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Membrane Topology and Heme Binding of the Histidine Kinases HrrS and ChrS in Corynebacterium glutamicum.

ABSTRACT: The HrrSA and the ChrSA two-component systems play a central role in the coordination of heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum and the prominent pathogen Corynebacterium diphtheriae, both members of the Corynebacteriaceae. In this study, we have performed a comparative analysis of the membrane topology and heme-binding characteristics of the histidine kinases HrrS and ChrS of C. glutamicum. While the cytoplasmic catalytic domains are highly conserved between HrrS and ChrS, the N-terminal sensing parts share only minor sequence similarity. PhoA and LacZ fusions of the N-terminal sensor domains of HrrS and ChrS revealed that both proteins are embedded into the cytoplasmic membrane via six ?-helices. Although the overall membrane topology appeared to be conserved, target gene profiling indicated a higher sensitivity of the ChrS system to low heme levels (< 1 ?M). In vitro, solubilized and purified full-length proteins bound heme in a 1:1 stoichiometry per monomer. Alanine-scanning of conserved amino acid residues in the N-terminal sensor domain revealed three aromatic residues (Y112, F115, and F118), which apparently contribute to heme binding of HrrS. Exchange of either one or all three residues resulted in an almost abolished heme binding of HrrS in vitro. In contrast, ChrS mutants only displayed a red shift of the soret band from 406 to 418 nm suggesting an altered set of ligands in the triple mutant. In line with target gene profiling, these in vitro studies suggest distinct differences in the heme-protein interface of HrrS and ChrS. Since the membrane topology mapping displayed no extensive loop regions and alanine-scanning revealed potential heme-binding residues in ?-helix number four, we propose an intramembrane sensing mechanism for both proteins. Overall, we present a first comparative analysis of the ChrS and HrrS kinases functioning as transient heme sensors in the Corynebacteriaceae.

SUBMITTER: Keppel M

PROVIDER: S-EPMC5812335 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Membrane Topology and Heme Binding of the Histidine Kinases HrrS and ChrS in Corynebacterium glutamicum.

Keppel Marc M Davoudi Eva E Gätgens Cornelia C Frunzke Julia J

Frontiers in microbiology 20180209

The HrrSA and the ChrSA two-component systems play a central role in the coordination of heme homeostasis in the Gram-positive soil bacterium Corynebacterium glutamicum and the prominent pathogen Corynebacterium diphtheriae, both members of the Corynebacteriaceae. In this study, we have performed a comparative analysis of the membrane topology and heme-binding characteristics of the histidine kinases HrrS and ChrS of C. glutamicum. While the cytoplasmic catalytic doma ...[more]

PMID: 29479345

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Project description:The response regulator HrrA belonging to the HrrSA two-component system (previously named CgtSR11) is known to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Sequence analysis indicated an involvement of the HrrSA system in heme-dependent gene expression. Growth experiments revealed that the non-pathogenic soil bacterium C. glutamicum is able to use hemin or hemoglobin as sole iron source. In DNA microarray analyses a putative operon encoding the hemin-binding protein HtaA and the putative hemin ABC transporter HmuTUV showed a strong upregulation in heme-grown cells. Deletion of the hmu operon clearly affects heme utilization, but does not completely abolish growth on heme or hemoglobin. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of a hrrA deletion mutant and C. glutamicum wild type in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase (hmuO), an enzyme involved in the utilization of heme as iron source. Besides hmuO, HrrA was shown to activate the expression of heme-containing components of the respiratory chain, namely ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa3 oxidase and three subunits of the cytochrome bc1 complex. Furthermore, HrrA represses almost all genes involved in heme biosynthesis, including glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Thus, our data clearly emphasize a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum. Three biological replicates of each experiment were performed. Experiment 1: Transcriptome comparison of wild type grown und FeSO4 or heme as iron source; Exp. 2: WT vs. hrrA deletion mutant grown on FeSO4; Exp. 3: WT vs. hrrA mutant grown on heme. For analysis via DNA microarraysose RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either 2.5 uM FeSO4 or 2.5 uM heme as iron source.

Dataset Information

Membrane Topology and Heme Binding of the Histidine Kinases HrrS and ChrS in Corynebacterium glutamicum.

Publications

Membrane Topology and Heme Binding of the Histidine Kinases HrrS and ChrS in <i>Corynebacterium glutamicum</i>.

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