HrrSA orchestrates a systemic response to heme and determines prioritization of terminal cytochrome oxidase expression.

Heme is a multifaceted molecule. While serving as a prosthetic group for many important proteins, elevated levels are toxic to cells. The complexity of this stimulus has shaped bacterial network evolution. However, only a small number of targets controlled by heme-responsive regulators have been described to date. Here, we performed chromatin affinity purification and sequencing to provide genome-wide insights into in vivo promoter occupancy of HrrA, the response regulator of the heme-regulated two-component system HrrSA of Corynebacterium glutamicum. Time-resolved profiling revealed dynamic binding of HrrA to more than 200 different genomic targets encoding proteins associated with heme biosynthesis, the respiratory chain, oxidative stress response and cell envelope remodeling. By repression of the extracytoplasmic function sigma factor sigC, which activates the cydABCD operon, HrrA prioritizes the expression of genes encoding the cytochrome bc1-aa3 supercomplex. This is also reflected by a significantly decreased activity of the cytochrome aa3 oxidase in the ΔhrrA mutant. Furthermore, our data reveal that HrrA also integrates the response to heme-induced oxidative stress by activating katA encoding the catalase. These data provide detailed insights in the systemic strategy that bacteria have evolved to respond to the versatile signaling molecule heme.

The copper-deprivation stimulon of Corynebacterium glutamicum comprises proteins for biogenesis of the actinobacterial cytochrome bc1-aa3 supercomplex.

Aerobic respiration in Corynebacterium glutamicum involves a cytochrome bc1-aa3 supercomplex with a diheme cytochrome c1, which is the only c-type cytochrome in this species. This organization is considered as typical for aerobic Actinobacteria. Whereas the biogenesis of heme-copper type oxidases like cytochrome aa3 has been studied extensively in α-proteobacteria, yeast, and mammals, nothing is known about this process in Actinobacteria. Here, we searched for assembly proteins of the supercomplex by identifying the copper-deprivation stimulon, which might include proteins that insert copper into cytochrome aa3 Using gene expression profiling, we found two copper starvation-induced proteins for supercomplex formation. The Cg2699 protein, named CtiP, contained 16 predicted transmembrane helices, and its sequence was similar to that of the copper importer CopD of Pseudomonas syringae in the N-terminal half and to the cytochrome oxidase maturation protein CtaG of Bacillus subtilis in its C-terminal half. CtiP deletion caused a growth defect similar to that produced by deletion of subunit I of cytochrome aa3, increased copper tolerance, triggered expression of the copper-deprivation stimulon under copper sufficiency, and prevented co-purification of the supercomplex subunits. The secreted Cg1884 protein, named CopC, had a C-terminal transmembrane helix and contained a Cu(II)-binding motif. Its absence caused a conditional growth defect, increased copper tolerance, and also prevented co-purification of the supercomplex subunits. CtiP and CopC are conserved among aerobic Actinobacteria, and we propose a model of their functions in cytochrome aa3 biogenesis. Furthermore, we found that the copper-deprivation response involves additional regulators besides the ECF sigma factor SigC.

A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum.

Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B • Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control • Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.

Identification of Surf1 as an assembly factor of the cytochrome bc1-aa3 supercomplex of Actinobacteria.

Respiration in aerobic Actinobacteria involves a cytochrome bc1-aa3 supercomplex with a diheme cytochrome c1, first isolated from Corynebacterium glutamicum. Synthesis of a functional cytochrome c oxidase requires incorporation of CuA, CuB, heme a, and heme a3. In contrast to eukaryotes and α-proteobacteria, this process is poorly understood in Actinobacteria. Here, we analyzed the role of a Surf1 homolog of C. glutamicum in the formation of a functional bc1-aa3 supercomplex. Deletion of the surf1 gene (cg2460) in C. glutamicum caused a growth defect and cytochrome spectra revealed reduced levels of cytochrome c and a and an increased level of cytochrome d. Membranes of the Δsurf1 strain had lost the ability to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine, suggesting that Surf1 is essential for the formation of a functional cytochrome aa3 oxidase. In contrast to the wild type, a bc1-aa3 supercomplex could not be purified from solubilized membranes of the Δsurf1 mutant. A transcriptome comparison revealed that the genes of the SigC regulon including those for cytochrome bd oxidase were upregulated in the Δsurf1 strain as well as the copper deprivation-inducible gene ctiP. Complementation studies showed that the Surf1 homologs of Corynebacterium diphtheriae, Mycobacterium smegmatis and Mycobacterium tuberculosis could at least partially abolish the growth defect of the C. glutamicum Δsurf1 mutant, suggesting that Surf1 is a conserved assembly factor for actinobacterial cytochrome aa3 oxidase.