Paleomicrobiology to investigate copper resistance in bacteria: isolation and description of Cupriavidus necator B9 in the soil of a medieval foundry.

Remains of a medieval foundry were excavated by archaeologists in 2013 in Verdun (France). Ancient workshops specialized in brass and copper alloys were found with an activity between 13th to 16th c. Levels of Cu, Zn and Pb reached 20000, 7000 and 6000 mg kg-1 (dw), respectively, in several soil horizons. The objective of the present work was to examine the microbial community in this contaminated site. A total of 8-22 106 reads were obtained by shotgun metagenomics in four soil horizons. Bioinformatic analyses suggest the presence of complex bacterial communities dominated by Proteobacteria. The structure of the community was not affected by metals, contrary to the set of metal-resistance genes. Using selective media, a novel strain of Cupriavidus necator (eutrophus), strain B9, was isolated. Its genome was sequenced and a novel metal resistance gene cluster with Hg resistance genes (merRTPCA) followed by 24 copper-resistance genes (actP, cusCBAF, silP, copK1, copH4QLOFGJH3IDCBARS, copH2H1, copK2) was found. This cluster is partly homologous to the cop genes of Cupriavidus gilardii CR3 and C. metallidurans CH34. Proteomics indicated that the four copH genes were differentially expressed: CopH1 and CopH2 were mostly induced by Cd while CopH4 was highly expressed by Cu.

Modelled microgravity cultivation modulates N-acylhomoserine lactone production in Rhodospirillum rubrum S1H independently of cell density.

The photosynthetic alphaproteobacterium Rhodospirillum rubrum S1H is part of the Micro-Ecological Life Support System Alternative (MELiSSA) project that is aiming to develop a closed life support system for oxygen, water and food production to support human life in space in forthcoming long-term space exploration missions. In the present study, R. rubrum S1H was cultured in a rotating wall vessel (RWV), simulating partial microgravity conditions on Earth. The bacterium showed a significant response to cultivation in simulated microgravity at the transcriptomic, proteomic and metabolic levels. In simulated microgravity conditions three N-acyl-l-homoserine lactones (C10-HSL, C12-HSL and 3-OH-C14-HSL) were detected in concentrations that were twice those detected under normal gravity, while no differences in cell density was detected. In addition, R. rubrum cultivated in modelled microgravity showed higher pigmentation than the normal gravity control, without change in culture oxygenation. When compared to randomized microgravity cultivation using a random positioning machine, significant overlap for the top differentially expressed genes and proteins was observed. Cultivation in this new artificial environment of simulated microgravity showed new properties of this well-known bacterium, including its first, to our knowledge, complete quorum-sensing-related N-acylhomoserine lactone profile.

Variation in genomic islands contribute to genome plasticity in Cupriavidus metallidurans.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Purification and characterization of the acetone carboxylase of Cupriavidus metallidurans strain CH34.

Acetone carboxylase (Acx) is a key enzyme involved in the biodegradation of acetone by bacteria. Except for the Helicobacteraceae family, genome analyses revealed that bacteria that possess an Acx, such as Cupriavidus metallidurans strain CH34, are associated with soil. The Acx of CH34 forms the heterohexameric complex α(2)ß(2)γ(2) and can carboxylate only acetone and 2-butanone in an ATP-dependent reaction to acetoacetate and 3-keto-2-methylbutyrate, respectively.

Mechanisms of gold biomineralization in the bacterium Cupriavidus metallidurans.

While the role of microorganisms as main drivers of metal mobility and mineral formation under Earth surface conditions is now widely accepted, the formation of secondary gold (Au) is commonly attributed to abiotic processes. Here we report that the biomineralization of Au nanoparticles in the metallophillic bacterium Cupriavidus metallidurans CH34 is the result of Au-regulated gene expression leading to the energy-dependent reductive precipitation of toxic Au(III)-complexes. C. metallidurans, which forms biofilms on Au grains, rapidly accumulates Au(III)-complexes from solution. Bulk and microbeam synchrotron X-ray analyses revealed that cellular Au accumulation is coupled to the formation of Au(I)-S complexes. This process promotes Au toxicity and C. metallidurans reacts by inducing oxidative stress and metal resistances gene clusters (including a Au-specific operon) to promote cellular defense. As a result, Au detoxification is mediated by a combination of efflux, reduction, and possibly methylation of Au-complexes, leading to the formation of Au(I)-C-compounds and nanoparticulate Au(0). Similar particles were observed in bacterial biofilms on Au grains, suggesting that bacteria actively contribute to the formation of Au grains in surface environments. The recognition of specific genetic responses to Au opens the way for the development of bioexploration and bioprocessing tools.

Structural and metal binding characterization of the C-terminal metallochaperone domain of membrane fusion protein SilB from Cupriavidus metallidurans CH34.

Detoxification of heavy metal ions in Proteobacteria is tightly controlled by various systems regulating their sequestration and transport. In Cupriavidus metallidurans CH34, a model organism for heavy metal resistance studies, the sil determinant is potentially involved in the efflux of silver and copper ions. Proteins SilA, SilB, and SilC form a resistance nodulation cell division (RND)-based transport system in which SilB is the periplasmic adaptor protein belonging to the membrane fusion protein (MFP) family. In addition to the four domains typical of known MFPs, SilB has a fifth additional C-terminal domain, called SilB(440-521), which is characterized here. Structure and backbone dynamics of SilB(440-521) have been investigated using nuclear magnetic resonance, and the residues of the metal site were identified from (15)N- and (13)C-edited HSQC spectra. The solution structure and additional metal binding experiments demonstrated that this C-terminal domain folds independently of the rest of the protein and has a conformation and a Ag(+) and Cu(+) binding specificity similar to those determined for CusF from Escherichia coli. The small protein CusF plays a role in metal trafficking in the periplasm. The similarity with CusF suggests a potential metallochaperone role for SilB(440-521) that is discussed in the context of simultaneous expression of different determinants involved in copper resistance in C. metallidurans CH34.

Insertion sequence elements in Cupriavidus metallidurans CH34: distribution and role in adaptation.

Cupriavidus metallidurans CH34 is a ß-proteobacterium well equipped to cope with harsh environmental conditions such as heavy metal pollution. The strain carries two megaplasmids specialized in the response to heavy metals and a considerable number of genomic islands, transposons and insertion sequence (IS) elements. The latter were characterized in detail in this study, which revealed nine new IS elements totaling to 21 distinct IS elements from 10 different IS families and reaching a total of 57 intact IS copies in CH34. Analysis of all fully sequenced bacterial genomes revealed that relatives of these IS elements were mostly found in the Burkholderiaceae family (ß-proteobacteria) to which C. metallidurans belongs. Three IS elements were 100% conserved in other bacteria suggesting recent interaction and horizontal transfer between these strains. In addition, a number of these IS elements were associated with genomic islands, gene inactivation or rearrangements that alter the autotrophic growth capacities of CH34. The latter rearrangements gave the first molecular evidence for the mutator phenotype that is characteristic for various C. metallidurans strains. Furthermore, differential expression of some IS elements (or adjacent genes in the same strand orientation) was found under heavy metal stress, an environmental stress to which C. metallidurans CH34 is well adapted. These observations indicate that these IS elements play an active role in C. metallidurans CH34 lifestyle, including its metabolic potential and adaptation under selective pressure.

Heavy metal resistance in Cupriavidus metallidurans CH34 is governed by an intricate transcriptional network.

The soil bacterium Cupriavidus metallidurans CH34 contains a high number of heavy metal resistance genes making it an interesting model organism to study microbial responses to heavy metals. In this study the transcriptional response of strain CH34 was measured when challenged to sub-lethal concentrations of various essential or toxic metals. Based on the global transcriptional responses for each challenge and the overlap in upregulated genes between different metal responses, the sixteen metals were clustered in three groups. In addition, the transcriptional response of already known metal resistance genes was assessed, and new metal response gene clusters were identified. The majority of the studied metal response loci showed similar expression profiles when cells were exposed to different metals, suggesting complex interplay at transcriptional level between the different metal responses. The pronounced redundancy of these metal resistant regions-as illustrated by the large number of paralogous genes-combined with the phylogenetic distribution of these metal response regions within either evolutionary related or other metal resistant bacteria, provides important insights on the recent evolutionary forces shaping this naturally soil-dwelling bacterium into a highly metal-resistant strain well adapted to harsh and anthropogenic environments.

Cupriavidus metallidurans CH34, a historical perspective on its discovery, characterization and metal resistance.

Cupriavidus metallidurans, and in particular type strain CH34, became a model bacterium to study bacterial resistance to metals. Although nowadays the routine use of a wide variety of omics and molecular techniques allow refining, deepening and expanding our knowledge on adaptation and resistance to metals, these were not available at the onset of C. metallidurans research starting from its isolation in 1976. This minireview describes the early research and legacy tools used to study its metal resistance determinants, characteristic megaplasmids, ecological niches and environmental applications.

Differential proteomic analysis using isotope-coded protein-labeling strategies: comparison, improvements and application to simulated microgravity effect on Cupriavidus metallidurans CH34.

Among differential proteomic methods based on stable isotopic labeling, isotope-coded protein labeling (ICPL) is a recent non-isobaric technique devised to label primary amines found in proteins. ICPL overcomes some of the disadvantages found in other chemical-labeling techniques, such as iTRAQ or ICAT. However, previous analyses revealed that more than 30% of the proteins identified in regular ICPL generally remain unquantified. In this study, we describe a modified version of ICPL, named Post-digest ICPL, that makes it possible to label and thus to quantify all the peptides in a sample (bottom-up approach). Optimization and validation of this Post-digest ICPL approach were performed using a standard protein mixture and complex protein samples. Using this strategy, the number of proteins that were identified and quantified was greatly increased in comparison with regular ICPL and cICAT approaches. The pros and cons of this improvement are discussed. This complementary approach to traditional ICPL was applied to the analysis of modification of protein abundances in the model bacterium Cupriavidus metallidurans CH34 after cultivation under simulated microgravity. In this context, two different systems - a 2-D clinorotation and 3-D random positioning device - were used and the results were compared and discussed.

CopK from Cupriavidus metallidurans CH34 binds Cu(I) in a tetrathioether site: characterization by X-ray absorption and NMR spectroscopy.

Cupriavidus metallidurans CH34 is a bacterium that is resistant to high metal concentrations in the environment. Increased copper resistance is associated with the cop cluster on the large plasmid pMOL30 that is composed of at least 21 genes. The copK gene encodes a 74 residue periplasmic protein whose expression is strongly upregulated in the presence of copper. CopK was previously shown to cooperatively bind Cu(I) and Cu(II) in distinct, specific sites. The solution structure of Cu(I)-CopK and the characterization of the Cu(I) site by X-ray absorption spectroscopy and NMR are reported here. EXAFS spectra are in agreement with a tetrathioether Cu(I) site, providing so far unique spectral information on a 4S-coordinated Cu(I) in a protein. The methionine residues forming the Cu(I) site, M28, M38, M44, and M54, are identified by NMR. We propose the chemical shift of the methionine C(epsilon) as a new and sensitive probe for the detection of Cu(I) bound to thioether groups. The solution structure of Cu(I)-CopK demonstrates that Cu(I) binding induces a complete structural modification with the disruption of the second beta-sheet and a rotation of the C-terminal part of nearly 180 degrees around a hinge formed by asparagine 57. This conformational change is directly related to the loss of the dimer interface and most probably to the formation of the Cu(II) site involving histidine 70. The solution structure of Cu(I)-CopK therefore provides the molecular basis for the understanding of the Cu(I)/Cu(II) binding cooperativity.

Toxicogenomic response of Rhodospirillum rubrum S1H to the micropollutant triclosan.

In the framework of the Micro-Ecological Life Support System Alternative (MELiSSA) project, a pilot study was performed to identify the effects of triclosan on the MELiSSA carbon-mineralizing microorganism Rhodospirillum rubrum S1H. Triclosan is a biocide that is commonly found in human excrement and is considered an emerging pollutant in wastewater and the environment. Chronic exposure to MELiSSA-relevant concentrations (> or =25 microg liter(-1)) of triclosan resulted in a significant extension of the lag phase of this organism but hardly affected the growth rate. Analytical determinations gave no indication of triclosan biodegradation during the growth experiment, and flow cytometric viability analyses revealed that triclosan is bacteriostatic and only slightly toxic to R. rubrum S1H. Using microarray analyses, the genetic mechanisms supporting the reversibility of triclosan-induced inhibition were scrutinized. An extremely triclosan-responsive cluster of four small adjacent genes was identified, for which there was up to 34-fold induction with 25 microg liter(-1) triclosan. These four genes, for which the designation microf (micropollutant-upregulated factor) is proposed, appear to be unique to R. rubrum and are shown here for the first time to be involved in the response to stress. Moreover, numerous other systems that are associated with the proton motive force were shown to be responsive to triclosan, but they were never as highly upregulated as the microf genes. In response to triclosan, R. rubrum S1H induced transcription of the phage shock protein operon (pspABC), numerous efflux systems, cell envelope consolidation mechanisms, the oxidative stress response, beta-oxidation, and carbonic anhydrase, while there was downregulation of bacterial conjugation and carboxysome synthesis genes. The microf genes and three efflux-related genes showed the most potential to be low-dose biomarkers.

Evaluation of the airborne bacterial population in the periodically confined Antarctic base Concordia.

The environmental airborne bacterial population in relation to human confinement was investigated over a period of 1 year in the Concordia Research Station, which is located on the Eastern Antarctic plateau. The unique location of the station makes it suitable for different research domains such as glaciology, atmospheric sciences, astronomy, etc. Furthermore, it is used as a test bed for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. A total of 96 samples were collected at eight different locations in the station at regular intervals. The airborne bacterial contamination was for 90% of the samples lower than 10.0 x 10(2) colony-forming units per cubic meter of air (CFU/m(3)) and the total bacterial contamination increased over time during confinement but diminished after re-opening of the base. Viable airborne bacteria with different morphology were identified by biochemical analyses. The predominant microflora was identified as Staphylococcus sp. (24.9% of total) and Bacillus sp. (11.6% of total) and was associated with human activity, but also environmental species such as Sphingomonas paucimobilis (belonging to the alpha-Proteobacteria) could establish themselves in the airborne population. A few opportunistic pathogens (6%) were also identified.

New mobile genetic elements in Cupriavidus metallidurans CH34, their possible roles and occurrence in other bacteria.

Cupriavidus metallidurans strain CH34 is a beta-Proteobacterium that thrives in low concentrations of heavy metals. The genetic determinants of resistance to heavy metals are located on its two chromosomes, and are particularly abundant in the two megaplasmids, pMOL28 and pMOL30. We explored the involvement of mobile genetic elements in acquiring these and others traits that might be advantageous in this strain using genome comparison of Cupriavidus/Ralstonia strains and related beta-Proteobacteria. At least eleven genomic islands were identified on the main replicon, three on pMOL28 and two on pMOL30. Multiple islands contained genes for heavy metal resistance or other genetic determinants putatively responding to harsh environmental conditions. However, cryptic elements also were noted. New mobile genetic elements (or variations of known ones) were identified through synteny analysis, allowing the detection of mobile genetic elements outside the bias of a selectable marker. Tn4371-like conjugative transposons involved in chemolithotrophy and degradation of aromatic compounds were identified in strain CH34, while similar elements involved in heavy metal resistance were found in Delftia acidovorans SPH-1 and Bordetella petrii DSM12804. We defined new transposons, viz., Tn6048 putatively involved in the response to heavy metals and Tn6050 carrying accessory genes not classically associated with transposons. Syntenic analysis also revealed new transposons carrying metal response genes in Burkholderia xenovorans LB400, and other bacteria. Finally, other putative mobile elements, which were previously unnoticed but apparently common in several bacteria, were also revealed. This was the case for triads of tyrosine-based site-specific recombinases and for an int gene paired with a putative repressor and associated with chromate resistance.

Lead(II) resistance in Cupriavidus metallidurans CH34: interplay between plasmid and chromosomally-located functions.

Proteome and transcriptome analysis, combined with mutagenesis, were used to better understand the response of Cupriavidus metallidurans CH34 against lead(II). Structural Pb(II)-resistance genes of the pMOL30-encoded pbrUTRABCD operon formed the major line of defense against Pb(II). However, several general stress response mechanisms under the control of alternative sigma factors such as sigma24/rpoK, sigma32/rpoH and sigma28/fliA were also induced. In addition, the expression of the pbrR(2) cadA pbrC(2) operon of the CMGI-1 region and the chromosomally encoded zntA were clearly induced in the presence of Pb(II), although their respective gene products were not detected via proteomics. After inactivation of the pbrA, pbrB or pbrD genes, the expression of the pbrR(2) cadA pbrC(2) operon went up considerably. This points towards synergistic interactions between pbrUTRABCD and pbrR(2) cadA pbrC(2) to maintain a low intracellular Pb(II) concentration, where pbrR(2) cadA pbrC(2) gene functions can complement and compensate for the mutations in the pbrA and pbrD genes. This role of zntA and cadA to complement for the loss of pbrA was further confirmed by mutation analysis. The pbrB:: colonsTn(Km2) mutation resulted in the most significant decrease of Pb(II) resistance, indicating that Pb(II) sequestration, avoiding re-entry of this toxic metal ion, forms a critical step in the pbr-encoded Pb(II) resistance mechanism.

The response of Cupriavidus metallidurans CH34 to spaceflight in the international space station.

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.

ArsR arsenic-resistance regulatory protein from Cupriavidus metallidurans CH34.

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).

Plasmids captured in C. metallidurans CH34: defining the PromA family of broad-host-range plasmids.

The self-transmissible, broad-host-range (BHR) plasmid pMOL98 was previously isolated from polluted soil using a triparental plasmid capture approach and shown to possess a replicon similar to that of the BHR plasmids pSB102 and pIPO2. Here, complete sequence analysis and comparative genomics reveal that the 55.5 kb nucleotide sequence of pMOL98 shows extensive sequence similarity and synteny with the BHR plasmid family that now includes pIPO2, pSB102, pTER331, and pMRAD02. They share a plasmid backbone comprising replication, partitioning and conjugative transfer functions. Comparison of the variable accessory regions of these plasmids shows that the majority of natural transposons, as well as the mini-transposon used to mark the plasmids, are inserted in the parA locus. The transposon unique to pMOL98 appears to have inserted from the chromosome of the recipient strain used in the plasmid capture procedure. This demonstrates the necessity for careful screening of plasmids and host chromosomes to avoid mis-interpretation of plasmid genome content. The presence of very similar BHR plasmids with different accessory genes in geographically distinct locations suggests an important role in horizontal gene exchange and bacterial adaptation for this recently defined plasmid group, which we propose to name "PromA".

From the deep sea to the stars: human life support through minimal communities.

Support of human life during long-distance exploratory space travel or in the creation of human habitats in extreme environments can be accomplished using the action of microbial consortia inhabiting interconnected bioreactors, designed for the purpose of reconversion of solid, liquid and gaseous wastes produced by the human crew or by one of the compartments of the bioregenerative loop, into nutritional biomass, oxygen and potable water. The microorganisms responsible for bioregenerative life support are part of Earth's own geomicrobial reconversion cycle. Depending on the resources and conditions available, minimal life support systems can be assembled using appropriately selected microorganisms that possess metabolic routes for each specific purpose in the transformation cycle. Under control of an engineered system, a reliable life-support system can hence be provided for.

Spontaneous mutation in the AgrRS two-component regulatory system of Cupriavidus metallidurans results in enhanced silver resistance.

The uncontrolled and widespread use of (nano)silver compounds has led to the increased release of these compounds into the environment, raising concerns about their negative impact on ecosystems. Concomitantly, silver resistance determinants are widely spread among environmental and clinically relevant bacteria although the underlying mechanisms are not yet fully understood. We show that Cupriavidus metallidurans is able to adapt to toxic silver concentrations. However, none of the known silver resistance determinants present in C. metallidurans are involved in the adaptative response. Instead, increased silver resistance is achieved by the concerted action of a two-component system AgrR-AgrS, previously not associated with metal resistance, and two periplasmic proteins PrsQ1 and PrsQ2. Both proteins belong to an unique group of small, uncharacterized, secreted proteins restricted to the genera Cupriavidus and Ralstonia. This system gives C. metallidurans the ability to withstand much higher silver concentrations. The latter could be facilitated by the accumulation of silver ions and the formation of silver nanoparticles.