Project description:Cupriavidus metallidurans CH34 is a metal resistant beta-proteobacterium. The genome of this bacterium contain many genes involved in heavy metal resistance. Gene expression of C. metallidurans was studied after the addition of of Zn(II), Cd(II), Cu(II), Ni(II), Pb(II), Hg(II) or Co(II). Keywords: Heavy metal stress response Cultures of C. metallidurans CH34 were grown at 30°C until OD reached 0.6 (mid- exponential phase cultures). Heavy metals (0.8 mM of Zn(II), 0.5 mM of Cd(II), 0.1 mM of Cu(II), 0.6 mM of Ni(II), 0.4 mM of Pb(II), 5 uM of Hg(II) and 0.5 mM of Co(II)) were added to the culture for 30 minutes induction time. Total RNA was extracted, reverse-transcribed and labeled with Cy3-dCTP for the control (without metal) and with Cy5-dCTP for each conditions (challenged with one metal). Labeled cDNA were (control and one condition) added to a spotted slide for overnight hybridization at 42°C. Slides were scanned with a laser at 532 and 635 nm.
Project description:Different Cupriavidus metallidurans strains isolated from metal-contaminated and other anthropogenic environments were genotypically and phenotypically compared with C. metallidurans type strain CH34. The latter is well-studied for its resistance to a wide range of metals, which is carried for a substantial part by its two megaplasmids pMOL28 and pMOL30. Comparative genomic hybridization (CGH) indicated that the extensive arsenal of determinants involved in metal resistance was well conserved among the different C. metallidurans strains. Contrary, the mobile genetic elements identified in type strain CH34 were not present in all strains but clearly showed a pattern, although, not directly related to a particular biotope nor location (geographical). One group of strains carried almost all mobile genetic elements, while these were much less abundant in the second group. This occurrence was also reflected in their ability to degrade toluene and grow autotrophically on hydrogen gas and carbon dioxide, which are two traits linked to separate genomic islands of the Tn4371-family. In addition, the clear pattern of genomic islands distribution allowed to identify new putative genomic islands on chromosome 1 and 2 of C. metallidurans CH34. Metal resistance determinants are shared by all C. metallidurans strains and their occurrence is apparently irrespective of the strain's isolation type and place. Cupriavidus metallidurans strains do display substantial differences in the diversity and size of their mobile gene pool, which may be extensive in some (including the type strain) while marginal in others. Comparative genome hybridization experiments. Comparing genomic DNA samples of different strains with a common reference strain (CH34).
Project description:Response of Cupriavidus metallidurans AE104 and delta zur mutant to Zn stress Overall design: In this study Zn-treatment was performed to acquire expression profiles of the total genome of Cupriavidus metallidurans
Project description:Background: The high number of heavy metal resistance genes in the soil bacterium Cupriavidus metallidurans CH34 makes it an interesting model organism to study microbial responses to heavy metals. Results: In this study the transcriptional response of this bacterium was measured after challenging it to a wide range of sub-lethal concentrations of various essential or toxic metals. Considering the global transcriptional responses for each challenge as well as by identifying the overlap in upregulated genes between different metal responses, the sixteen metals could be clustered in three different groups. Additionally, next to the assessment of the transcriptional response of already known metal resistance genes, new metal response gene clusters were identified. The majority of the metal response loci showed similar expression profiles when cells were exposed to different metals, suggesting complex cross-talk at transcriptional level between the different metal responses. The highly redundant nature of these metal resistant regions – illustrated by the large number of paralogous genes – combined with the phylogenetic distribution of these metal response regions within evolutionary related and other metal resistant bacteria, provides important insights on the recent evolution of this naturally soil dwelling bacterium towards a highly metal-resistant strain found in harsh and anthropogenic environments. Conclusions: The metal-resistant soil bacterium Cupriavidus metallidurans CH34 displays myriads of gene expression patterns when exposed to a wide range of heavy metals at non-lethal concentrations. The interplay between the different gene expression clusters points towards a complex cross-regulated regulatory network governing heavy metal resistance in C. metallidurans CH34. Keywords: Cupriavidus metallidurans CH34, transcriptional regulation, heavy metal resistance Two-condition experiments. Comparing samples after induction with heavy metals versus non-induced samples. Biological duplicate or triplicate. Each array contains 3 or 4 technical replicates.
Project description:Response of Cupriavidus metallidurans AE104(delta zupT) mutant to Zn/EDTA stress In this study 2 different treatments were performed to acquire expression profiles of the total genome of Cupriavidus metallidurans
Project description:Response of Cupriavidus metallidurans CH34, AE104 and delta zupT mutant to Zn/EDTA stress In this study 3 different treatments were performed to acquire expression profiles of the total genome of Cupriavidus metallidurans
Project description:The physiological and transcriptomic response of the metal resistant bacterium Cupriavidus metallidurans strain CH34 in response to stable (non-radioactive) strontium ions (Sr) was investigated. C. metallidurans CH34 could survive and proliferate in the presence of relatively high concentrations of SrCl2 (D10 is 70mM, MIC is 120 mM). Precipitation of Sr as strontium carbonate was observed in the culture during aerobic growth of CH34 in the presence of 60 mM SrCl2. To identify the cellular mechanisms involved in the bioprecipitation process, gene expression in the cells was analyzed after short-time (30 min) exposure to low (5 mM) and high (60 mM) concentrations of SrCl2. The transcription of the gene clusters annotated as hmyFCBA and czcCBADRS, coding for ion efflux pumps, was significantly induced following exposure to Sr, and not with Ca. There were also significant changes is the transcription of the genes encoding TctCBA proteins involved in citrate uptake and two hypothetical porin coding genes following exposure Sr and Ca. These results highlight a specific molecular response of bacterium Cupriavidus metallidurans CH34 to Sr, including the identification of putative Sr specific efflux pumps, and thus the potential of this bacterium to distinguish Sr from Ca. These findings will help to better understand natural Sr (and Ca) microbial weathering or mineralization processes in the environment, and could be of interest for bioremediation or bioprocessing of (radioactive) Sr-containing water, soil or waste. Two-condition experiments. Comparing samples after induction with metals (Sr, Ca) versus non-induced samples. Biological triplicate. Each array contains 3 technical replicates.
Project description:The weathering of volcanic minerals makes a significant contribution to the global silicate weathering budget, influencing carbon dioxide drawdown and climate control. Basalt rocks may account for over 30% of the global carbon dioxide drawdown in silicate weathering. Yet the genetics of biological rock weathering are unknown. For the first time, we apply a DNA microarray to investigate the genes involved in weathering by the heavy metal resistant organism, Cupriavidus metallidurans CH34; in particular we investigate the sequestering of iron. The results show that the bacterium sequesters iron in the ferrous state (FeII); therefore, not requiring siderophores. Instead an energy efficient process involving upregulation of large porins is employed concomitantly with genes associated with biofilm formation. We hypothesise that rock weathering is induced by changes in chemical equilibrium at the microbe-mineral interface, reducing the saturation state of iron. We also demonstrate that low concentrations of metals in the basalt induce heavy metal resistant genes. Volcanic environments are analogous to some of the earliest environments on Earth. These results not only elucidate the mechanisms by which microorganisms might have sequestered nutrients on the early Earth but they also provide an explanation for the evolution of multiple heavy metal resistance genes long before the creation of contaminated industrial biotopes by human activity. Cultures of Cupriavidus metallidurans CH34 were grown in Tris buffered medium MM284 media (with iron), MM284 without iron and MM284 without iron with sterilized basalt at 25 rpm, 30°C until mid-log phase. RNA was extracted from the cells. Three biological replicates of both samples were differentially labeled (resp. Cy3 and Cy5) and hybridized to three CH34 60-mer oligonucleotide glass-spotted microarray carrying three technical repeats.
Project description:Various environmental bacteria were adapted to the presence of toxic and recalcitrant organic solvents. Cupriavidus metallidurans CH34, a β-proteobacterium that was found in industrial environments is known as a bacterial model to study heavy metal resistance. Interestingly, genome screening of CH34 also reveals the existence of genes involved in the degradation of recalcitrant organic solvents, such acetone and aromatic compounds. Here, we showed that this bacterium could resist a large variety of organic solvents, and was also able to metabolize some of them. In particular, investigations were focused on acetone and isopropanol catabolism. Integrative studies based on transcriptomic (DNA microarrays), proteomic (2D-DIGE and Isotope-Coded Protein Label technology) and biochemical analyses (enzyme purification and characterization) showed a similar catabolic pathway for both molecules which involved the AcxABC acetone carboxylase. First, isopropanol is oxidized into acetone by the Adh alcohol dehydrogenase. Acetoacetate production from acetone is then catalyzed by the acetone carboxylase. The generated acetoacetate molecules are then transformed by the PacIJ 3-oxoacid CoA-transferase, to acetoacetyl-CoA and succinate. Finally, an acetyl-CoA acetyltransferase catalyses the hydrolysis of acetoacetyl-CoA into 2 acetyl-CoA that are introduced into the glyoxylate cycle. As demonstrated, key enzymes of the acetone/isopropanol catabolism (encoded by acx and ald genes) are under the control of a σ54-dependent RNA polymerase. Moreover, the catabolic pathway involved in acetone and isopropanol consumption was repressed when gluconate was given as alternative carbon substrate, displaying a new example of diauxie. The results presented here provide a comprehensive picture of acetone and isopropanol biodegradation in Cupriavidus metallidurans CH34 and strongly support the fact that CH34 can be considered as a solvent tolerant bacterium. Two-condition experiments. Comparing samples after induction with acetone versus non-induced samples. Biological triplicate. Each array contains 3 technical replicates.