Copper Response Regulator Research Articles

Distinct function of Chlamydomonas CTRA-CTR transporters in Cu assimilation and intracellular mobilization.

Regulation of Cu uptake and sequestration by members of the CTR family of proteins in Chlamydomonas.

MSMEG_0918 is not Essential for the Growth of Mycobacteriumsmegmatis.

Copper homeostasis plays a crucial role in mycobacteria. In Mycobacterium tuberculosis , Rv0474 is a copper-responsive regulator with a copper-binding motif but its homolog in Mycobacterium smegmatis , MSMEG_0918, lacks the copper-binding motif. We generated MSMEG_0918 knockdown strains of M. smegmatis using CRISPRi. We confirmed the strains had varying levels of MSMEG_0918 expression using RT-PCR. We demonstrated that MSMEG_0918 under-expression did not alter the growth of M. smegmatis in standard aerobic culture as compared to the wild-type. Our knockdown strains (CHROME1 and CHROME2) could be further used towards understanding the role of MSMEG_0918 in M. smegmatis .

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

A Role for Mycobacterium tuberculosis Sigma Factor C in Copper Nutritional Immunity.

Sigma factor C (SigC) contributes to Mycobacterium tuberculosis virulence in various animal models, but the stress response coordinated by this transcription factor was undefined. The results presented here indicate that SigC prevents copper starvation. Whole genome expression studies demonstrate short-term (4-h) induction of sigC, controlled from a tetracycline-inducible promoter, upregulates ctpB and genes in the nonribosomal peptide synthase (nrp) operon. These genes are expressed at higher levels after 48-h sigC induction, but also elevated are genes encoding copper-responsive regulator RicR and RicR-regulated copper toxicity response operon genes rv0846–rv0850, suggesting prolonged sigC induction results in excessive copper uptake. No growth and global transcriptional differences are observed between a sigC null mutant relative to its parent strain in 7H9 medium. In a copper-deficient medium, however, growth of the sigC deletion strain lags the parent, and 40 genes (including those in the nrp operon) are differentially expressed. Copper supplementation reverses the growth defect and silences most transcriptional differences. Together, these data support SigC as a transcriptional regulator of copper acquisition when the metal is scarce. Attenuation of sigC mutants in severe combined immunodeficient mice is consistent with an inability to overcome innate host defenses that sequester copper ions to deprive invading microbes of this essential micronutrient.

New Plant Growth-Promoting, Chromium-Detoxifying Microbacterium Species Isolated From a Tannery Wastewater: Performance and Genomic Insights.

Hexavalent chromium [Cr(VI)], widely generated by tannery activities, is considered among the most toxic substances and causes a serious damage for the environment and for human health. Interestingly, some microorganisms have a potential of bioremediation of chromium-contaminated wastewaters and soils through the reduction of Cr(VI) (soluble and harmful form) into Cr(III) (stable and non-toxic form). Here, we present the full genome sequence of a novel heavy-metal-resistant, plant growth-promoting bacterium (PGPB), Microbacterium metallidurans TL13, which was isolated from a Tunisian leather industry. The strain TL13 was resistant to many heavy metals, such as chromium, copper, nickel, cobalt, and arsenic. The 50% TL13 growth inhibitory concentration (IC50) values of HgCl2, CoCl2, K2Cr2O7, CuSO4, NiCl2, FeSO4, and Na2HAsO4 are 368, 445, 676, 1,590, 1,680, 4,403, and 7,007 mg/L, respectively, with the following toxicity order: HgCl2 > CoCl2 > K2Cr2O7 > CuSO4 > NiCl2 > FeSO4 > Na2HAsO4. This new strain was also able to promote the growth of the hybrid tomato (Elika F1) under chromium metal stress. Its whole genome sequence length was estimated to be 3,587,460 bp (3,393 coding sequences) with a G + C content of 70.7%. Functional annotation of the genome of TL13 revealed the presence of open reading frames (ORFs) involved in adaptation to metal stress, such as the chromate transport protein, cobalt–zinc–cadmium resistance protein, copper resistance protein, copper responsive transcriptional regulator, multidrug resistance transporters, arsenical resistance operon repressor, arsenate reductase, arsenic resistance protein, mercuric resistance operon regulatory protein, mercuric ion reductase, and organomercurial lyase. Moreover, genes for the production of glutathione peroxidase, catalase, superoxide dismutase, and thioredoxin reductase, which confer a higher tolerance to oxidative/metal stresses, were identified in TL13 genome. In addition, genes for heat shock tolerance, cold shock tolerance, glycine-betaine production, mineral phosphate solubilization, ammonia assimilation, siderophores, exopolysaccharides, polyketides, and lytic enzymes (cellulase, chitinase, and proteases) production that enable bacteria to survive biotic/abiotic stress and to promote plant growth and health were also revealed. Based on genome analysis and experimental approaches, strain TL13 appears to have evolved from various metabolic strategies and could play a role in ensuring sustainable environmental and agricultural systems.

Discovery of CRR1-targeted copper deficiency response in Chlamydomonas reinhardtii exposed to silver nanoparticles

The molecular mechanisms behind the adaptive responses for interactions between organisms and nanoparticles, such as silver nanoparticles (AgNPs), are of great concern. In this study, the transcriptome of freshwater alga Chlamydomonas reinhardtii was characterized via RNA sequencing (RNA-seq) after exposure to a nontoxic concentration of AgNPs (0.5 mg/L). The cytochrome c6 (CYC6) and ferredoxin-5 (FDX5) genes were identified with the greatest increase in expression level, which were indications of the copper deficiency in the algae. Gene set enrichment analysis also showed significant enrichment of copper deficiency responsive genes in the transcriptome of algae exposed to AgNPs. These results indicated that AgNPs induced a copper deficiency response in algae, and the excessive intracellular copper content suggested this was due to functional copper deficiency. This deficiency response was further validated to be regulated by transcription factor CRR1 (copper response regulator 1) according to the assays on the mutant strain with defect of CRR1. To the best of our knowledge, this is the first corroboration of a CRR1-targeted copper deficiency response in algae following AgNP exposure. Given the function of copper in fundamental metabolic pathways, such as photosynthesis and respiration, we propose a potential role of CRR1-targeted copper deficiency as an adaptation of algae after exposure to AgNPs.

Regulation of sulfur deprivation-induced expression of the ferredoxin-encoding FDX5 gene Chlamydomonas reinhardtii in aerobic conditions

The unicellular green alga Chlamydomonas reinhardtii reacts to sulfur (S) starvation with the increased expression of numerous genes. One gene which is induced in illuminated anaerobic S-deprived cells is the ferredoxin-5 gene (FDX5). To test FDX5 transcriptional regulation in aerobic cultures, we used a real-time PCR analysis and an artificial microRNA approach. We demonstrated that FDX5 gene is controlled by S deprivation independently of anoxia-treatment. The Ser/Thr kinase SNRK2.1 is necessary for expression of FDX5 during deprivation to S. Copper response regulator 1 (CRR1) is not involved in FDX5 up-regulation in S-deficient cells under aerobic conditions. Furthermore, expression of FDX5 is negatively regulated by nitric oxide (NO). Moreover, truncated hemoglobin 1 (THB1) underexpression resulted in the decrease in FDX5 transcript abundance in S-deficient cells under aerobic conditions. Together, our results imply that the FDX5 gene is controlled by NO in THB1-dependent pathway under conditions of depleted S supply.

New insights into AOX2 transcriptional regulation in Chlamydomonas reinhardtii

A feature of the mitochondrial electron transport chain in plants, some protists and many fungi is the presence of two terminal oxidases, the energy-conserving cytochrome oxidase and another termed alternative oxidase (AOX). AOX branches from the main respiratory chain, directly coupling the oxidation of ubiquinol with reduction of oxygen to water. The AOX genes can be divided into two discrete subfamilies, AOX1 and AOX2. Although AOX has been proposed to play essential roles in stress tolerance of organisms, the role of subfamily AOX2 is largely unknown. In the model green alga Chlamydomonas reinhardtii, two genes have been identified that encode for AOX, AOX1 and AOX2. To test AOX2 transcriptional regulation in this alga, we used a real-time PCR analysis and an artificial microRNA approach. The C. reinhardtii AOX2 gene is up-regulated by oxygen or copper deprivation. Moreover, in dark-adapted unstressed cells, AOX2 is induced. Together, our results imply that the AOX2 gene is a stress-inducible and is regulated by the copper response regulator 1 (CRR1), probably together with yet-unknown regulatory factor(s).

Copper economy in Chlamydomonas: Prioritized allocation and reallocation of copper to respiration vs. photosynthesis

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retention of genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.

Zinc Deficiency Impacts CO2 Assimilation and Disrupts Copper Homeostasis in Chlamydomonas reinhardtii

Zinc is an essential nutrient because of its role in catalysis and in protein stabilization, but excess zinc is deleterious. We distinguished four nutritional zinc states in the alga Chlamydomonas reinhardtii: toxic, replete, deficient, and limited. Growth is inhibited in zinc-limited and zinc-toxic cells relative to zinc-replete cells, whereas zinc deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition-responsive changes in gene expression. We identified genes encoding zinc-handling components, including ZIP family transporters and candidate chaperones. Additionally, we noted an impact on two other regulatory pathways, the carbon-concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc deficiency, probably due to reduced carbonic anhydrase activity, validated by quantitative proteomics and immunoblot analysis of Cah1, Cah3, and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in zinc-limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. The Crr1 regulon responds to copper limitation and is turned on in zinc deficiency, and Crr1 is required for growth in zinc-limiting conditions. Zinc-deficient cells are functionally copper-deficient, although they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester copper in a biounavailable form, perhaps to prevent mismetallation of critical zinc sites.

Anaerobic Expression of the Ferredoxin-Encoding FDX5 Gene of Chlamydomonas reinhardtii Is Regulated by the Crr1 Transcription Factor

The unicellular green alga Chlamydomonas reinhardtii has a complex anaerobic metabolism and reacts to hypoxic or anaerobic conditions with the induced expression of many genes. One gene which is upregulated particularly strongly is the FDX5 gene, encoding one of at least six ferredoxin isoforms in C. reinhardtii. Fdx5 is a typical plant-type 2Fe2S protein that is located in the chloroplast. The FDX5 promoter region contains three GTAC motifs, which are known to be the binding sites for copper response regulator 1 (Crr1) and other SQUAMOSA promoter binding proteins (SBPs). This study shows that two of these GTAC sites are essential to confer oxygen and also copper responsiveness to a reporter gene. The SBP domain of Crr1 is able to bind to both of these GTAC sites in in vitro binding assays. Moreover, in a Crr1-deficient C. reinhardtii strain, FDX5 is not expressed. These results clearly indicate that Crr1 is involved in the transcriptional regulation of the FDX5 gene in the absence of oxygen or copper.

Molecular characterization of a conserved archaeal copper resistance (cop) gene cluster and its copper-responsive regulator in Sulfolobus solfataricus P2

Using a comparative genomics approach, a copper resistance gene cluster has been identified in multiple archaeal genomes. The cop cluster is predicted to encode a metallochaperone (CopM), a P-type copper-exporting ATPase (CopA) and a novel, archaea-specific transcriptional regulator (CopT) which might control the expression of the cop genes. Sequence analysis revealed that CopT has an N-terminal DNA-binding helix-turn-helix domain and a C-terminal TRASH domain; TRASH is a novel domain which has recently been proposed to be uniquely involved in metal-binding in sensors, transporters and trafficking proteins in prokaryotes. The present study describes the molecular characterization of the cop gene cluster in the thermoacidophilic crenarchaeon Sulfolobus solfataricus. The polycistronic copMA transcript was found to accumulate in response to growth-inhibiting copper concentrations, whereas copT transcript abundance appeared to be constitutive. DNA-binding assays revealed that CopT binds to the copMA promoter at multiple sites, both upstream and downstream of the predicted TATA-BRE site. Copper was found to specifically modulate the affinity of DNA binding by CopT. This study describes a copper-responsive operon in archaea, a new family of archaeal DNA-binding proteins, and supports the idea that this domain plays a prominent role in the archaeal copper response. A model is proposed for copper-responsive transcriptional regulation of the copMA gene cluster.

A regulator of nutritional copper signaling in Chlamydomonas is an SBP domain protein that recognizes the GTAC core of copper response element

The CRR1 (Copper Response Regulator) locus, required for both activating and repressing target genes of a copper- and hypoxia-sensing pathway in Chlamydomonas, encodes a 1,232-residue candidate transcription factor with a plant-specific DNA-binding domain named SBP, ankyrin repeats, and a C-terminal Cys-rich region, with similarity to a Drosophila metallothionein. The recombinant SBP domain of Crr1 shows zinc-dependent binding to functionally defined copper-response elements associated with the CYC6 and CPX1 promoters that contain a critical GTAC core sequence. Competition experiments indicate equivalent selectivity for copper-response elements from either promoter and 10-fold greater selectivity for the wild-type sequence vs. a sequence carrying a single mutation in the GTAC core. The SBP domain of Chlamydomonas Crr1 binds also to a related GTAC-containing sequence in the Arabidopsis AP1 promoter that is the binding site of a defining member of the SBP family of DNA-binding proteins. Chlamydomonas Crr1 is most similar to a subset of the Arabidopsis SBP domain proteins, which include SPL1, SPL7, and SPL12. The abundance of the CRR1 mRNA is only marginally copper-responsive, and although two mRNAs that differ with respect to splicing of the first intron are detected, there is no indication that the splicing event is regulated by metal nutrition or hypoxia. It is likely that the dramatic copper-responsive action of Crr1 occurs at the level of the polypeptide.

Genetic Dissection of Nutritional Copper Signaling in Chlamydomonas Distinguishes Regulatory and Target Genes

A genetic screen for Chlamydomonas reinhardtii mutants with copper-dependent growth or nonphotosynthetic phenotypes revealed three loci, COPPER RESPONSE REGULATOR 1 (CRR1), COPPER RESPONSE DEFECT 1 (CRD1), and COPPER RESPONSE DEFECT 2 (CRD2), distinguished as regulatory or target genes on the basis of phenotype. CRR1 was shown previously to be required for transcriptional activation of target genes like CYC6, CPX1, and CRD1, encoding, respectively, cytochrome c(6) (which is a heme-containing substitute for copper-containing plastocyanin), coproporphyrinogen III oxidase, and Mg-protoporphyrin IX monomethylester cyclase. We show here that CRR1 is required also for normal accumulation of copper proteins like plastocyanin and ferroxidase in copper-replete medium and for apoplastocyanin degradation in copper-deficient medium, indicating that a single pathway controls nutritional copper homeostasis at multiple levels. CRR1 is linked to the SUPPRESSOR OF PCY1-AC208 13 (SOP13) locus, which corresponds to a gain-of-function mutation resulting in copper-independent expression of CYC6. CRR1 is required also for hypoxic growth, pointing to a physiologically meaningful regulatory connection between copper deficiency and hypoxia. The growth phenotype of crr1 strains results primarily from secondary iron deficiency owing to reduced ferroxidase abundance, suggesting a role for CRR1 in copper distribution to a multicopper ferroxidase involved in iron assimilation. Mutations at the CRD2 locus also result in copper-conditional iron deficiency, which is consistent with a function for CRD2 in a pathway for copper delivery to the ferroxidase. Taken together, the observations argue for a specialized copper-deficiency adaptation for iron uptake in Chlamydomonas.

Copper response element and Crr1-dependent Ni(2+)-responsive promoter for induced, reversible gene expression in Chlamydomonas reinhardtii.

The Cpx1 and Cyc6 genes of Chlamydomonas reinhardtii are activated in copper-deficient cells via a signal transduction pathway that requires copper response elements (CuREs) and a copper response regulator defined by the CRR1 locus. The two genes can also be activated by provision of nickel or cobalt ions in the medium. The response to nickel ions requires at least one CuRE and also CRR1 function, suggesting that nickel interferes with a component in the nutritional copper signal transduction pathway. Nickel does not act by preventing copper uptake/utilization because (i) holoplastocyanin formation is unaffected in Ni(2+)-treated cells and (ii) provision of excess copper cannot reverse the Ni-dependent activation of the target genes. The CuRE is sufficient for conferring Ni-responsive expression to a reporter gene, which suggests that the system has practical application as a vehicle for inducible gene expression. The inducer can be removed either by replacing the medium or by chelating the inducer with excess EDTA, either of which treatments reverses the activation of the target genes.

The sctR of Salmonella enterica serova Typhimurium encoding a homologue of MerR protein is involved in the copper-responsive regulation of cuiD

We have identified the cuiD gene in Salmonella enterica serova Typhimurium that codes for a putative multicopper oxidase. Expression of cuiD is induced by copper ion and its promoter/operator has sequence similarity to the promoters controlled by the transcriptional regulators of the MerR family. We also identified and isolated a gene from S. enterica serova Typhimurium that encodes a 138-amino acid residue protein, sctR, a new member of the MerR family of transcriptional regulators. Transposon-insertional disruption of sctR shows sensitivity to copper ion and no response of cuiD expression. Copper-responsive induction and copper tolerance were restored by providing sctR in trans, suggesting that SctR plays an important role in intracellular copper detoxification.