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Bacteria are increasingly used for biotechnological applications such as bioremediation, biorecovery, bioproduction, and biosensing. The development of strains suited for such applications requires a thorough understanding of their behavior, with a key role for their transcriptomic landscape. We present a thorough analysis of the transcriptome of Cupriavidus metallidurans CH34 cells acutely exposed to copper by tagRNA-sequencing. C. metallidurans CH34 is a model organism for metal resistance, and its potential as a biosensor and candidate for metal bioremediation has been demonstrated in multiple studies. Several metabolic pathways were impacted by Cu exposure, and a broad spectrum of metal resistance mechanisms, not limited to copper-specific clusters, was overexpressed. In addition, several gene clusters involved in the oxidative stress response and the cysteine-sulfur metabolism were induced. In total, 7500 transcription start sites (TSSs) were annotated and classified with respect to their location relative to coding sequences (CDSs). Predicted TSSs were used to re-annotate 182 CDSs. The TSSs of 2422 CDSs were detected, and consensus promotor logos were derived. Interestingly, many leaderless messenger RNAs (mRNAs) were found. In addition, many mRNAs were transcribed from multiple alternative TSSs. We observed pervasive intragenic TSSs both in sense and antisense to CDSs. Antisense transcripts were enriched near the 5' end of mRNAs, indicating a functional role in post-transcriptional regulation. In total, 578 TSSs were detected in intergenic regions, of which 35 were identified as putative small regulatory RNAs. Finally, we provide a detailed analysis of the main copper resistance clusters in CH34, which include many intragenic and antisense transcripts. These results clearly highlight the ubiquity of noncoding transcripts in the CH34 transcriptome, many of which are putatively involved in the regulation of metal resistance.

The metal-resistant β-proteobacterium Cupriavidus metallidurans drives gold (Au) biomineralisation and the (trans)formation of Au nuggets largely via unknown biochemical processes, ultimately leading to the reductive precipitation of mobile, toxic Au(i/iii)-complexes. In this study proteomic responses of C. metallidurans CH34 to mobile, toxic Au(iii)-chloride are investigated. Cells were grown in the presence of 10 and 50 μM Au(iii)-chloride, 50 μM Cu(ii)-chloride and without additional metals. Differentially expressed proteins were detected by difference gel electrophoresis and identified by liquid chromatography coupled mass spectrometry. Proteins that were more abundant in the presence of Au(iii)-chloride are involved in a range of important cellular functions, e.g., metabolic activities, transcriptional regulation, efflux and metal transport. To identify Au-binding proteins, protein extracts were separated by native 2D gel electrophoresis and Au in protein spots was detected by laser absorption inductively coupled plasma mass spectrometry. A chaperon protein commonly understood to bind copper (Cu), CupC, was identified and shown to bind Au. This indicates that it forms part of a multi-metal detoxification system and suggests that similar/shared detoxification pathways for Au and Cu exist. Overall, this means that C. metallidurans CH34 is able to mollify the toxic effects of cytoplasmic Au(iii) by sequestering this Au-species. This effect may in the future be used to develop CupC-based biosensing capabilities for the in-field detection of Au in exploration samples.

The weathering of volcanic minerals makes a significant contribution to the global silicate weathering budget, influencing carbon dioxide drawdown and long-term climate control. Basalt rocks may account for over 30% of the global carbon dioxide drawdown in silicate weathering. Micro-organisms are known to play a role in rock weathering yet the genomics and genetics of biological rock weathering are unknown. We apply DNA microarray technology to determine putative genes involved in weathering using the heavy metal-resistant organism, Cupriavidus metallidurans CH34; in particular we investigate the sequestering of iron. The results show that the bacterium does not depend on siderophores. Instead, the up-regulation of porins and transporters which are employed concomitantly with genes associated with biofilm formation suggests that novel passive and active iron uptake systems are involved. We hypothesize that these mechanisms induce rock weathering 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. Some of the earliest environments on the Earth were volcanic. Therefore, these results not only elucidate the mechanisms by which micro-organisms might have sequestered nutrients on the early Earth but also provide an explanation for the evolution of multiple heavy metal resistance genes long before the creation of contaminated industrial biotopes by human activity., (© 2010 Blackwell Publishing Ltd.)

The Cupriavidus metallidurans CH34 arsR gene, which is part of the arsRIC(2)BC(1)HP operon, and its putative arsenic-resistance regulatory protein were identified and characterized. The arsenic-induced transcriptome of C. metallidurans CH34 showed that the genes most upregulated in the presence of arsenate were all located within the ars operon, with none of the other numerous heavy metal resistance systems present in CH34 being induced. A transcriptional fusion between the luxCDABE operon and the arsR promoter/operator (P/O) region was used to confirm the in vivo induction of the ars operon by arsenite and arsenate. The arsR gene was cloned into expression vectors allowing for the overexpression of the ArsR protein as either his-tagged or untagged protein. The ability of the purified ArsR proteins to bind to the ars P/O region was analyzed in vitro by gel mobility shift assays. ArsR showed an affinity almost exclusively to its own ars P/O region. Dissociation of ArsR and its P/O region was metal dependent, and based on decreasing degrees of dissociation three groups of heavy metals could be distinguished: As(III), Bi(III), Co(II), Cu(II), Ni(II); Cd(II); Pb(II) and Zn(II), while no dissociation was observed in the presence of As(V).

Cupriavidus metallidurans CH34 possesses a multitude of metal efflux systems. Here, the function of the novel P(IB4)-type ATPase CzcP is characterized, which belongs to the plasmid pMOL30-mediated cobalt-zinc-cadmium (Czc) resistance system. Contribution of CzcP to transition metal resistance in C. metallidurans was compared with that of three P(IB2)-type ATPases (CadA, ZntA, PrbA) and to other efflux proteins by construction and characterization of multiple deletion mutants. These data also yielded additional evidence for an export of metal cations from the periplasm to the outside of the cell rather than from the cytoplasm to the outside. Moreover, metal-sensitive Escherichia coli strains were functionally substituted in trans with CzcP and the three P(IB2)-type ATPases. Metal transport kinetics performed with inside-out vesicles identified the main substrates for these four exporters, the K(m) values and apparent turn-over numbers. In combination with the mutant data, transport kinetics indicated that CzcP functions as 'resistance enhancer': this P(IB4)-type ATPase exports transition metals Zn(2+), Cd(2+) and Co(2+) much more rapidly than the three P(IB2)-type proteins. However, a basic resistance level has to be provided by the P(IB2)-type efflux pumps because CzcP may not be able to reach all different speciations of these metals in the cytoplasm.

The survival and behavior of Cupriavidus metallidurans strain CH34 were tested in space. In three spaceflight experiments, during three separate visits to the 'International Space Station' (ISS), strain CH34 was grown for 10-12 days at ambient temperature on mineral agar medium. Space- and earth-grown cells were compared post-flight by flow cytometry and using 2D-gel protein analysis. Pre-, in- and post-flight incubation conditions and experiment design had a significant impact on the survival and growth of CH34 in space. In the CH34 cells returning from spaceflight, 16 proteins were identified which were present in higher concentration in cells developed in spaceflight conditions. These proteins were involved in a specific response of CH34 to carbon limitation and oxidative stress, and included an acetone carboxylase subunit, fructose biphosphate aldolase, a DNA protection during starvation protein, chaperone protein, universal stress protein, and alkyl hydroperoxide reductase. The reproducible observation of the over-expression of these same proteins in multiple flight experiments, indicated that the CH34 cells could experience a substrate limitation and oxidative stress in spaceflight where cells and substrates are exposed to lower levels of gravity and higher doses of ionizing radiation. Bacterium C. metallidurans CH34 was able to grow normally under spaceflight conditions with very minor to no effects on cell physiology, but nevertheless specifically altered the expression of a few proteins in response to the environmental changes.

Metal responsive MerR family transcriptional regulators are widespread in bacteria and activate the transcription of genes involved in metal ion detoxification, efflux, or homeostasis, in response to the presence of cognate metal species in the cytoplasm. MerR family regulators recognize and bind to dyad symmetrical DNA sequences in specific promoters that have a spacer region between the -35 and -10 sequences which is longer than the canonical 16-18 bp spacer for other sigma(70)-dependent promoters. In this study we report beta-galactosidase assays of MerR family-regulated gene expression in the multiple metal resistant bacterium Cupriavidus metallidurans. A series of pMU2385 reporter plasmid derivatives containing cloned MerR family-activated promoters were used to determine metal ion-induced responses from different MerR family regulated promoters, as well as regulators cloned with the cognate promoter into pMU2385. Mercuric ion-responsive MerR and lead ion-responsive PbrR activity was confirmed using this assay system as well as MerR family activator activity on heterologous promoters PcopA, PcadA, and Pzcc from Escherichia coli, Pseudomonas aeruginosa and Bordetella pertussis, respectively. In C. metallidurans CH34, transcription from these promoters was activated by MerR family regulators encoded on the chromosome or megaplasmids in response to copper (PcopA), and lead (PcadA and PzccA), showing that MerR family activators in C. metallidurans can act on MerR family promoters from other organisms, which have sequence differences to the predicted C. metallidurans promoters.