HtaA Is an Iron-Regulated Hemin Binding Protein Involved in the Utilization of Heme Iron in
            <i>Corynebacterium diphtheriae</i>

Allen, Courtni E.; Schmitt, Michael

doi:10.1128/jb.01784-08

Cited by 68 publications

(103 citation statements)

References 49 publications

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“…For haem utilization, C. glutamicum, as well as its pathogenic relative Corynebacterium diphtheriae, depends on a haem uptake apparatus composed of the ABC transporter HmuTUV, several cell surface haem-binding proteins (Allen & Schmitt, 2009Drazek et al, 2000;Frunzke et al, 2011) and a haem oxygenase (HmuO), which catalyses the intracellular degradation of the tetrapyrrol ring to abiliverdin, free iron (Fe 3+ ) and carbon monoxide Schmitt, 1997;Wilks & Schmitt, 1998). Acquisition of haem, however, exposes the respective organism to the toxicity associated with high levels of haem.…”

Section: Introductionmentioning

confidence: 99%

The two-component system ChrSA is crucial for haem tolerance and interferes with HrrSA in haem-dependent gene regulation in Corynebacterium glutamicum

et al. 2012

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The two-component system ChrSA is crucial for haem tolerance and interferes with HrrSA in haem-dependent gene regulation in Corynebacterium glutamicum

et al. 2012

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“…1A) (1, 18). The hmuTUV and htaA genes constitute a single operon, while htaB and htaC are independently transcribed (1,18). Mutations in the hmuTUV genes or htaA result in a reduced ability to use hemin and Hb as iron sources, suggesting that the ABC transporter and HtaA most likely function in the uptake of hemin (1).…”

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confidence: 99%

“…The hmuTUV and htaA genes constitute a single operon, while htaB and htaC are independently transcribed (1,18). Mutations in the hmuTUV genes or htaA result in a reduced ability to use hemin and Hb as iron sources, suggesting that the ABC transporter and HtaA most likely function in the uptake of hemin (1). Deletion of the entire hmu operon does not abolish the use of hemin and Hb as iron sources, which suggests that additional hemin uptake systems are present in C. diphtheriae (1).…”

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confidence: 99%

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Novel Hemin Binding Domains in the Corynebacterium diphtheriae HtaA Protein Interact with Hemoglobin and Are Critical for Heme Iron Utilization by HtaA

Allen

Schmitt

2011

J Bacteriol

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The human pathogen Corynebacterium diphtheriae utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. The use of hemin iron in C. diphtheriae involves the dtxR-and iron-regulated hmu hemin uptake locus, which encodes an ABC hemin transporter, and the surface-anchored hemin binding proteins HtaA and HtaB. Sequence analysis of HtaA and HtaB identified a conserved region (CR) of approximately 150 amino acids that is duplicated in HtaA and present in a single copy in HtaB. The two conserved regions in HtaA, designated CR1 and CR2, were used to construct glutathione S-transferase (GST) fusion proteins (GST-CR1 and GST-CR2) to assess hemin binding by UV-visual spectroscopy. These studies showed that both domains were able to bind hemin, suggesting that the conserved sequences are responsible for the hemin binding property previously ascribed to HtaA. HtaA and the CR2 domain were also shown to be able to bind hemoglobin (Hb) by the use of an enzyme-linked immunosorbent assay (ELISA) method in which Hb was immobilized on a microtiter plate. The CR1 domain exhibited a weak interaction with Hb in the ELISA system, while HtaB showed no significant binding to Hb. Competitive binding studies demonstrated that soluble hemin and Hb were able to inhibit the binding of HtaA and the CR domains to immobilized Hb. Moreover, HtaA was unable to bind to Hb from which the hemin had been chemically removed. Alignment of the amino acid sequences of CR domains from various Corynebacterium species revealed several conserved residues, including two highly conserved tyrosine (Y) residues and one histidine (H) residue. Site-directed mutagenesis studies showed that Y361 and H412 were critical for the binding to hemin and Hb by the CR2 domain. Biological assays showed that Y361 was essential for the hemin iron utilization function of HtaA. Hemin transfer experiments demonstrated that HtaA was able to acquire hemin from Hb and that hemin bound to HtaA could be transferred to HtaB. These findings are consistent with a proposed mechanism of hemin uptake in C. diphtheriae in which hemin is initially obtained from Hb by HtaA and then transferred between surfaceanchored proteins, with hemin ultimately transported into the cytosol by an ABC transporter.

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“…diphtheriae utilizes the hmu system for heme acquisition, which includes the heme-specific ABC transporter HmuTUV and the cell surface hemin binding protein HtaA (1,2,13). After entering the cytosol, heme is degraded by the heme oxygenase HmuO, which results in the release of the heme-associated iron (33,45).…”

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The ChrA Response Regulator in Corynebacterium diphtheriae Controls Hemin-Regulated Gene Expression through Binding to the hmuO and hrtAB Promoter Regions

Burgos

Schmitt

2012

J Bacteriol

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Corynebacterium diphtheriae, the etiologic agent of diphtheria, utilizes heme and hemoglobin (Hb) as iron sources for growth. Heme-iron utilization involves HmuO, a heme oxygenase that degrades cytosolic heme, resulting in the release of heme-associated iron. Expression of the hmuO promoter is under dual regulation, in which transcription is repressed by DtxR and iron and activated by a heme source, such as hemin or Hb. Hemin-dependent activation is mediated primarily by the ChrAS two-component system, in which ChrS is a putative heme-responsive sensor kinase while ChrA is proposed to serve as a response regulator that activates transcription. It was recently shown that the ChrAS system similarly regulates the hrtAB genes, which encode an ABC transporter involved in the protection of C. diphtheriae from hemin toxicity. In this study, we characterized the phosphorelay mechanism in the ChrAS system and provide evidence for the direct regulation of the hmuO and hrtAB promoters by ChrA. A fluorescence staining method was used to show that ChrS undergoes autophosphorylation and that the phosphate moiety is subsequently transferred to ChrA. Promoter fusion studies identified regions upstream of the hmuO and hrtAB promoters that are critical for the heme-dependent regulation by ChrA. Electrophoretic mobility shift assays revealed that ChrA specifically binds at the hmuO and hrtAB promoter regions and that binding is phosphorylation dependent. A phosphorylation-defective mutant of ChrA [ChrA(D50A)] exhibited significantly diminished binding to the hmuO promoter region relative to that of wildtype ChrA. DNase I footprint analysis further defined the sequences in the hmuO and hrtAB promoters that are involved in ChrA binding, and this analysis revealed that the DtxR binding site at the hmuO promoter partially overlaps the binding site for ChrA. DNase I protection studies as well as promoter fusion analysis suggest that ChrA and DtxR compete for binding at the hmuO promoter. Collectively, these data demonstrate that the ChrA response regulator directly controls the expression of hmuO and the hrtAB genes and the binding activity of ChrA is dependent on phosphorylation by its cognate sensor kinase ChrS.

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HtaA Is an Iron-Regulated Hemin Binding Protein Involved in the Utilization of Heme Iron in Corynebacterium diphtheriae

Cited by 68 publications

References 49 publications

The two-component system ChrSA is crucial for haem tolerance and interferes with HrrSA in haem-dependent gene regulation in Corynebacterium glutamicum

The two-component system ChrSA is crucial for haem tolerance and interferes with HrrSA in haem-dependent gene regulation in Corynebacterium glutamicum

Novel Hemin Binding Domains in the Corynebacterium diphtheriae HtaA Protein Interact with Hemoglobin and Are Critical for Heme Iron Utilization by HtaA

The ChrA Response Regulator in Corynebacterium diphtheriae Controls Hemin-Regulated Gene Expression through Binding to the hmuO and hrtAB Promoter Regions

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