Specificity of phosphatase activity and interaction of heme-responsive two-component systems in Corynebacterium glutamicum
Final Report Abstract
Heme (iron bound protoporphyrin IX) is a versatile molecule that serves as the prosthetic group of various proteins and plays an essential role in many cellular processes, such as electron transfer, respiration and oxygen metabolism. High intracellular heme levels are, however, highly toxic. To cope with this Janus-faced nature of heme, sophisticated regulatory systems have evolved to tightly balance heme uptake, synthesis and utilization. Remarkably, in several corynebacterial species, e.g. Corynebacterium glutamicum and Corynebacterium diphtheriae, two paralogous, heme-responsive two-component systems (TCSs), HrrSA and ChrSA, are dedicated to the coordinated control of heme homeostasis. While the heme detoxification system ChrSA activates a heme exporter hrtBA the paralogous system HrrSA mediates utilization of heme as an alternative iron source by the activation of the heme-oxygenase hmuO. In this project we studied the complex interaction between the systems to understand how the particular network architecture shapes the adaptation to the versatile signaling molecule heme. The proposed dissection of the RR-HK interface via random mutagenesis was not yet successful as only a low library diversity was obtained and chimera construction of HKs was not conclusive. We furthermore faced challenges with the structural analysis of the RR-HK complexed due to the impaired solubility of single components. Consequently, this project focused on the question of how the particular network architecture shapes the adaptation to the versatile signaling molecule heme. Key results are summarized in three manuscripts describing (1) the identification of the heme-protein interface in ChrS and HrrS, (2) the analysis of the temporal dynamics and network architecture of the TCSs and (3) the genome-wide profiling of HrrA in response to heme: (1) While N-terminal sensing parts of HrrS and ChrS share only minor sequence similarity, the comparative analysis of the membrane topology and the heme-binding characteristics of the HKs revealed that, both proteins are embedded into the cytoplasmic membrane via six α-helices and directly bind heme in a 1:1 stoichiometry per monomer. Alanine-scanning of conserved amino acid residues in the N-terminal sensor domain of HrrS revealed three aromatic residues (Y112, F115, F118), which apparently contribute to heme binding and suggest an intra-membrane sensing mechanism of this HK. Exchange of the corresponding residues in ChrS and the resulting red shift of the soret band of the heme-protein complex indicated, that in this HK, an altered set of ligands might contribute to heme binding in the triple mutant. (2) To understand how the particular regulatory network architecture of HrrSA and ChrSA shapes the dynamic response to heme, experimental reporter profiling was combined with a quantitative mathematical model (cooperation with Dr. Fritz, Synmikro Marburg). We found, that both HKs contribute to the fast onset of the detoxification response (hrtBA) upon stimulus perception and that the instant deactivation of the hrtBA promoter is achieved by a strong ChrS phosphatase activity upon stimulus decline. While the activation of the detoxification response is uncoupled from further factors, heme utilization (hmuO) is additionally governed by the global iron regulator DtxR, which integrates information on iron availability into the regulatory network. (3) As part of an additional workpackage (WP4), we performed a time-resolved and genome-wide monitoring of in-vivo promoter occupancy of HrrA via chromatin affinity purification and sequencing (ChAP-Seq). This approach revealed HrrA binding to more than 250 different genomic targets, encoding proteins associated with heme biosynthesis, the respiratory chain, oxidative stress response and cell envelope remodeling. By repression of sigC, encoding an activator of the cydABCD operon, HrrA prioritizes the expression of genes encoding the cytochrome bc1-aa3 supercomplex. Given the many properties of heme, the complexity of this response is actually not surprising but paves the way for further functional analysis of HrrA targets with so far unknown functions. Altogether, the results obtained by this project provide comprehensive insights into the regulatory interplay of the HrrSA and ChrSA TCSs shaping a systemic response to the versatile signaling molecule heme.
Publications
- (2020) HrrSA orchestrates a systemic response to heme and determines prioritization of terminal cytochrome oxidase expression. Nucleic acids research 48 (12) 6547–6562
Keppel, Marc; Hünnefeld, Max; Filipchyk, Andrei; Viets, Ulrike; Davoudi, Cedric-Farhad; Krüger, Aileen; Mack, Christina; Pfeifer, Eugen; Polen, Tino; Baumgart, Meike; Bott, Michael; Frunzke, Julia
(See online at https://doi.org/10.1093/nar/gkaa415) - (2018) Membrane Topology and Heme Binding of the Histidine Kinases HrrS and ChrS in Corynebacterium glutamicum. Front Microbiol. 9:183
Keppel M., Davoudi E., Gätgens C. and Frunzke J.
(See online at https://doi.org/10.3389/fmicb.2018.00183) - (2019) Toxic but tasty - Temporal dynamics and network architecture of heme-responsive two-component signalling in Corynebacterium glutamicum. Mol Microbiol, 111:1367-1381
Keppel M, Piepenbreier H, Gätgens C, Fritz G, Frunzke J
(See online at https://doi.org/10.1111/mmi.14226)
