catalyzing the common step for biosynthesis of TAFC and FC) and sidC (non-ribosomal peptide synthetase required for FC synthesis) (Figure 1B). The proposed siderophore biosynthetic pathway is provided in Figure 1E. As shown previously (Oberegger et al, 2002b), expression of sreA is repressed during −Fe conditions, whereas that of the SreA target genes mirB, sidA, and sidC is repressed during +Fe conditions. Deletion of hapX or of hapC caused derepression of sreA during −Fe conditions (Figure 1B). In turn, as SreA represses siderophore biosynthesis and uptake, it was conceivable that SreA-regulated genes were repressed during −Fe conditions in these strains. However, regulation of mirB and sidA was unaffected in both ΔhapX and ΔhapC and, therefore, does not explain the reduced TAFC production. These data suggest that SreA-mediated repression requires either post-translational activation, and/or other additional factors. In contrast, the transcript levels of sidC were elevated in ΔhapX and ΔhapC during −Fe conditions, which agrees with the increased FC accumulation. Notably, sidC transcripts are approximately 15 kb in length and are therefore preferentially subject to physical degradation during RNA preparation. The upregulation of sidC expression in ΔhapX was also confirmed by dot blot analysis (data not shown). HapX and the CBC are required to repress iron-dependent pathways during –Fe conditions sreA belongs to a class of genes, which are downregulated during iron starvation. Most members of this class encode proteins requiring iron-containing cofactors, such as cycA, which encodes the heme-protein cytochrome c, as well as acoA and lysF, which encode the iron–sulfur cluster proteins aconitase and homoaconitase, respectively (Oberegger et al, 2002a). Another example is hemA, which codes for 5-aminolevulinate synthase (Bradshaw et al, 1993). HemA does not require iron by itself, but catalyzes the first committed step in heme biosynthesis. Deletion of hapX or of CBC subunit-encoding genes resulted in derepressed expression of all four genes during –Fe conditions (Figure 1C). In agreement with PpIX accumulation (Figure 2C), hemA expression was not only derepressed, but highly upregulated in both ΔhapX and ΔhapC (Figure 1C). A hypothetical explanation for the latter would be the lack of feedback inhibition of hemA expression by heme. Remarkably, hapC deletion resulted not only in derepression of iron-dependent pathways during −Fe conditions, but additionally in their upregulation during +Fe growth, in particular that of cycA (Figure 1C). Notably, neither sreA (Figure 1A) nor hapX deletion affected the hapC transcript levels, and hapC deletion did not affect hapX expression (data not shown). The A. nidulans wild-type BPU used is auxotrophic for uracil (pyrG89) and pyridoxamine (pyroA4). With respect to siderophore production and PpIX accumulation, BPU did not show any difference to A. nidulans strain TRAN, which is prototrophic for uracil and pyridoxamine, demonstrating that these auxotrophies do not influence iron metabolism (data not shown). Consistently, regulation of siderophore biosynthesis and iron-dependent pathways in BPU was as previously shown in TRAN (Oberegger et al, 2002a, 2002b). Interaction of HapX with DNA-bound CBC is abolished by iron To investigate putative HapX/CBC interaction in vivo, we applied bimolecular fluorescence complementation (BiFC) assays, previously shown to be valuable to define in vivo protein interaction (Hink et al, 2002; Hu and Kerppola, 2003; Hoff and Kück, 2005). BiFC was detected between enhanced-yellow-fluorescent-protein (eYFP) split fragments fused to HapX and HapB in strain yXB, under −Fe (Figure 3B) but not +Fe conditions (Figure 3A). BiFC could not be detected between HapX and HapB in the ΔhapC strain yXBΔC (Figure 3C), but was reconstituted by complementation with the hapC gene in strain yXBCc (Figure 3D). Northern analysis confirmed constitutive expression of the two eYFP split fragment-encoding genes in the used strains (Supplementary Figure 1). These data indicate that the entire CBC is required for in vivo interaction with HapX. Figure 3.HapX and HapB interact in vivo. The interaction was observed after 24 h of growth, using BiFC in A. nidulans strains producing HapX and HapB fused with the C-terminal and N-terminal split fragments of eYFP, respectively. Panels 1, light microscopy; panels 2 and 3, fluorescence microscopy of DAPI-stained nuclei and BiFC, respectively. HapX and HapB interact during −Fe (B) but not +Fe (A) conditions in strain yXB. (C) HapX/HapB interaction is abolished by deletion of hapC in strain yXBΔC and is (D) reconstituted after complementation of yXBΔC with hapC in yXBCc. Download figure Download PowerPoint HapE contains two evolutionary conserved regions (Figure 4A). Domain B is conserved among all HapE orthologs and is essential for the assembly of the CBC, as shown for S. cerevisiae Hap5p (McNabb et al, 1997). In contrast, domain A is only conserved among fungal HapE orthologs and has been shown to be required for the recruitment of Hap4p in S. cerevisiae. Recently, Tanoue et al (2006) reported that only the B-domain is required for CBC-mediated transcriptional activation in A. nidulans. Here, we found that deletion of the non-conserved N-terminal region and the A-domain phenocopies hapX deletion, that is, wild type-like growth during +Fe conditions, but decreased growth rate, reduced TAFC production, and increased accumulation of FC and PpIX during −Fe conditions (Figure 4B). In contrast, truncation of the non-conserved C-terminal domain of HapE had no effect (Figure 4B). These data suggest that the region encompassing the N-terminus and the A-domain of HapE is involved in interaction with HapX in vivo. Figure 4.Deletion of the non-conserved N-terminal region or the conserved A-domain of HapE phenocopies hapX deletion. (A) Schematic representation of the HapE versions investigated. (B) Growth rates and production of TAFC, FC and PpIX after growth for 48 h during+Fe and −Fe conditions. For induction of amylase promoter-driven genes (Tanoue et al, 2006), strains were grown in medium containing starch as the sole carbon source. During +Fe conditions, production of TAFC, FC and PpIX was wild type-like in all strains (data not shown). Download figure Download PowerPoint The 5′-upstream regions of the putative HapX/CBC target genes cycA, acoA, lysF, hemA and sreA all contain CCAAT boxes (Supplementary Figure 2). To study interaction of the CBC with the CCAAT sequences from promoter regions of iron-induced genes, we overproduced recombinant HapB, HapC, HapE, HapE-ΔNΔA and HapX in Escherichia coli and purified the proteins to homogeneity (Figure 5A). The CBC was reconstituted by mixing equimolar amounts of the CBC subunits. Real-time protein–DNA interaction analysis was performed with two 50-bp DNA duplexes immobilized on flow cells of a surface plasmon resonance (SPR) biosensor. The two sequences used covered the CCAAT box at position –1235 of the sreA promoter, which perfectly matches the consensus CBC-binding sequence (Mantovani, 1998), and the CCAAT box at position –181 of the lysF promoter, respectively. The apparent dissociation constants of the CBC were 1.8 and 4.6 nM for the CCAAT boxes from sreA and lysF, respectively, indicating specific and high-affinity binding. In contrast, HapX alone bound non-specifically, and with low affinity to sensor-bound DNA. (Figure 5B). Figure 5.SPR analysis of iron-regulated HapX binding to DNA-bound CBC. (A) SDS–PAGE analysis of 1.5 μg of purified HapB, HapC, HapE, HapE-ΔNΔA and HapX proteins. (B) Concentration-dependent, steady-state binding of the CBC to biosensor-bound CCAAT boxes derived from the 5′-upstream regions of sreA and lysF, respectively. (C) Schematic representation of the SPR analysis of HapX/CBC interaction. HapX was injected onto preformed CBC/DNA complexes after reaching the steady-state level (D) Concentration-dependent association of HapX (12.5, 25, 50 and 100 nM, respectively) to the CBC (6.25 nM) bound to the biosensor-linked sreA CCAAT box. ‘CBC(−HapX)’ shows the steady-state association of the CBC to the CCAAT box without application of HapX. ‘(−CBC)’ shows the unspecific interaction of HapX (50 nM) with sensor-bound DNA. (E) Comparison of the interaction of HapX (100 nM) with the CBC and with the CBC containing HapE-ΔNΔA (CBC*). Note that 12.5 nM CBC* was necessary to reach an equilibrium response equivalent to 6.25 nM CBC. (F) Interaction of HapX (100 nM) after preincubation with iron (1, 2.5, 5 and 10 μM FeCl3, respectively) or without iron (−Fe), with the DNA-bound CBC (6.25 nM). (G) Interaction of HapX (100 nM) after preincubation with 10 μM FeCl3, 10 μM (NH4)2Fe(SO4)2, 10 μM CuCl2, or without any metal (−metal), with DNA-bound CBC (6.25 nM). (H) Proposed model for HapX/CBC-mediated regulation of iron-dependent pathways and sreA. Download figure Download PowerPoint To investigate HapX/CBC interaction in vitro, HapX was injected onto the preformed CBC/DNA complex after reaching the steady-state level, as represented schematically in Figure 5C. Apart from its unspecific DNA binding, HapX bound with a remarkably high affinity to the CBC bound to the CCAAT box from the sreA promoter (Figure 5D). However, HapX did not bind to a CBC containing a HapE version that lacks the N-terminal region and the A-domain (HapE-ΔNΔA), that is, the measured response did not exceed the unspecific interaction of HapX with CBC-free DNA (Figure 5E). The latter agrees with the in vivo requirement for the N- and A-domains of HapE for repression of iron-dependent pathways during −Fe co
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