Putative DNA-binding Domain Research Articles

DNA Binding of L1 Is Required for Human Papillomavirus Morphogenesis in Vivo

The role of putative DNA-binding domains of human papillomavirus (HPV) capsid proteins for DNA encapsidation in vivo is still unknown. We have now analyzed mutants of the major capsid protein L1 of HPV type 33, which are defective for DNA binding, for their ability to encapsidate DNA using an in vivo packaging approach. Since the DNA-binding domain and the nuclear localization signal (NLS) of L1 overlap, both a carboxy-terminal deletion mutant (L1-1/470) and a substitution mutant (L1-1/477M9) were analyzed. L1-1/477M9 has the classical NLS replaced by a noncanonical NLS taken from the human hnRNP protein A1. The mutant proteins were defective for DNA binding in contrast to wild-type (wt) L1 proteins of several HPV types. L1-1/470 localized to the cytoplasm when expressed in human cell lines, whereas L1-1/477M9 was found mainly in the nucleus. When coexpressed with wt L2, mutant L1 proteins colocalized with L2 in nuclear domains 10 (ND10) similar to wt L1 and assembled into virus-like particles incorporating L2. Wt L1 and L2 but not mutant L1 proteins and wt L2 efficiently encapsidated DNA and transferred it into cells, suggesting that physical interaction of L1 with DNA is essential for DNA inclusion into VLPs. We also compared the infectivity of histone-free DNA-containing pseudovirions generated in vitro with the infectivity of pseudovirions generated in vivo, which most likely harbor DNA in the form of chromatin. Pseudovirions generated in vivo were, on a per genome basis, four to five times more infectious than pseudovirions generated in vitro, even though no structural or biochemical difference was obvious. This indicates that chromatin may enhance the infectivity of pseudovirions.

AG4sequence withinPHR1promoter acts as a gate for cross‐talks between damage‐signaling pathway and multi‐stress response

Rph1 and Gis1 are damage‐responsive repressors involved in PHR1 expression. They have two C2H2 zinc finger motifs as putative DNA binding domains and N‐terminal conserved domain with unknown function. They are also found in the human retinoblastoma binding protein 2 and the mouse jumonji‐ encoded protein. The repressors are able to bind to AG4 sequence within a 39‐bp sequence called upstream repressing sequence of PHR1 promoter (URSPHR1 ) responsible for the damage‐response of PHR1. We report here that Rph1 is predominantly localized in the nucleus as examined by fluorescence microscopic analysis with GFP‐Rph1 fusion protein. On the basis of the fact that the AG4 sequence that is recognized by Rph1 and Gis1 is also recognized by Msn2 and Msn4 in a process of stress response, we also tried to examine the in vivo function of AG4 and the role of Msn2 and Msn4 in PHR1 expression. Our results demonstrate that Msn2 and Msn4 are actually required for the basal transcription of PHR1 expression but not for its damage induction. When AG4 sequence was inserted into the minimal promoter of the cyc1‐LacZ reporter, the increased LacZ expression was observed, indicating its involvement in transcriptional activation. The data suggest that the AG4 is primarily required for basal trans criptional activation of PHR1 or CYC1 promoter through the possible involvement of Msn2 and Msn4. However, since the AG4 is also involved in the repression of PHR1 via Rph1 and Gis1, it is proposed that AG4 functions as either URS or upstream activating sequence (UAS) depending on the promoter context.

Characterization of a Novel GT-box Binding Protein from Arabidopsis.

We isolated a cDNA encoding a novel GT-box binding protein from Arabidopsis, designated GT-4. The predicted open reading frame encodes a protein of 372 amino acids and the predicted protein sequence revealed the presence of a putative DNA-binding domain with 80% homology to the trihelix region of previously described light-responsive element binding protein, GT-1. Reverse transcription-PCR analysis showed that GT-4 transcripts are present in all tissues tested in light-grown plants, but light-regulated expression of GT-4 mRNA was observed only in etiolated seedlings. Electro-mobility shift assay using recombinant protein revealed that the GT-box-binding specificity of GT-4 is almost identical to that of GT-1. Transient expression of GT-4::GFP fusion protein in onion epidermal cells revealed the presence of a nuclear localization signal within the GT-4 protein. These results suggest the possibility that GT-4 is involved in GT-box-mediated gene expression by recognizing target sequences closely related to GT-1 binding sites.

Expression of Dax-1 during gonadal development of the frog

Dax-1, a member of the nuclear hormone receptor superfamily of transcription factors, is known to be involved in gonadal development in mammals. To date, Dax-1 has only been isolated in reptiles, birds and mammals. The expression of Dax-1 is down-regulated in the developing testis, but persists in the ovary of mice (Swain et al., Nat. Genet. 12 (1996) 404) and chicken (Smith et al., J. Mol. Endocrinol. 24 (2000) 23). Curiously, there is no sex difference in the expression patterns of Dax-1 in the American alligator (Western et al., Gene 241 (2000) 223). To understand its role(s) in gonadal development in vertebrates, molecular cloning of Dax-1 in amphibians is required. In this study, we cloned an amphibian Dax-1 homologue of the frog Rana rugosa and examined its expression profile during gonadal development. Cloned Dax-1 cDNA encoded a protein of 287 amino acids. Unlike mammalians that possess the three and one half repeat elements representing the putative DNA binding domain in the predicted sequence of Dax-1 protein, the frog had a single poorly conserved copy of the repeat unit. By RT-PCR analysis, the Dax-1 mRNA was detected in the liver and pancreas, but not in the testis and ovary of adult frogs. However, Dax-1 expression was seen first in the embryo at stage 12 and became stronger in tadpoles until stage X. The Dax-1 was transcribed in the testis stronger than in the ovary of frogs at stage XXV (just after completion of metamorphosis). In the gonad of frogs 2 months after metamorphosis (at this stage postmeiotic cells can be seen in the seminiferous tubules), the Dax-1 was expressed only in males. In addition, the Dax-1 transcription declined gradually as ovarian development proceeded, but its expression was down-regulated and then up-regulated rapidly when female-to-male sex reversal was caused by administration of testosterone into female tadpoles. Taken together, the results suggest that the Dax-1 may be closely involved in testicular development of amphibians.

The Autoimmune Regulator (AIRE) Is a DNA-binding Protein

The autoimmune regulator (AIRE) protein is a putative transcription regulator with two plant homeodomain-type zinc fingers, a putative DNA-binding domain (SAND), and four nuclear receptor binding LXXLL motifs. We have shown here that in vitro, recombinant AIRE can form homodimers and homotetramers that were also detected in thymic protein extracts. Recombinant AIRE also oligomerizes spontaneously upon phosphorylation by cAMP dependent protein kinase A or protein kinase C. Similarly, thymic AIRE protein is phosphorylated at the tyrosine and serine/threonine residues. AIRE dimers and tetramers, but not the monomers, can bind to G-doublets with the ATTGGTTA motif and the TTATTA-box. Competition assays revealed that sequences with one TTATTA motif and two tandem repeats of ATTGGTTA had the highest binding affinity. These findings demonstrate that AIRE is an important DNA binding molecule involved in immune regulation.

Cloning and expression of human HBP1, a high mobility group protein that enhances myeloperoxidase (MPO) promoter activity.

Factors which regulate transcription in immature myeloid cells are of great current interest for the light they may shed upon myeloid differentiation. In the course of screening for transcription factors which interact with the human myeloperoxidase (MPO) promoter we, for the first time, identified and cloned the cDNA and genomic DNA for human HBP1 (HMG-Box containing protein 1), a member of the high mobility group of non-histone chromosomal proteins. HBP1 cDNA was initially cloned from rat brain in 1994, but its presence in human cells or in myeloid tissue had not been described previously. The sequence of human HBP1 cDNA shows 84% overall homology with the rat HBP1 cDNA sequence. We have subsequently cloned the gene, which is present as a single copy, 25 kbp in length. Northern blotting reveals a single 2.6 kb mRNA transcript which is expressed at higher levels in human myeloid and B lymphoid cell lines than in T cell lines tested and is present in several non-myeloid human cell lines. Comparison of the mRNA and genomic sequences reveals the gene to contain 10 exons and 9 introns. The sequence of human HBP1 mRNA contains a single open reading frame, which codes for a protein 514 amino acids in length. The amino acid sequence specified by the coding region shows 95% homology with the rat HBP1 protein. The human protein sequence exhibits a putative DNA-binding domain similar to that seen in rat HBP1 and shows homology with the activation and repressor domains previously demonstrated in the rat protein. We have expressed human HBP1 protein both in vitro and in prokaryotic and eukaryotic cells. The expressed fusion protein binds to a sequence in a functionally important region within the basal human MPO promoter. In transient co-transfection experiments HBP1 enhances MPO promoter activity. Human HBP1 appears to be a novel transcription factor which is likely to play an important role in regulating transcription in developing myeloid cells.

Expression of the Avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is regulated by the global nitrogen response factor NRF1.

Here we describe the role of the Cladosporium fulvum nitrogen response factor 1 (Nrf1) gene in regulation of the expression of avirulence gene Avr9 and virulence on tomato. The Nrf1 gene, which was isolated by a polymerase chain reaction-based strategy, is predicted to encode a protein of 918 amino acid residues. The protein contains a putative zinc finger DNA-binding domain that shares 98% amino acid identity with the zinc finger of the major nitrogen regulatory proteins AREA and NIT2 of Aspergillus nidulans and Neurospora crassa, respectively. Functional equivalence of Nrf1 to areA was demonstrated by complementation of an A. nidulans areA loss-of-function mutant with Nrf1. Nrf1-deficient transformants of C. fulvum obtained by homologous recombination were unable to utilize nitrate and nitrite as a nitrogen source. In contrast to what was observed in the C. fulvum wild-type, the Avr9 gene was no longer induced under nitrogen-starvation conditions in Nrf1-deficient strains. On susceptible tomato plants, the Nrf1-deficient strains were as virulent as wild-type strains of C. fulvum, although the expression of the Avr9 gene was strongly reduced. In addition, Nrf1-deficient strains were still avirulent on tomato plants containing the functional Cf-9 resistance gene, indicating that in planta, apparently sufficient quantities of stable AVR9 elicitor are produced. Our results suggest that the NRF1 protein is a major regulator of the Avr9 gene.

Mutational analysis of conserved residues in the putative DNA-binding domain of the response regulator Spo0A of Bacillus subtilis.

The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation. Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation. The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A. Previous studies identified a region of the C-terminal half of Spo0A that is highly conserved among species of endospore-forming Bacillus and Clostridium and which encodes a putative helix-turn-helix DNA-binding domain. To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis. We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation. The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in spoIIA and spoIIE promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation. These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.

Point Mutations within AT-Hook Domains of the HMGI Homologue HMGIYL1 Affect Binding to Gene Promoter but Not to Four-Way Junction DNA

High-mobility group I/Y (HMGI/Y) proteins are chromosomal proteins involved in gene and chromatin regulation. Elevated levels of HMGI/Y proteins were reported in diverse malignant tumors, and rearrangements of their genes are casually involved in the development of benign tumors. In humans, the chromosomal locus Xp22 has been often found to be affected in diverse benign mesenchymal tumors. Recent studies revealed that this region contains a retropseudogene HMGIYL1 which potentially can be activated in a way of "exonization" upon aberrations involving this region. The coding sequence of the HMGIY-L1 is highly homologous to the HMGI(Y) gene. On the protein level, both HMGIYL1 and HMGI differ at few amino acid residues, including their putative DNA-binding domains (DBDs). Here we have approached the question of whether the HMGIYL1 product would be able to adopt a role of HMGI in the context of binding to gene promoters and chromatin. Comparative binding studies, employing protein footprinting technique, revealed that HMGIYL1 has lost the ability to bind to the promoter of the interferon beta gene, but retained its high affinity for the four-way junction DNA. Our results stress the importance of particular residues within the DBDs for DNA binding and demonstrate that tight binding of HMGI/Y proteins to the four-way junction DNA can be achieved in alternative ways. The binding of HMGIYL1 to four-way junction DNA suggests that activation of the HMGIYL1 gene would yield a protein sharing some binding properties with HMG1-box proteins and histone H1. Thus, the HMGIYL1 could interplay together with these components in chromatin regulation.

Regulation of vibrio cholerae genes required for acid tolerance by a member of the "ToxR-like" family of transcriptional regulators.

The ability of the intestinal pathogen Vibrio cholerae to undergo an adaptive stress response, known as the acid tolerance response (ATR), was previously shown to enhance virulence. An essential component of the ATR is CadA-mediated lysine decarboxylation. CadA is encoded by the acid- and infection-induced gene cadA. Herein, cadA is shown to be the second gene in an operon with cadB, encoding a lysine/cadaverine antiporter. cadC, which is 5' of cadB, encodes an acid-responsive, positive transcriptional regulator of cadBA. Unlike in Escherichia coli, V. cholerae cadB and cadA are also transcribed monocistronically. Of note, bicistronic cadBA is transcribed at low constitutive levels in an acid- and CadC-independent manner. CadC represents a new member of the "ToxR-like" family of transcriptional regulators in V. cholerae and, in addition, exhibits extensive amino acid and functional similarity to E. coli CadC. The amino-terminal, putative DNA binding domains of ToxR and CadC are highly conserved, as are the putative promoter elements recognized by these transcription factors.

Nerfin-1 and -2, novel Drosophila Zn-finger transcription factor genes expressed in the developing nervous system

To gain insight into the regulatory networks controlling Drosophila neural-identity decisions, we have identified new neuronal precursor genes by performing an in situ hybridization screen of differentially selected embryonic head cDNAs. Here, we describe the molecular characteristics and expression profile of nerfin-1, a novel pan-neural precursor gene. This paper also documents the embryonic expression of another structurally related gene, nerfin-2. During early CNS development, nerfin-1 gene expression is activated in neuroblasts (NBs) prior to lineage formation. However, after early sublineage development, nerfin-1 expression shifts from NBs to ganglion mother cells (GMCs) but is not expressed in neurons or glia. Differing from nerfin-1, nerfin-2 is expressed only in a subset of brain neurons. Possessing a conserved putative DNA-binding domain, the predicted Nerfin-1 and -2 proteins belong to a subfamily of Zn-finger transcription factors with cognates identified in nematode, mouse and man.

Molecular characterization of the first non-mammalian p73 cDNA

The p53 gene is believed to be mutated or deficient in over 50% of human tumours, and is therefore considered to be instrumental in the process of carcinogenesis. Recently in humans, homologues of p53 (such as p73 and p63) have been isolated. In our studies in fish, we have been isolating tumour suppressor genes with a view to their potential use to study genotoxins in the aquatic environment. In this paper, we report the characterisation of the first non-mammalian p73 cDNA, isolated from barbel (Barbus barbus), a freshwater cyprinid fish indigenous to UK rivers. The deduced barbel p73 amino acid sequence has a high homology with human p73α: the proteins are 641 and 636 aa in length, respectively, and there is a 72% identity over the entire sequence length of the protein (over 90% in the putative DNA binding domain). The level of conservancy for p73 is considerably higher across class (from man to fish), than for p53 and it may therefore have particular value in studies on environmental mutagenesis. Northern analysis showed expression of three p73 mRNA transcripts/ homologues. The patterns of p73 tissue expression in the barbel differed from the expression of p53 mRNA, suggesting specific functional roles for the two genes.

ArgRII, a component of the ArgR-Mcm1 complex involved in the control of arginine metabolism in Saccharomyces cerevisiae, is the sensor of arginine.

Repression of arginine anabolic genes and induction of arginine catabolic genes are mediated by a three-component protein complex, interacting with specific DNA sequences in the presence of arginine. Although ArgRI and Mcm1, two MADS-box proteins, and ArgRII, a zinc cluster protein, contain putative DNA binding domains, alone they are unable to bind the arginine boxes in vitro. Using purified glutathione S-transferase fusion proteins, we demonstrate that ArgRI and ArgRII1-180 or Mcm1 and ArgRII1-180 are able to reconstitute an arginine-dependent binding activity in mobility shift analysis. Binding efficiency is enhanced when the three recombinant proteins are present simultaneously. At physiological concentration, the full-length ArgRII is required to fulfill its functions; however, when ArgRII is overexpressed, the first 180 amino acids are sufficient to interact with ArgRI, Mcm1, and arginine, leading to the formation of an ArgR-Mcm1-DNA complex. Several lines of evidence indicate that ArgRII is the sensor of the effector arginine and that the binding site of arginine would be the region downstream from the zinc cluster, sharing some identity with the arginine binding domain of bacterial arginine repressors.

A novel snail-related transcription factor Smuc regulates basic helix-loop-helix transcription factor activities via specific E-box motifs.

Snail family proteins are zinc finger transcriptional regulators first identified in Drosophila which play critical roles in cell fate determination. We identified a novel Snail -related gene from murine skeletalmusclecells designated Smuc. Northern blot analysis showed that Smuc was highly expressed in skeletal muscle and thymus. Smuc contains five putative DNA-binding zinc finger domains in its C-terminal half. In electrophoretic mobility shift assays, recombinant zinc finger domains of Smuc specifically bound to CAGGTG and CACCTG E-box motifs (CANNTG). Because basic helix-loop-helix transcription factors (bHLH) bind to the same E-box sequences, we examined whether Smuc competes with the myogenic bHLH factor MyoD for DNA binding. Smuc inhibited the binding of a MyoD-E12 complex to the CACCTG E-box sequence in a dose-dependent manner and suppressed the transcriptional activity of MyoD-E12. When heterologously targeted to the thymidine kinase promoter as fusion proteins with the GAL4 DNA-binding domain, the non-zinc finger domain of Smuc acted as a transcriptional repressor. Furthermore, overexpression of Smuc in myoblasts repressed transactivation of muscle differentiation marker Troponin T. Thus, Smuc might regulate bHLH transcription factors by zinc finger domains competing for E-box binding, and non-zinc finger repressor domains might also confer transcriptional repression to control differentiation processes.

Hormone-independent Transcriptional Activation and Coactivator Binding by Novel Orphan Nuclear Receptor ERR3

Orphan nuclear receptors share sequence homology with members of the nuclear receptor superfamily, but ligands are unknown or unnecessary. A novel orphan receptor, estrogen receptor-related protein 3 (ERR3), was identified by yeast two-hybrid screening, using the transcriptional coactivator glucocorticoid receptor interacting protein 1 (GRIP1) as bait. The putative full-length mouse ERR3 contains 458 amino acids and is closely related to two known orphan receptors ERR1 and ERR2. All the ERR family members share an almost identical DNA-binding domain, which has 68% amino acid identity with that of estrogen receptor. ERR3 bound specifically to an estrogen response element and activated reporter genes controlled by estrogen response elements, both in yeast and in mammalian cells, in the absence of any added ligand. A conserved AF-2 activation domain located in the hormone-binding domain of ERR3 was primarily responsible for transcriptional activation. The ERR3 AF-2 domain bound GRIP1 in a ligand-independent manner both in vitro and in vivo, through the LXXLL motifs of GRIP1, and GRIP1 functioned as a transcriptional coactivator for ERR3 in both yeast and mammalian cells. Expression of ERR3 in adult mouse was restricted; highest expression was observed in heart, kidney, and brain. In the mouse embryo no expression was observed at day 7, and highest expression occurred around the 11-15 day stages. Although ERR3 is much more closely related to ERR2 than to ERR1, the expression pattern for ERR3 was similar to that of ERR1 and distinct from that for ERR2, suggesting a unique role for ERR3 in development.

Expression and purification of a novel ZNF191 zinc finger protein

ZNF191 is a new zinc finger gene which has a 1107 bp open reading frame (ORF) and encodes a 368 amino acid protein including a putative DNA-binding domain of four Cys2His2 zinc finger motifs at its C-terminal region. The ZNF191 clDNA is located in the chromosome 18q12.1 region where it is known that a variety of hereditary diseases are related to. Probably, this protein has potential function of stimulating the gene transcription.

TATA box-binding protein (TBP)-related factor 2 (TRF2), a third member of the TBP family.

The TATA box-binding protein (TBP) is an essential component of the RNA polymerase II transcription apparatus in eukaryotic cells. Until recently, it was thought that the general transcriptional machinery was largely invariant and relied on a single TBP, whereas a large and diverse collection of activators and repressors were primarily responsible for imparting specificity to transcription initiation. However, it now appears that the "basal" transcriptional machinery also contributes to specificity via tissue-specific versions of TBP-associated factors as well as a tissue-specific TBP-related factor (TRF1) responsible for gene selectivity in Drosophila. Here we report the cloning of a TBP-related factor (TRF2) that is found in humans, Drosophila, Caenorhabditis elegans, and other metazoans. Like TRF1 and TBP, TRF2 binds transcription factor IIA (TFIIA) and TFIIB and appears to be part of a larger protein complex. TRF2's primary amino acid structure suggests divergence in the putative DNA binding domain, and not surprisingly, it fails to bind to DNA containing canonical TATA boxes. Most importantly, TRF2 is associated with loci on Drosophila chromosomes distinct from either TBP or TRF1, so it may have different promoter specificity and regulate a select subset of genes. These findings suggest that metazoans have evolved multiple TBPs to accommodate the vast increase in genes and expression patterns during development and cellular differentiation.

Repression of Human Fibroblast Growth Factor 2 by a Novel Transcription Factor

Here we describe the cloning of the regulator of fibroblast growth factor 2 (FGF-2) transcription (RFT) using a yeast one-hybrid screening with a defined motif in FGF-2 promoter as a target sequence. Overexpression of human RFT (RFT-A) reduces FGF-2 RNA and protein levels in both normal and tumor cell lines. Its splice variants, RFT-A' and RFT-B, have deletions in the putative DNA binding domain and fail to bind FGF-2 promoter and repress FGF-2 gene expression. The ratios of RFT isoforms differ between normal and tumor cells, with the splice variants dominating in tumor cells. Overexpression of RFT-A induces glioma cell death. Our data suggest that regulation of FGF-2 by RFT is important for cellular functions and may be impaired in certain tumors.

Cloning of the APECED gene provides new insight into human autoimmunity

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is the only autoimmune disease characterized so far that is caused by a defect in a single gene. We have recently isolated the defective gene in this disease by positional cloning and have identified several different mutations in APECED patients. This novel gene, AIRE, contains two plant homeodomain (PHD)-type zinc finger motifs and a newly described putative DNA-binding domain SAND. We have further shown that the protein encoded by the AIRE gene is localized to the nuclear body-like structures of cell nuclei. Similar discrete speckles within the nucleus have been suggested to be involved in the regulation of transcription, oncogenesis and differentiation of cells. Together with the predicted structural features of the APECED protein the new data obtained both in vitro and ex vivo suggest that this protein participates in the regulation of gene expression in a restricted set of tissues and cells.

The Aspergillus nidulans GATA Factor SREA Is Involved in Regulation of Siderophore Biosynthesis and Control of Iron Uptake

A gene encoding a new GATA factor from Aspergillus nidulans, sreA, was isolated and characterized. SREA displays homology to two fungal regulators of siderophore biosynthesis: about 30% overall identity to SRE from Neurospora crassa and about 50% identity to URBS1 from Ustilago maydis over a stretch of 200 amino acid residues containing two GATA-type zinc finger motifs and a cysteine-rich region. This putative DNA binding domain, expressed as a fusion protein in Escherichia coli, specifically binds to GATA sequence motifs. Deletion of sreA results in derepression of L-ornithine-N5-oxygenase activity and consequently in derepression of the biosynthesis of the hydroxamate siderophore N,N',N"-triacetyl fusarinine under sufficient iron supply in A. nidulans. Transcription of sreA is confined to high iron conditions, underscoring the function of SREA as a repressor of siderophore biosynthesis under sufficient iron supply. Nevertheless, overexpression of sreA does not result in repression of siderophore synthesis under low iron conditions, suggesting additional mechanisms involved in this regulatory circuit. Consistent with increased sensitivity to the iron-activated antibiotics phleomycin and streptonigrin, the sreA deletion mutant displays increased accumulation of 59Fe. These results demonstrate that SREA plays a central role in iron uptake in addition to siderophore biosynthesis.