Cupriavidus Metallidurans CH34 Research Articles

Engineered Bacteria Achieve Breakthrough 91% Lead Removal in Bioremediation

Background: Heavy metal pollutants pose a significant environmental and public health threat, necessitating effective remediation strategies. Genetically modified microorganisms, Shewanella oneidensis MR-1 and Cupriavidus metallidurans CH34, show promise in mitigating contamination. Specific Background: Engineered bacteria have been designed to improve metal absorption, specifically lead, mercury, cadmium, and zinc, but further research is needed to understand their effectiveness in different pH levels and water conditions. Knowledge Gap: Few studies have investigated the pH-dependent efficiency of genetically engineered microbes in heavy metal absorption or their performance under wastewater conditions, despite numerous studies on microbial bioremediation. Aims: The study evaluates the efficacy of genetically modified Shewanella oneidensis and Cupriavidus metallidurans in removing heavy metals from contaminated water at various pH levels. Results: The study found that both bacterial strains effectively removed 91% of lead at pH 7, with optimal levels for lead and cadmium absorption depending on the specific pH. Novelty: CRISPR/Cas9 technology is used for genetic modification of strains, enhancing microbial engineering for environmental remediation. Study provides new data on pH-specific absorption rates.. Implications: These findings can be applied to enhance the design of bioreactors and biofiltration systems, offering a sustainable solution to heavy metal contamination in wastewater, with broad potential for industrial and environmental applications. Highlights: Modified bacteria remove 91% of lead at neutral pH. CRISPR/Cas9 enhances bacteria for efficient heavy metal removal. pH optimization is key for effective heavy metal absorption. Keywords: Heavy metals, Bioremediation, Genetically modified bacteria, Shewanella oneidensis, Cupriavidus metallidurans

Proteomic and morphological insights into the exposure of Cupriavidus metallidurans CH34 planktonic cells and biofilms to aluminium

Aluminium (Al) is one of the most popular materials for industrial and domestic use. Nevertheless, research has proven that this metal can be toxic to most organisms. This light metal has no known biological function and to date very few aluminium-specific biological pathways have been identified. In addition, information about the impact of this metal on microbial life is scarce. Here, we aimed to study the effect of aluminium on the metal-resistant soil bacterium Cupriavidus metallidurans CH34 in different growth modes, i.e. planktonic cells, adhered cells and mature biofilms. Our results indicated that despite a significant tolerance to aluminium (minimal inhibitory concentration of 6.25 mM Al₂(SO₄)₃.18H₂O), the exposure of C. metallidurans to a sub-inhibitory dose (0.78 mM) caused early oxidative stress and an increase in hydrolytic activity. Changes in the outer membrane surface of planktonic cells were observed, in addition to a rapid disruption of mature biofilms. On protein level, aluminium exposure increased the expression of proteins involved in metabolic activity such as pyruvate kinase, formate dehydrogenase and poly(3-hydroxybutyrate) polymerase, whereas proteins involved in chemotaxis, and the production and transport of iron scavenging siderophores were significantly downregulated.

Chromate resistance in Cupriavidus metallidurans CH34: molecular modeling from ChrC superoxide dismutase

Chromate has become an environmental pollutant present in different ecosystems due to its use in industry. Bacteria have evolved to resist stress produced by chromate. Among chromate-resistance mechanisms we can list Reactive Oxygen Species detoxification systems. CmeSOD (ChrC) protein from Cupriavidus metallidurans CH34 is a superoxide dismutase that mitigate oxidative stress caused by chromate. Cme-SOD protein belongs to Fe and Mn-dependent SOD family (pfam02777). The objective of this study was to analyze the threedimensional structure of the Cme-SOD protein, for which monomer and tetramer models of the enzyme were built. In the monomer model it was observed that Cme-SOD has a characteristic two-domain structure from iron-dependent SOD, additionally, Cme-SOD has an iron-binding site formed by conserved residues H26 and H75 in the Nterminal domain, and D157 and H161 in the domain Cterminal domain. It was show that chromate stress response SODs have a non-conserved residues in the active site (R37, N59, S71, D143 and Y164). These findings suggest the presence of a novel active site in this family of enzymes.

Five copper homeostasis gene clusters encode the Cu-efflux resistome of the highly copper-tolerant Methylorubrum extorquens AM1.

In the last decade, the use of copper has reemerged as a potential strategy to limit healthcare-associated infections and to control the spread of multidrug-resistant pathogens. Numerous environmental studies have proposed that most opportunistic pathogens have acquired antimicrobial resistance in their nonclinical primary habitat. Thus, it can be presumed that copper-resistant bacteria inhabiting a primary commensal niche might potentially colonize clinical environments and negatively affect the bactericidal efficacy of Cu-based treatments. The use of copper in agricultural fields is one of the most important sources of Cu pollution that may exert selection pressure for the increase of copper resistance in soil and plant-associated bacteria. To assess the emergence of copper-resistant bacteria in natural habitats, we surveyed a laboratory collection of bacterial strains belonging to the order Rhizobiales. This study proposes that Methylorubrum extorquens AM1 is an environmental isolate well adapted to thrive in copper-rich environments that could act as a reservoir of copper resistance genes. The minimal inhibitory concentrations (MICs) of CuCl2 were used to estimate the copper tolerance of eight plant-associated facultative diazotrophs (PAFD) and five pink-pigmented facultative methylotrophs (PPFM) belonging to the order Rhizobiales presumed to come from nonclinical and nonmetal-polluted natural habitats based on their reported source of isolation. Their sequenced genomes were used to infer the occurrence and diversity of Cu-ATPases and the copper efflux resistome of Mr. extorquens AM1. These bacteria exhibited minimal inhibitory concentrations (MICs) of CuCl2 ranging between 0.020 and 1.9 mM. The presence of multiple and quite divergent Cu-ATPases per genome was a prevalent characteristic. The highest copper tolerance exhibited by Mr. extorquens AM1 (highest MIC of 1.9 mM) was similar to that found in the multimetal-resistant model bacterium Cupriavidus metallidurans CH34 and in clinical isolates of Acinetobacter baumannii. The genome-predicted copper efflux resistome of Mr. extorquens AM1 consists of five large (6.7 to 25.7 kb) Cu homeostasis gene clusters, three clusters share genes encoding Cu-ATPases, CusAB transporters, numerous CopZ chaperones, and enzymes involved in DNA transfer and persistence. The high copper tolerance and the presence of a complex Cu efflux resistome suggest the presence of relatively high copper tolerance in environmental isolates of Mr. extorquens.

Insight into biosorption of hexavalent chromium using isolated species Aspergillus Proliferans LA: A systemic and In silico studies

Background: The wastewater disposal into the water bodies without removing the toxic heavy metals and other industrial impurities is a major issue these days. These heavy metals cause serious health issues to the human and animal life and also harm the environment and reduce the productivity of crops. A potent microorganism resistant to hexavalent chromium was isolated. The activity of this isolated strain was analyzed using in silico studies. Methods: In this study, a chromium-resistant fungus was isolated from the soil of the dumping sites of the tanneries in Kanpur, UP, India, followed by isolation by serial dilution, plating method, and finally, genome sequencing. It was identified as Aspergillus proliferans LA that is submitted to the National Collection for Industrial Microorganisms (NCIM) database with accession no. NCIM-1473. In the current study, the comparative analysis of the protein sequence of A. proliferans (NCIM-1473) was done against the known 53 protein sequences of the fungus and bacterial strains already reported for their chromium-resistant nature. The physical and chemical parameters of the known and isolated chromium-resistant proteins were analyzed using the ProtParam tool. The comparative study on the sequence and structural alignment of known and isolated chromium-resistant protein was done using EMBOSS-NEEDLE and FATCAT, respectively. Results: In this analysis, the top 10 strains showing similarity with A. proliferans (NCIM-1473) were reported and among which ChrI, chromium regulatory protein Cupriavidus metallidurans CH34 was showing maximum similarity with isolated chromium resistant protein for all the analysis, namely ProtParam, sequence, and FATCAT analysis. This strain, Cupriavidus metallidurans CH34, has been reported resistant against eight heavy metals, one of which is chromate, and was first identified in the heavy-metal contaminated sludge in a settling tank of Belgium. Conclusion: These studies conclude that the strain isolated in our laboratory (accession no. NCIM-1473) is potentially chromium resistant and a unique strain.

Synthetic Biology Toolbox, Including a Single-Plasmid CRISPR-Cas9 System to Biologically Engineer the Electrogenic, Metal-Resistant Bacterium Cupriavidus metallidurans CH34.

Cupriavidus metallidurans CH34 exhibits extraordinary metabolic versatility, including chemolithoautotrophic growth; degradation of BTEX (benzene, toluene, ethylbenzene, xylene); high resistance to numerous metals; biomineralization of gold, platinum, silver, and uranium; and accumulation of polyhydroxybutyrate (PHB). These qualities make it a valuable host for biotechnological applications such as bioremediation, bioprocessing, and the generation of bioelectricity in microbial fuel cells (MFCs). However, the lack of genetic tools for strain development and studying its fundamental physiology represents a bottleneck to boosting its commercial applications. In this study, inducible and constitutive promoter libraries were built and characterized, providing the first comprehensive list of biological parts that can be used to regulate protein expression and optimize the CRISPR-Cas9 genome editing tools for this host. A single-plasmid CRISPR-Cas9 system that can be delivered by both conjugation and electroporation was developed, and its efficiency was demonstrated by successfully targeting the pyrE locus. The CRISPR-Cas9 system was next used to target candidate genes encoding type IV pili, hypothesized by us to be involved in extracellular electron transfer (EET) in this organism. Single and double deletion strains (ΔpilA, ΔpilE, and ΔpilAE) were successfully generated. Additionally, the CRISPR-Cas9 tool was validated for constructing genomic insertions (ΔpilAE::gfp and ΔpilAE::λPrgfp). Finally, as type IV pili are believed to play an important role in extracellular electron transfer to solid surfaces, C. metallidurans CH34 ΔpilAE was further studied by means of cyclic voltammetry using disposable screen-printed carbon electrodes. Under these conditions, we demonstrated that C. metallidurans CH34 could generate extracellular currents; however, no difference in the intensity of the current peaks was found in the ΔpilAE double deletion strain when compared to the wild type. This finding suggests that the deleted type IV pili candidate genes are not involved in extracellular electron transfer under these conditions. Nevertheless, these experiments revealed the presence of different redox centers likely to be involved in both mediated electron transfer (MET) and direct electron transfer (DET), the first interpretation of extracellular electron transfer mechanisms in C. metallidurans CH34.

Iron reducing sludge as a source of electroactive bacteria: assessing iron reduction in biofilm bacteria, planktonic cells and isolates from a microbial fuel cell

In this study, bacteria from a microbial fuel cell (MFC) and isolates were evaluated on their Fe3+ reduction capability at different concentrations of iron using acetate as the sole source of carbon. The results demonstrated that the planktonic cells can reach an iron reduction up to 60% at 27mmol Fe3+. Azospira oryzae (µ 0.89 ± 0.27 d-1) and Cupriavidus metallidurans CH34 (µ 2.34 ± 0.81 d-1) presented 55 and 62% of Fe3+ reduction, respectively, at 16mmoll-1. Enterobacter bugandensis (µ 0.4 ± 0.01 d-1) 40% Fe3+ at 27mmoll-1, Citrobacter freundii ATCC 8090 (µ 0.23 ± 0.05 d-1) and Citrobacter murliniae CDC2970-59 (µ 0.34 ± 0.02 d-1) reduced Fe3+ in ~ 50%, at 55mmoll-1. This is the first report on these bacteria on a percentage of iron reduction. These results may be useful for anode design to contribute to a higher energy generation in MFCs.

Individual competence predominates over host nutritional status in Arabidopsis root exudate-mediated bacterial enrichment in a combination of four Burkholderiaceae species

BackgroundRhizosphere microorganisms play a crucial role in plant health and development. Plant root exudates (PRE) are a complex mixture of organic molecules and provide nutritional and signaling information to rhizosphere microorganisms. Burkholderiaceae species are non-abundant in the rhizosphere but exhibit a wide range of plant-growth-promoting and plant-health-protection effects. Most of these plant-associated microorganisms have been studied in isolation under laboratory conditions, whereas in nature, they interact in competition or cooperation with each other. To improve our understanding of the factors driving growth dynamics of low-abundant bacterial species in the rhizosphere, we hypothesized that the growth and survival of four Burkholderiaceae strains (Paraburkholderia phytofirmans PsJN, Cupriavidus metallidurans CH34, C. pinatubonensis JMP134 and C. taiwanensis LMG19424) in Arabidopsis thaliana PRE is affected by the presence of each other.ResultsDifferential growth abilities of each strain were found depending on plant age and whether PRE was obtained after growth on N limitation conditions. The best-adapted strain to grow in PRE was P. phytofirmans PsJN, with C. pinatubonensis JMP134 growing better than the other two Cupriavidus strains. Individual strain behavior changed when they succeeded in combinations. Clustering analysis showed that the 4-member co-culture grouped with one of the best-adapted strains, either P. phytofirmans PsJN or C. pinatubonensis JMP134, depending on the PRE used. Sequential transference experiments showed that the behavior of the 4-member co-culture relies on the type of PRE provided for growth.ConclusionsThe results suggest that individual strain behavior changed when they grew in combinations of two, three, or four members, and those changes are determined first by the inherent characteristics of each strain and secondly by the environment.

Bacillus megaterium HgT21: a Promising Metal Multiresistant Plant Growth-Promoting Bacteria for Soil Biorestoration.

ABSTRACTThe environmental deterioration produced by heavy metals derived from anthropogenic activities has gradually increased. The worldwide dissemination of toxic metals in crop soils represents a threat for sustainability and biosafety in agriculture and requires strategies for the recovery of metal-polluted crop soils. The biorestoration of metal-polluted soils using technologies that combine plants and microorganisms has gained attention in recent decades due to the beneficial and synergistic effects produced by its biotic interactions. In this context, native and heavy metal-resistant plant growth-promoting bacteria (PGPB) play a crucial role in the development of strategies for sustainable biorestoration of metal-contaminated soils. In this study, we present a genomic analysis and characterization of the rhizospheric bacterium Bacillus megaterium HgT21 isolated from metal-polluted soil from Zacatecas, Mexico. The results reveal that this autochthonous bacterium contains an important set of genes related to a variety of operons associated with mercury, arsenic, copper, cobalt, cadmium, zinc and aluminum resistance. Additionally, halotolerance-, beta-lactam resistance-, phosphate solubilization-, and plant growth-promotion-related genes were identified. The analysis of resistance to metal ions revealed resistance to mercury (HgII+), arsenate [AsO4]³–, cobalt (Co2+), zinc (Zn2+), and copper (Cu2+). Moreover, the ability of the HgT21 strain to produce indole acetic acid (a phytohormone) and promote the growth of Arabidopsis thaliana seedlings in vitro was also demonstrated. The genotype and phenotype of Bacillus megaterium HgT21 reveal its potential to be used as a model of both plant growth-promoting and metal multiresistant bacteria.IMPORTANCE Metal-polluted environments are natural sources of a wide variety of PGPB adapted to cope with toxic metal concentrations. In this work, the bacterial strain Bacillus megaterium HgT21 was isolated from metal-contaminated soil and is proposed as a model for the study of metal multiresistance in spore-forming Gram-positive bacteria due to the presence of a variety of metal resistance-associated genes similar to those encountered in the metal multiresistant Gram-negative Cupriavidus metallidurans CH34. The ability of B. megaterium HgT21 to promote the growth of plants also makes it suitable for the study of plant-bacteria interactions in metal-polluted environments, which is key for the development of techniques for the biorestoration of metal-contaminated soils used for agriculture.

Oxygen-Tolerant RAFT Polymerization Initiated by Living Bacteria.

Living organisms can synthesize a wide range of macromolecules from a small set of natural building blocks, yet there is potential for even greater materials diversity by exploiting biochemical processes to convert unnatural feedstocks into new abiotic polymers. Ultimately, the synthesis of these polymers in situ might aid the coupling of organisms with synthetic matrices, and the generation of biohybrids or engineered living materials. The key step in biohybrid materials preparation is to harness the relevant biological pathways to produce synthetic polymers with predictable molar masses and defined architectures under ambient conditions. Accordingly, we report an aqueous, oxygen-tolerant RAFT polymerization platform based on a modified Fenton reaction, which is initiated by Cupriavidus metallidurans CH34, a bacterial species with iron-reducing capabilities. We show the synthesis of a range of water-soluble polymers under normoxic conditions, with control over the molar mass distribution, and also the production of block copolymer nanoparticles via polymerization-induced self-assembly. Finally, we highlight the benefits of using a bacterial initiation system by recycling the cells for multiple polymerizations. Overall, our method represents a highly versatile approach to producing well-defined polymeric materials within a hybrid natural-synthetic polymerization platform and in engineered living materials with properties beyond those of biotic macromolecules.

Cupriavidus metallidurans CH34 Possesses Aromatic Catabolic Versatility and Degrades Benzene in the Presence of Mercury and Cadmium.

Heavy metal co-contamination in crude oil-polluted environments may inhibit microbial bioremediation of hydrocarbons. The model heavy metal-resistant bacterium Cupriavidus metallidurans CH34 possesses cadmium and mercury resistance, as well as genes related to the catabolism of hazardous BTEX aromatic hydrocarbons. The aims of this study were to analyze the aromatic catabolic potential of C. metallidurans CH34 and to determine the functionality of the predicted benzene catabolic pathway and the influence of cadmium and mercury on benzene degradation. Three chromosome-encoded bacterial multicomponent monooxygenases (BMMs) are involved in benzene catabolic pathways. Growth assessment, intermediates identification, and gene expression analysis indicate the functionality of the benzene catabolic pathway. Strain CH34 degraded benzene via phenol and 2-hydroxymuconic semialdehyde. Transcriptional analyses revealed a transition from the expression of catechol 2,3-dioxygenase (tomB) in the early exponential phase to catechol 1,2-dioxygenase (catA1 and catA2) in the late exponential phase. The minimum inhibitory concentration to Hg (II) and Cd (II) was significantly lower in the presence of benzene, demonstrating the effect of co-contamination on bacterial growth. Notably, this study showed that C. metallidurans CH34 degraded benzene in the presence of Hg (II) or Cd (II).

Generation of electrical energy in a microbial fuel cell coupling acetate oxidation to Fe3+ reduction and isolation of the involved bacteria.

An iron reducing enrichment was obtained from sulfate reducing sludge and was evaluated on the capability of reducing Fe3+ coupled to acetate oxidation in a microbial fuel cell (MFC). Three molar ratios for acetate/Fe3+ were evaluated (2/16, 3.4/27 and 6.9/55mM). The percentages of Fe3+ reduction were in a range of 80-90, 60-70 and 40-50% for the MFCs at closed circuit for the molar ratios of 2/16, 3.4/27 and 6.9/55mM, respectively. Acetate consumption was in a range of 80-90% in all cases. The results obtained at closed circuit for current density were: 11.37mA/m2, 4.5mA/m2 and 7.37mA/m2 for the molar ratios of 2/16, 3.4/27 and 6.9/55mM, respectively. Some microorganisms that were isolated and identified in the MFCs were Azospira oryzae, Cupriavidus metallidurans CH34, Enterobacter bugandensis 247BMC, Citrobacter freundii ATCC8090 and Citrobacter murliniae CDC2970-59, these bacteria have been reported as exoelectrogens in MFC and in MFC involving metals removal but not all of them have been reported to utilize acetate as preferred substrate. The results demonstrate that the isolates can utilize acetate as the sole source of carbon and suggest that Fe3+ reduction was carried out by a combination of different mechanisms (direct contact and redox mediators) utilized by the bacteria identified in the MFC. Storage of the energy generated from the 2/16mM MFC system arranged in a series of three demonstrated that it is possible to utilize the energy to charge a battery.

The multi metal-resistant bacterium Cupriavidus metallidurans CH34 affects growth and metal mobilization in Arabidopsis thaliana plants exposed to copper.

Copper (Cu) is important for plant growth, but high concentrations can lead to detrimental effects such as primary root length inhibition, vegetative tissue chlorosis, and even plant death. The interaction between plant-soil microbiota and roots can potentially affect metal mobility and availability, and, therefore, overall plant metal concentration. Cupriavidus metallidurans CH34 is a multi metal-resistant bacterial model that alters metal mobility and bioavailability through ion pumping, metal complexation, and reduction processes. The interactions between strain CH34 and plants may affect the growth, metal uptake, and translocation of Arabidopsis thaliana plants that are exposed to or not exposed to Cu. In this study, we looked also at the specific gene expression changes in C. metallidurans when co-cultured with Cu-exposed A. thaliana. We found that A. thaliana’s rosette area, primary and secondary root growth, and dry weight were affected by strain CH34, and that beneficial or detrimental effects depended on Cu concentration. An increase in some plant growth parameters was observed at copper concentrations lower than 50 µM and significant detrimental effects were found at concentrations higher than 50 µM Cu. We also observed up to a 90% increase and 60% decrease in metal accumulation and mobilization in inoculated A. thaliana. In turn, copper-stressed A. thaliana altered C. metallidurans colonization, and cop genes that encoded copper resistance in strain CH34 were induced by the combination of A. thaliana and Cu. These results reveal the complexity of the plant-bacteria-metal triad and will contribute to our understanding of their applications in plant growth promotion, protection, and phytoremediation strategies.

Adaptation of Cupriavidus metallidurans CH34 to Toxic Zinc Concentrations Involves an Uncharacterized ABC-Type Transporter.

Cupriavidus metallidurans CH34 is a well-studied metal-resistant β-proteobacterium and contains a battery of genes participating in metal metabolism and resistance. Here, we generated a mutant (CH34ZnR) adapted to high zinc concentrations in order to study how CH34 could adaptively further increase its resistance against this metal. Characterization of CH34ZnR revealed that it was also more resistant to cadmium, and that it incurred seven insertion sequence-mediated mutations. Among these, an IS1088 disruption of the glpR gene (encoding a DeoR-type transcriptional repressor) resulted in the constitutive expression of the neighboring ATP-binding cassette (ABC)-type transporter. GlpR and the adjacent ABC transporter are highly similar to the glycerol operon regulator and ATP-driven glycerol importer of Rhizobium leguminosarum bv. viciae VF39, respectively. Deletion of glpR or the ABC transporter and complementation of CH34ZnR with the parental glpR gene further demonstrated that loss of GlpR function and concomitant derepression of the adjacent ABC transporter is pivotal for the observed resistance phenotype. Importantly, addition of glycerol, presumably by glycerol-mediated attenuation of GlpR activity, also promoted increased zinc and cadmium resistance in the parental CH34 strain. Upregulation of this ABC-type transporter is therefore proposed as a new adaptation route towards metal resistance.

A genomic perspective of metal-resistant bacteria from gold particles: Possible survival mechanisms during gold biogeochemical cycling.

A bacterial consortium was enriched from gold particles that 'experienced' ca. 80 years of biotransformation within waste-rock piles (Australia). This bacterial consortium was exposed to 10 µM AuCl3 to obtain Au-tolerant bacteria. From these isolates, Serratia sp. and Stenotrophomonas sp. were the most Au-tolerant and reduced soluble Au as pure gold nanoparticles, indicating that passive mineralisation is a mechanism for mediating the toxic effect of soluble Au produced during particle dissolution. Genome-wide analysis demonstrated that these isolates also possessed various genes that could provide cellular defence enabling survival under heavy-metal stressed condition by mediating the toxicity of heavy metals through active efflux/reduction. Diverse metal-resistant genes or genes clusters (cop, cus, czc, zntand ars) were detected, which could confer resistance to soluble Au. Comparative genome analysis revealed that the majority of detected heavy-metal resistant genes were similar (i.e. orthologous) to those genes of Cupriavidus metallidurans CH34. The detection of heavy-metal resistance, nutrient cycling and biofilm formation genes (pgaABCD, bsmAandhmpS) may have indirect yet important roles when dealing with soluble Au during particle dissolution. In conclusion, the physiological and genomic results suggest that bacteria living on gold particles would likely use various genes to ensure survival during Au-biogeochemical cycling.

Copper Resistance Mediates Long-Term Survival of Cupriavidus metallidurans in Wet Contact With Metallic Copper.

Metallic copper to combat bacterial proliferation in drinking water systems is being investigated as an attractive alternative to existing strategies. A potential obstacle to this approach is the induction of metal resistance mechanisms in contaminating bacteria, that could severely impact inactivation efficacy. Thus far, the role of these resistance mechanisms has not been studied in conditions relevant to drinking water systems. Therefore, we evaluated the inactivation kinetics of Cupriavidus metallidurans CH34 in contact with metallic copper in drinking water. Viability and membrane permeability were examined for 9 days through viable counts and flow cytometry. After an initial drop in viable count, a significant recovery was observed starting after 48 h. This behavior could be explained by either a recovery from an injured/viable-but-non-culturable state or regrowth of surviving cells metabolizing lysed cells. Either hypothesis would necessitate an induction of copper resistance mechanisms, since no recovery was seen in a CH34 mutant strain lacking metal resistance mechanisms, while being more pronounced when copper resistance mechanisms were pre-induced. Interestingly, no biofilms were formed on the copper surface, while extensive biofilm formation was observed on the stainless steel control plates. When CH34 cells in water were supplied with CuSO4, a similar initial decrease in viable counts was observed, but cells recovered fully after 7 days. In conclusion, we have shown that long-term bacterial survival in the presence of a copper surface is possible upon the induction of metal resistance mechanisms. This observation may have important consequences in the context of the increasing use of copper as an antimicrobial surface, especially in light of potential co-selection for metal and antimicrobial resistance.

Determinants of the Lead(II) Affinity in pbrR Protein: A Computational Study.

Investigation of the diverse evolutionary developed mechanisms enabling bacteria to maintain homeostasis and to be resistant to lead is crucial for the discovery of novel strategies for isolation of this highly toxic metal and its subsequent elimination from contaminated environments. The metalloregulatory protein pbrR and its homologues that were identified in the Cupriavidus metallidurans CH34 chromosome are the only characterized natural metalloproteins that have a special affinity toward Pb(II) and that bind it with at least a 1000-fold selectivity over other heavy metals. The X-ray structures of apo and Pb(II)-bound pbrR have been recently reported. In the present study, the binding of Pb(II) at pbrR was investigated by means of multiscale computational modeling. Molecular dynamics simulations substantiated how conformations amenable for the Pb(II) complexation through the tris-cysteine motif are formed from the antiparallel coiled-coil packing interaction of two dimerization helices of two pbrR monomers, and the phase space of apo-pbrR has been extensively sampled. Hybrid quantum mechanics/molecular mechanics (QM/MM) calculations on metal-bound structures of pbrR also allowed us to determine the most probable protonation state for the lead binding motif and evaluate the structural features mostly affecting the Pb(II) coordination in this protein. In agreement with available experimental data, we found that pbrR may control its Pb(II) affinity, probably, by conformational changes that affect the distance between Cys78' and Cys122 and their protonation states, thus being able to switch on the Pb(II) sequestration/release-prone states in response to external stimuli. The protein structure enveloping the metal binding motif favors the thiol-thiolate-thiolate protonation state of Pb(II)-pbrR, thus probably enhancing the binding selectivity for Pb(II), compared to other metal ions.

The Response of Cupriavidus metallidurans CH34 to Cadmium Involves Inhibition of the Initiation of Biofilm Formation, Decrease in Intracellular c-di-GMP Levels, and a Novel Metal Regulated Phosphodiesterase.

Cadmium is a highly toxic heavy metal for biological systems. Cupriavidus metallidurans CH34 is a model strain to study heavy metal resistance and bioremediation as it is able to deal with high heavy metal concentrations. Biofilm formation by bacteria is mediated by the second messenger bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). The aim of this study was to characterize the response of C. metallidurans CH34 planktonic and biofilm cells to cadmium including their c-di-GMP regulatory pathway. Inhibition of the initiation of biofilm formation and EPS production by C. metallidurans CH34 correlates with increased concentration of cadmium. Planktonic and biofilm cells showed similar tolerance to cadmium. During exposure to cadmium an acute decrease of c-di-GMP levels in planktonic and biofilm cells was observed. Transcription analysis by RT-qPCR showed that cadmium exposure to planktonic and biofilm cells induced the expression of the urf2 gene and the mercuric reductase encoding merA gene, which belong to the Tn501/Tn21 mer operon. After exposure to cadmium, the cadA gene involved in cadmium resistance was equally upregulated in both lifestyles. Bioinformatic analysis and complementation assays indicated that the protein encoded by the urf2 gene is a functional phosphodiesterase (PDE) involved in the c-di-GMP metabolism. We propose to rename the urf2 gene as mrp gene for metal regulated PDE. An increase of the second messenger c-di-GMP content by the heterologous expression of the constitutively active diguanylate cyclase PleD correlated with an increase in biofilm formation and cadmium susceptibility. These results indicate that the response to cadmium in C. metallidurans CH34 inhibits the initiation of biofilm lifestyle and involves a decrease in c-di-GMP levels and a novel metal regulated PDE.

Recombinant expression and purification of a functional bacterial metallo-chaperone PbrD-fusion construct as a potential biosorbent for Pb(II)

PbrD is a lead (II) binding protein encoded by the pbr lead resistance operon found exclusively in Cupriavidus metallidurans CH34. Its ability to sequester Pb(II) shows potential for it to be developed as a biosorbent for Pb in the bioremediation of contaminated wastewaters. In this study the pbrD gene from C. metallidurans CH34 was transformed and overexpressed in Escherichia coli BL21 (DE3) using the pET32 Xa/Lic vector. Optimal expression of recombinant (r)PbrD (∼50 kDa) was achieved post-induction with IPTG within inclusion bodies (IBs). Inclusion bodies were solubilised by denaturation and purified by Ni-NTA affinity chromatography. The purified denatured protein containing the N-terminal Trx•Tag™, His•Tag® and S®Tag™ was refolded in vitro via dialysis to a biologically functional form. Circular dichroism spectra of refolded rPbrD-fusion protein indicated a high degree of turns, β-sheets and 310 helices content and tryptophan fluorescence showed a structural conformational change in the presence of Pb(II). Refolded rPbrD-fusion protein bound 99.7% of Pb(II) when mixed with lead nitrate in ten-fold increasing concentrations. Adsorption isotherms including Langmuir, Freundlich, Temkin and Dubinin-Radushkevich models were applied to determine the biosorption mechanism. A biologically functional rPbrD-fusion protein has potential application in the development of a biosorbent for remediation of Pb(II) from wastewater.

Toluene degradation by Cupriavidus metallidurans CH34 in nitrate-reducing conditions and in Bioelectrochemical Systems.

Bioelectrochemical remediation of hydrocarbons is a technology that exploits the ability of specific microorganisms to use as electron acceptor an electrode, thus potentially lowering the operational costs related to classical bioremediation. Several well-characterized hydrocarbonoclastic strains might be electroactive, thus their biodegradation performances in Bioelectrochemical Systems should be studied. Cupriavidus metallidurans CH34 is a model metal-resistant strain whose capacity to degrade benzene aerobically has recently been described. In this study, toluene degradation under anaerobic conditions and the exoelectrogenic capacity of Cupriavidus metallidurans CH34 were determined. Strain CH34 was grown anaerobically with toluene as sole carbon source in sealed serum bottles and then inoculated in a Microbial Electrolysis Cell (MEC) to assess its exoelectrogenic capacity. It was demonstrated for the first time that strain CH34 is able to degrade toluene under nitrate-reducing conditions (up to 45 mgtoluene/L were removed within 17 days, corresponding to 73% of toluene amended). Nitrate consumption and cellular growth were observed during toluene removal. In the MEC, toluene degradation was linked to current production, showing current peaks after every toluene addition (maximum current density 48 mA/m2). Coulombic efficiency of the toluene biodegradation process increased with time, from 11% (first batch cycle), up to 77% (last batch cycle).