Purification and structural characterization of siderophore (corynebactin) from Corynebacterium diphtheriae

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin.     

Regulation of toxinogenesis in Corynebacterium diphtheriae. I. Mutations in bacteriophage beta that alter the effects of iron on toxin production

Diphtherial toxin is produced in maximal yields by Corynebacterium diphtheriae (C7(beta tox+) only when iron is present in growth-limiting amounts. Toxin production is markedly decreased under high-iron conditions. We studied the role of the bacteriophage beta genome in this apparent regulation of toxin production by iron. Using a passive immune hemolysis assay to detect toxin antigen production in individual plaques, we identified rare phage mutants that were toxinogenic in high-iron medium. Lysogenic derivatives of C. diphtheriae C7 harboring such phage mutants were constructed. The lysogens were compared with wild-type strain C7(beta) for their ability to produce toxin in deferrated liquid medium containing varying amounts of added iron. Quantitative tests for extracellular toxin were performed by competitive-binding radioimmunoassays. We identified phenotypically distinct mutant strains that produced slightly, moderately, or greatly increased yields of toxin antigen under high-iron conditions. The toxin produced by the mutant lysogens was biologically active and immunochemically indistinguishable from wild-type toxin. Complementation experiments demonstrated that the phage mutation designated tox-201 had a cis-dominant effect on the expression of the toxin structural gene of phage beta. The characteristics of the tox-201 mutation suggest that it defines a regulatory locus of phage beta that is involved in control of toxinogenesis by iron in C. diphtheriae.     

Regulation of toxinogenesis in Corynebacterium diphtheriae: mutations in the bacterial genome that alter the effects of iron on toxin production

Mutants of Corynebacterium diphtheriae C7(beta) that are resistant to the inhibitory effects of iron on toxinogenesis were identified by their ability to form colonies surrounded by toxin-antitoxin halos on agar medium containing both antitoxin and a high concentration of iron. Chromosomal mutations were essential for the altered phenotypes of four independently isolated mutant strains. During growth in deferrated liquid medium containing various amounts of added iron, these mutants differed from wild-type C. diphtheriae C7(beta) in several ways. Their growth rates were slower under low-iron conditions and were stimulated to various degrees under high-iron conditions. The concentrations of iron at which optimal toxin production occurred were higher for the mutants than for wild-type C. diphtheriae C7(beta). Toxin production by the mutants during growth in low-iron medium occurred throughout the period of exponential growth at nearly constant rates that were proportional to the bacterial growth rates. In contrast, toxin production by wild-type C. diphtheriae C7(beta) in similar low-iron cultures occurred predominantly during the late exponential phase, when iron was a growth-limiting nutrient. Additional studies demonstrated that these mutants had severe defects in their transport systems for ferric iron. We propose that the altered regulation of toxinogenesis by iron in our mutants was caused by the severe defects in their iron transport systems. As a consequence, the mutants exhibited a low-iron phenotype during growth under conditions that permitted wild-type C. diphtheriae C7(beta) to exhibit a high-iron phenotype.     

Analysis of diphtheria toxin repressor-operator interactions and characterization of a mutant repressor with decreased binding activity for divalent metals

The diphtheria toxin repressor (DtxR) is an Fe²⁺-activated protein with sequence-specific DNA-binding activity for the diphtheria toxin (tox) operator. Under high-iron conditions in Corynebacterium diphtheriae, DtxR represses toxin and siderophore biosynthesis as well as iron uptake. DtxR and a mutant repressor with His–47 substituted for Arg–47, designated DtxR-R47H, were purified and compared. Six different divalent cations (Cd²⁺, Co²⁺, Fe²⁺, Mn²⁺, Ni²⁺, and Zn²⁺) activated the sequence-specific DNA-binding activity of DtxR and enabled it to protect the fox operator from DNase I digestion, but Cu²⁺ failed to activate DtxR. Hydroxyl radical footprinting experiments indicated that DtxR binds symmetrically about the dyad axis of the tox operator. Methylation protection experiments demonstrated that DtxR binding alters the susceptibility to methylation of three G residues within the AT-rich tox operator. These findings suggest that two or more monomers of DtxR are involved in binding to the tox operator, with symmetrical DNA-protein interactions occurring at each end of the palindromic operator. In this regard, DtxR resembles several other well-characterized prokaryotic repressor proteins but differs dramatically from the Fe²⁺-activated ferric uptake repressor protein (Fur) of Escherichia coli. The concentration of Co²⁺ required to activate DtxR-R47H was at least 10-foid greater than that needed to activate DtxR, but the sequence-specific DNA binding of activated DtxR-R47H was indistinguishable from that of wild-type DtxR. The markedly deficient repressor activity of DtxR-R47H is consistent with a significant decrease in its binding activity for divalent cations.     

Constitutive expression of the iron-enterochelin and ferrichrome uptake systems in a mutant strain of Salmonella typhimurium.

Two high-affinity iron uptake systems are known in Salmonella typhimurium, one utilizing iron-enterochelin and the other utilizing ferrichrome. It has been shown previously that expression of several elements of the iron-enterochelin uptake system are regulated by the iron content of the medium, with growth in high-iron medium resulting in repression of enzymes of enterochelin synthesis and degradation and of the ability of whole cells to take up iron-enterochelin. In this study we describe a mutant strain in which growth in high-iron medium was associated with constitutive expression of: (i) iron-enterochelin uptake by whole cells; (ii) ferrichrome uptake by whole cells; (iii) synthesis of enterochelin; (iv) intracellular degradation of iron-enterochelin; and (v) synthesis of three major outer membrane proteins (OM1, OM2, and OM3). In contrast, in the wild-type strain these properties were expressed only after growth in iron-deficient medium. It is proposed that the mutation affects a gene responsible for regulating expression of the structural genes for the components of the high-affinity iron uptake systems. The term fur, for iron (Fe) uptake regulation, is suggested for this new class of mutant.     

Molecular cloning, iron-regulation and mutagenesis of the irp2 gene encoding HMWP2, a protein specific for the highly pathogenic Yersinia

Under iron-starvation, the highly pathogenic Yersinia synthesize several iron-regulated proteins including two high-molecular-weight polypeptides (HMWP1 and HMWP2). From the chromosome of Yersinia enterocolitica serovar O:8 (strain Ye 8081), the genes coding for the HMWP2 (irp2) and its promoter were cloned into plasmid pUC18 (pIR2) and used as a probe. We show here that the irp2 gene is present only in the highly pathogenic strains and that its promoter is iron-regulated in Escherichia coli. After introduction of the pIR2 plasmid into a fur mutant of E. coli, both the iron-starved and the iron-replete bacteria expressed the HMWP2. Repressibility of irp2 by iron was restored by introduction of a plasmid carrying the fur gene. These results demonstrate that the irp2 promoter is controlled by the Fur repressor in E. coli. Mutagenesis of the chromosomal irp2 gene of Yersinia pseudotuberculosis was obtained by homologous recombination with a 1 kb fragment of this gene cloned on the suicide plasmid pJM703.1. Inactivation of irp2 resulted in the non-expression of both HMWPs, while introduction of plasmid pIR2 into the mutant strain led to the synthesis of the HMWP2 only. Therefore, it is probable that the genes coding for the HMWPs constitute an operon where irp2 is upstream of irp1. When comparing the virulence of the wild-type strain and of its irp2 mutant derivative, we found that the 50% lethality (LD50) for mice of the mutant strain was increased, whatever the route of infection, but more markedly when injected parenterally. Accordingly, these data demonstrate that a mutation in the irp2 gene alters the pathogenicity of Y. pseudotuberculosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen and iron regulation of iron regulatory protein 2

Iron regulatory protein 2 (IRP2) is a central regulator of cellular iron homeostasis due to its regulation of specific mRNAs encoding proteins of iron uptake and storage. Iron regulates IRP2 by mediating its rapid proteasomal degradation, where hypoxia and the hypoxia mimetics CoCl2 and desferrioxamine (DFO) stabilize it. Previous studies showed that iron-mediated degradation of IRP2 requires the presence of critical cysteines that reside within a 73-amino acid unique region. Here we show that a mutant IRP2 protein lacking this 73-amino acid region degraded at a rate similar to that of wild-type IRP2. In addition, DFO and hypoxia blocked the degradation of both the wild-type and mutant IRP2 proteins. Recently, members of the 2-oxoglutarate (2-OG)-dependent dioxygenase family have been shown to hydroxylate hypoxia-inducible factor-1 alpha (HIF-1 alpha), a modification required for its ubiquitination and proteasomal degradation. Since 2-OG-dependent dioxygenases require iron and oxygen, in addition to 2-OG, for substrate hydroxylation, we hypothesized that this activity may be involved in the regulation of IRP2 stability. To test this we used the 2-OG-dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG) and showed that it blocked iron-mediated IRP2 degradation. In addition, hypoxia, DFO and DMOG blocked IRP2 ubiquitination. These data indicate that the region of IRP2 that is involved in IRP2 iron-mediated degradation lies outside of the 73-amino acid unique region and suggest a model whereby 2-OG-dependent dioxygenase activity may be involved in the oxygen and iron regulation of IRP2 protein stability.     

Cloning of a Corynebacterium diphtheriae iron-repressible gene that shares sequence homology with the AhpC subunit of alkyl hydroperoxide reductase of Salmonella typhimurium.

To understand how Corynebacterium diphtheriae responds to iron limitation, we compared the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles of both wild-type cells and iron uptake mutants grown in either high- or low-iron medium. The removal of iron by ethylene diamine di-(o-hydroxy-phenyl acetic acid) from the growth medium of wild-type cells resulted in induction of at least 14 polypeptides. DirA, a major iron-repressible polypeptide, was purified from wild-type cells by preparative SDS-PAGE, and the dirA structural gene was isolated from a genomic library of nontoxigenic C. diphtheriae. The nucleotide sequence of dirA was determined, and the deduced amino acid sequence of DirA revealed strong homologies with the AhpC subunit of Salmonella typhimurium alkyl hydroperoxide reductase and polypeptides of other microorganisms associated with oxidation reduction activity. Like AhpC, cloned DirA reduced the susceptibility of an Escherichia coli ahp mutant to cumene hydroperoxide, suggesting that DirA has alkyl hydroperoxide reductase activity.     

Corynebacterium diphtheriae genes required for acquisition of iron from haemin and haemoglobin are homologous to ABC haemin transporters

Corynebacterium diphtheriae and Corynebacterium ulcerans use haemin and haemoglobin as essential sources of iron during growth in iron-depleted medium. C. diphtheriae and C. ulcerans mutants defective in haemin iron utilization were isolated and characterized. Four clones from a C. diphtheriae genomic library complemented several of the Corynebacteria haemin utilization mutants. The complementing plasmids shared an approximately 3 kb region, and the nucleotide sequence of one of the plasmids revealed five open reading frames that appeared to be organized in a single operon. The first three genes, which we have termed hmuT, hmuU and hmuV, shared striking homology with genes that are known to be required for haemin transport in Gram-negative bacteria and are proposed to be part of an ABC (ATP-binding cassette) transport system. The hmuT gene encodes a 37 kDa lipoprotein that is associated with the cytoplasmic membrane when expressed in Escherichi coli and C. diphtheriae. HmuT binds in vitro to haemin- and haemoglobin-agarose, suggesting that it is capable of binding both haemin and haemoglobin and may function as the haemin receptor in C. diphtheriae. This study reports the first genetic characterization of a transport system that is involved in the utilization of haemin and haemoglobin as iron sources by a Gram-positive bacterium.