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.
Project description:Heme is an essential cofactor and alternative iron source for almost all bacterial species but may cause severe toxicity upon elevated levels and consequently, regulatory mechanisms coordinating heme homeostasis represent an important fitness trait. A remarkable scenario is found in several corynebacterial species, e.g. Corynebacterium glutamicum and Corynebacterium diphtheriae, which dedicate two paralogous, heme-responsive two-component systems, HrrSA and ChrSA, to cope with the Janus nature of heme. Here, we combined experimental reporter profiling with a quantitative mathematical model to understand how this particular regulatory network architecture shapes the dynamic response to heme. Our data revealed an instantaneous activation of the detoxification response (hrtBA) upon stimulus perception and we found that kinase activity of both kinases contribute to this fast onset. Furthermore, instant deactivation of the PhrtBA promoter is achieved by a strong ChrS phosphatase activity upon stimulus decline. While the activation of detoxification response is uncoupled from further factors, heme utilization is additionally governed by the global iron regulator DtxR integrating information on iron availability into the regulatory network. Altogether, our data provide comprehensive insights how TCS cross-regulation and network hierarchy shape the temporal dynamics of detoxification (hrtBA) and utilization (hmuO) as part of a global homeostatic response to heme.
Project description:The MmpL family of proteins translocates complex (glyco)lipids and siderophores across the cell envelope of mycobacteria and closely related Corynebacteriaceae and plays important roles in the biogenesis of the outer membrane of these organisms. Despite their significance in the physiology and virulence of Mycobacterium tuberculosis, and from the perspective of developing novel antituberculosis agents, little is known about their structure and mechanism of translocation. In this study, the essential mycobacterial mycolic acid transporter, MmpL3, and its orthologue in Corynebacterium glutamicum, CmpL1, were investigated as prototypical MmpL proteins to gain insight into the transmembrane topology, tertiary and quaternary structures, and functional regions of this transporter family. The combined genetic, biochemical, and biophysical studies indicate that MmpL3 and CmpL1 are structurally similar to Gram-negative resistance-nodulation and division efflux pumps. They harbor 12 transmembrane segments interrupted by two large soluble periplasmic domains and function as homotrimers to export long-chain (C22-C90) mycolic acids, possibly in their acetylated form, esterified to trehalose. The mapping of a number of functional residues within the middle region of the transmembrane domain of MmpL3 shows a striking overlap with mutations associated with resistance to MmpL3 inhibitors. The results suggest that structurally diverse inhibitors of MmpL3 all target the proton translocation path of the transporter and that multiresistance to these inhibitors is enabled by conformational changes in MmpL3.
Project description:Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT) reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow.
Project description:Corynebacterium diphtheriae, the causative agent of diphtheria, utilizes various host compounds to acquire iron. The C. diphtheriae hmuO gene encodes a heme oxygenase that is involved in the utilization of heme and hemoglobin as iron sources. Transcription of the hmuO gene in C. diphtheriae is controlled under a dual regulatory mechanism in which the diphtheria toxin repressor protein (DtxR) and iron repress expression while either heme or hemoglobin is needed to activate transcription. In this study, two clones isolated from a C. diphtheriae chromosomal library were shown to activate transcription from the hmuO promoter in Escherichia coli. Sequence analysis revealed that these activator clones each carried distinct genes whose products had significant homology to response regulators of two-component signal transduction systems. Located upstream from each of these response regulator homologs are partial open reading frames that are predicted to encode the C-terminal portions of sensor kinases. The full-length sensor kinase gene for each of these systems was cloned from the C. diphtheriae chromosome, and constructs each carrying one complete sensor kinase gene and its cognate response regulator were constructed. One of these constructs, pTSB20, which carried the response regulator (chrA) and its cognate sensor kinase (chrS), was shown to strongly activate transcription from the hmuO promoter in a heme-dependent manner in E. coli. A mutation in chrA (chrAD50N), which changed a conserved aspartic acid residue at position 50, the presumed site of phosphorylation by ChrS, to an asparagine, abolished heme-dependent activation. These findings suggest that the sensor kinase ChrS is involved in the detection of heme and the transduction of this signal, via a phosphotransfer mechanism, to the response regulator ChrA, which then activates transcription of the hmuO promoter. This is the first report of a bacterial two-component signal transduction system that controls gene expression through a heme-responsive mechanism.
Project description:Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter in the solute carrier 29 (SLC29) family. Previous studies suggested that the major substrate recognition sites are located within transmembrane domains (TM) 1-6, and interaction of PMAT with organic cations may involve aromatic residues. In this study, we analyzed the roles of tyrosine and tryptophan residues located within TM1-6 with a goal of identifying potential residues involved in substrate recognition and translocation. The six tyrosines and one tryptophan in this region were each mutated to alanine followed by analysis of the mutant's membrane localization and transport activity toward 1-methyl-4-phenylpyridinium (MPP(+)), serotonin (5-HT), and dopamine. Two mutants, Y85A and Y112A, exhibited normal cell surface expressions but lost their transport activities toward organic cations. At position Y85, aromatic substitution with phenylalanine or tryptophan fully restored organic cation transport activity. Interestingly, at position Y112, phenylalanine substitution was not allowed. Tryptophan substitution at Y112 partially restored transport activity toward 5-HT and dopamine but severely impaired MPP(+) transport. Detailed kinetic analyses revealed that tryptophan substitution at Y85 and Y112 affected the apparent binding affinity (K(m)) and maximal transport velocity (V(max)) in a substrate-dependent manner. Together, our data suggest that Y85 and Y112 are important molecular determinants for PMAT function, and Y112 is indispensable for optimal interaction with organic cation substrates. Our analyses also suggest the involvement of transmembrane domains 1 and 2 in forming the substrate permeation pathway of PMAT.
Project description:The response regulator HrrA of the HrrSA two-component system (previously named CgtSR11) was recently found to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Here, we provide evidence that HrrA mediates heme-dependent gene regulation in this nonpathogenic soil bacterium. Growth experiments and DNA microarray analysis revealed that C. glutamicum is able to use hemin as an alternative iron source and emphasize the involvement of the putative hemin ABC transporter HmuTUV and heme oxygenase (HmuO) in heme utilization. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of an hrrA deletion mutant and C. glutamicum wild-type strain in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase. Based on our results, it can be furthermore deduced that HrrA activates the expression of heme-containing components of the respiratory chain, namely, ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa(3) oxidase and three subunits of the cytochrome bc(1) complex. In addition, HrrA was found to repress almost all genes involved in heme biosynthesis, including those for glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Growth experiments with an hrrA deletion mutant showed that this strain is significantly impaired in heme utilization. In summary, our results provide evidence for a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum.
Project description:Corynebacterium diphtheriae, the causative agent of the severe respiratory disease diphtheria, utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. Because of the toxicity of high levels of hemin and iron, these compounds are often tightly regulated in bacterial systems. In this report, we identify and characterize the C. diphtheriae hrtAB genes, which encode a putative ABC type transporter involved in conferring resistance to the toxic effects of hemin. Deletion of the hrtAB genes in C. diphtheriae produced increased sensitivity to hemin, which was complemented by a plasmid harboring the cloned hrtAB locus. The HrtAB system was not involved in the uptake and use of hemin as an iron source. The hrtAB genes are located on the C. diphtheriae genome upstream from the chrSA operon, which encodes a previously characterized two-component signal transduction system that regulates gene expression in a heme-dependent manner. The hrtB promoter is activated by the ChrAS system in the presence of hemin or hemoglobin, and mutations in the chrSA genes abolish heme-activated expression from the hrtB promoter. It was also observed that transcription from the hrtB promoter is reduced in a dtxR deletion mutant, suggesting that DtxR is required for optimal expression of hrtAB. Previous studies proposed that the ChrS sensor kinase may be responsive to an environmental signal, such as hemin. We show that specific point mutations in the ChrS N-terminal transmembrane domain result in a reduced ability to activate the hrtB promoter in the presence of a heme source, suggesting that this putative sensor region is essential for the detection of a signal produced in response to hemin exposure. This study shows that the HrtAB system is required for protection from hemin toxicity and that expression of the hrtAB genes is regulated by the ChrAS two-component system. This study demonstrates a direct correlation between the detection of heme or a heme-associated signal by the N-terminal sensor domain of ChrS and the transcriptional activation of the hrtAB genes.
Project description:Resistance to arsenite (As(III)) by cells is generally accomplished by arsenite efflux permeases from Acr3 or ArsB unrelated families. We analyzed the function of three Acr3 proteins from Corynebacterium glutamicum, CgAcr3-1, CgAcr3-2, and CgAcr3-3. CgAcr3-1 conferred the highest level of As(III) resistance and accumulation in vivo. CgAcr3-1 was also the most active when everted membranes vesicles from Escherichia coli or C. glutamicum mutants were assayed for efflux with different energy sources. As(III) and antimonite (Sb(III)) resistance and accumulation studies using E. coli or C. glutamicum arsenite permease mutants clearly show that CgAcr3-1 is specific for As(III). In everted membrane vesicles expressing CgAcr3-1, dissipation of either the membrane potential or the pH gradient of the proton motive force did not prevent As(III) uptake, whereas dissipation of both components eliminated uptake. Further, a mutagenesis study of CgAcr3-1 suggested that a conserved cysteine and glutamate are involved in active transport. Therefore, we propose that CgAcr3-1 is an antiporter that catalyzes arsenite-proton exchange with residues Cys129 and Glu305 involved in efflux.
Project description:ArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export of l-arginine (Arg) in Escherichia coli and l-lysine (Lys) and Arg in Corynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane of E. coli, we have employed a combination of cysteine accessibility in situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Cout configuration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO function in vivo and that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO function in vivo and their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomer in vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit.The orthologous proteins LysE of C. glutamicum and ArgO of E. coli function as exporters of the basic amino acids l-arginine and l-lysine and the basic amino acid l-arginine, respectively, and LysE can functionally substitute for ArgO when expressed in E. coli Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic and in silico studies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.
Project description:Background:Biliverdin, a prospective recyclable antioxidant and one of the most important precursors for optogenetics, has received growing attention. Biliverdin is currently produced by oxidation of bilirubin from mammalian bile using chemicals. However, unsustainable procedures of extraction, chemical oxidation, and isomer separation have prompted bio-based production using a microbial cell factory. Results:In vitro thermodynamic analysis was performed to show potential candidates of bottleneck enzymes in the pathway to produce biliverdin. Among the candidates, hemA and hemL were overexpressed in Corynebacterium glutamicum to produce heme, precursor of biliverdin. To increase precursor supply, we suggested a novel hemQ-mediated coproporphyrin dependent pathway rather than noted hemN-mediated protoporphyrin dependent pathway in C. glutamicum. After securing precursors, hmuO was overexpressed to pull the carbon flow to produce biliverdin. Through modular optimization using gene rearrangements of hemA, hemL, hemQ, and hmuO, engineered C. glutamicum BV004 produced 11.38 ± 0.47 mg/L of biliverdin at flask scale. Fed-batch fermentations performed in 5 L bioreactor with minimal medium using glucose as a sole carbon source resulted in the accumulation of 68.74 ± 4.97 mg/L of biliverdin, the highest titer to date to the best of our knowledge. Conclusions:We developed an eco-friendly microbial cell factory to produce biliverdin using C. glutamicum as a biosystem. Moreover, we suggested that C. glutamicum has the thermodynamically favorable coproporphyrin dependent pathway. This study indicated that C. glutamicum can work as a powerful platform to produce biliverdin as well as heme-related products based on the rational design with in vitro thermodynamic analysis.