Mobile genetic elements associated with utilization of dichloromethane and methanol as energy sources in Cupriavidus metallidurans.

Cupriavidus metallidurans strain PD11 isolated from laboratory waste drainage can use C1 compounds, such as dichloromethane (DCM) and methanol, as a sole carbon and energy source. However, strain CH34 (a type-strain) cannot grow in the medium supplemented with DCM. In the present study, we aimed to unravel the genetic elements underlying the utilization of C1 compounds by strain PD11. The genome subtraction approach indicated that only strain PD11 had several genes highly homologous to those of Herminiimonas arsenicoxydans strain ULPAs1. Moreover, a series of polymerase chain reaction (PCR) to detect the orthologs of H. arsenicoxydans genes and the comparative study of the genomes of three strains revealed that the 87.9 kb DNA fragment corresponding to HEAR1959 to HEAR2054 might be horizontally transferred to strain PD11. The 87.9 kb DNA fragment identified was found to contain three genes whose products were putatively involved in the metabolism of formaldehyde, a common intermediate of DCM and methanol. In addition, reverse transcription PCR analysis showed that all three genes were significantly expressed when strain PD11 was cultivated in the presence of DCM or methanol. These findings suggest that strain PD11 can effectively utilize the C1 compounds because of transfer of the mobile genetic elements from other bacterial species, for instance, from H. arsenicoxydans.

In a state of flux: new insight into the transport processes that maintain bacterial metal homeostasis.

A new study by Nies et al. (J Bacteriol 206:e00080-24, 2024, https://doi.org/10.1128/jb.00080-24) provides a rich, quantitative data set of zinc accumulation by cells of Cupriavidus metallidurans, including of mutant bacterial strains lacking import or efflux genes, and comparison of zinc accumulation by cells previously starved of metal with those of zinc-replete cells. The data surprisingly demonstrate the concomitant activity of both active metal import and metal efflux systems. They present a flow equilibrium model to describe zinc homeostasis in bacteria.

A flow equilibrium of zinc in cells of Cupriavidus metallidurans.

The hypothesis was tested that a kinetical flow equilibrium of uptake and efflux reactions is responsible for balancing the cellular zinc content. The experiments were done with the metal-resistant bacterium Cupriavidus metallidurans. In pulse-chase experiments, the cells were loaded with radioactive 65Zn and chased with the 100-fold concentration of non-radioactive zinc chloride. In parallel, the cells were loaded with isotope-enriched stable 67Zn and chased with non-enriched zinc to differentiate between zinc pools in the cell. The experiments demonstrated the existence of a kinetical flow equilibrium, resulting in a constant turnover of cell-bound zinc ions. The absence of the metal-binding cytoplasmic components, polyphosphate and glutathione, metal uptake, and metal efflux systems influenced the flow equilibrium. The experiments also revealed that not all zinc uptake and efflux systems are known in C. metallidurans. Cultivation of the cells under zinc-replete, zinc-, and zinc-magnesium-starvation conditions influenced zinc import and export rates. Here, magnesium starvation had a stronger influence compared to zinc starvation. Other metal cations, especially cobalt, affected the cellular zinc pools and zinc export during the chase reaction. In summary, the experiments with 65Zn and 67Zn demonstrated a constant turnover of cell-bound zinc. This indicated that simultaneously occurring import and export reactions in combination with cytoplasmic metal-binding components resulted in a kinetical flow equilibrium that was responsible for the adjustment of the cellular zinc content. IMPORTANCE: Understanding the biochemical action of a single enzyme or transport protein is the pre-requisite to obtain insight into its cellular function but this is only one half of the coin. The other side concerns the question of how central metabolic functions of a cell emerge from the interplay of different proteins and other macromolecules. This paper demonstrates that a flow equilibrium of zinc uptake and efflux reactions is at the core of cellular zinc homeostasis and identifies the most important contributors to this flow equilibrium: the uptake and efflux systems and metal-binding components of the cytoplasm.

A gold speciation that adds a second layer to synergistic gold-copper toxicity in Cupriavidus metallidurans.

The metal-resistant bacterium Cupriavidus metallidurans occurs in metal-rich environments. In auriferous soils, the bacterium is challenged by a mixture of copper ions and gold complexes, which exert synergistic toxicity. The previously used, self-made Au(III) solution caused a synergistic toxicity of copper and gold that was based on the inhibition of the CupA-mediated efflux of cytoplasmic Cu(I) by Au(I) in this cellular compartment. In this publication, the response of the bacterium to gold and copper was investigated by using a commercially available Au(III) solution instead of the self-made solution. The new solution was five times more toxic than the previously used one. Increased toxicity was accompanied by greater accumulation of gold atoms by the cells. The contribution of copper resistance determinants to the commercially available Au(III) solution and synergistic gold-copper toxicity was studied using single- and multiple-deletion mutants. The commercially available Au(III) solution inhibited periplasmic Cu(I) homeostasis, which is required for the allocation of copper ions to copper-dependent proteins in this compartment. The presence of the gene for the periplasmic Cu(I) and Au(I) oxidase, CopA, decreased the cellular copper and gold content. Transcriptional reporter gene fusions showed that up-regulation of gig, encoding a minor contributor to copper resistance, was strictly glutathione dependent. Glutathione was also required to resist synergistic gold-copper toxicity. The new data indicated a second layer of synergistic copper-gold toxicity caused by the commercial Au(III) solution, inhibition of the periplasmic copper homeostasis in addition to the cytoplasmic one.IMPORTANCEWhen living in auriferous soils, Cupriavidus metallidurans is not only confronted with synergistic toxicity of copper ions and gold complexes but also by different gold species. A previously used gold solution made by using aqua regia resulted in the formation of periplasmic gold nanoparticles, and the cells were protected against gold toxicity by the periplasmic Cu(I) and Au(I) oxidase CopA. To understand the role of different gold species in the environment, another Au(III) solution was commercially acquired. This compound was more toxic due to a higher accumulation of gold atoms by the cells and inhibition of periplasmic Cu(I) homeostasis. Thus, the geo-biochemical conditions might influence Au(III) speciation. The resulting Au(III) species may subsequently interact in different ways with C. metallidurans and its copper homeostasis system in the cytoplasm and periplasm. This study reveals that the geochemical conditions may decide whether bacteria are able to form gold nanoparticles or not.

Simultaneous removal of ammonia nitrogen, phosphate, zinc, and phenol by degradation of cellulose in composite mycelial pellet bioreactor: Enhanced performance and community co-assembly mechanism.

In this experiment, the prepared tea biochar-cellulose@LDH material (TB-CL@LDH) was combined with mycelium pellets to form the composite mycelial pellets (CMP), then assembled and immobilized with strains Pseudomonas sp. Y1 and Cupriavidus sp. ZY7 to construct a bioreactor. At the best operating parameters, the initial concentrations of phosphate (PO43--P), ammonia nitrogen (NH4+-N), chemical oxygen demand (COD), zinc (Zn2+), and phenol were 22.3, 25.0, 763.8, 1.0, and 1.0 mg L-1, the corresponding removal efficiencies were 80.4, 87.0, 83.4, 91.8, and 96.6%, respectively. Various characterization analyses demonstrated that the strain Y1 used the additional carbon source produced by the strain ZY7 degradation of cellulose to enhance the removal of composite pollutants and clarified the principle of Zn2+ and PO43--P removal by adsorption, co-precipitation and biomineralization. Pseudomonas and Cupriavidus were the dominant genera according to the high-throughput sequencing. As shown by KEGG results, nitrification and denitrification genes were affected by phenol. The study offers prospects for the simultaneous removal of complex pollutants consisting of NH4+-N, PO43--P, Zn2+, and phenol.

Protecting the Achilles heel: three FolE_I-type GTP-cyclohydrolases needed for full growth of metal-resistant Cupriavidus metallidurans under a variety of conditions.

In Cupriavidus metallidurans and other bacteria, biosynthesis of the essential biochemical cofactor tetrahydrofolate (THF) initiates from guanosine triphosphate (GTP). This step is catalyzed by FolE_I-type GTP cyclohydrolases, which are either zinc-dependent FolE_IA-type or metal-promiscuous FolE_IB-type enzymes. As THF is also essential for GTP biosynthesis, GTP and THF synthesis form a cooperative cycle, which may be influenced by the cellular homeostasis of zinc and other metal cations. Metal-resistant C. metallidurans harbors one FolE_IA-type and two FolE_IB-type enzymes. All three proteins were produced in Escherichia coli. FolE_IA was indeed zinc dependent and the two FolE_IB enzymes metal-promiscuous GTP cyclohydrolases in vitro, the latter, for example, functioning with iron, manganese, or cobalt. Single and double mutants of C. metallidurans with deletions in the folE_I genes were constructed to analyze the contribution of the individual FolE_I-type enzymes under various conditions. FolE_IA was required in the presence of cadmium, hydrogen peroxide, metal chelators, and under general metal starvation conditions. FolE_IB1 was important when zinc uptake was impaired in cells without the zinc importer ZupT (ZIP family) and in the presence of trimethoprim, an inhibitor of THF biosynthesis. FolE_IB2 was needed under conditions of low zinc and cobalt but high magnesium availability. Together, these data demonstrate that C. metallidurans requires all three enzymes to allow efficient growth under a variety of conditions.IMPORTANCETetrahydrofolate (THF) is an important cofactor in microbial biochemistry. This "Achilles heel" of metabolism has been exploited by anti-metabolites and antibiotics such as sulfonamide and trimethoprim. Since THF is essential for the synthesis of guanosine triphosphate (GTP) and THF biosynthesis starts from GTP, synthesis of both compounds forms a cooperative cycle. The first step of THF synthesis by GTP cyclohydrolases (FolEs) is metal dependent and catalyzed by zinc- or metal-promiscuous enzymes, so that the cooperative THF and GTP synthesis cycle may be influenced by the homeostasis of several metal cations, especially that of zinc. The metal-resistant bacterium C. metallidurans needs three FolEs to grow in environments with both high and low zinc and cadmium content. Consequently, bacterial metal homeostasis is required to guarantee THF biosynthesis.

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.

Extracellular proteins enhance Cupriavidus pauculus nickel tolerance and cell aggregate formation.

Heavy metal-resistant bacteria secrete extracellular proteins (e-PNs). However, the role of e-PNs in heavy metal resistance remains elusive. Here Fourier Transform Infrared Spectroscopy implied that N-H, C = O and NH2-R played a crucial role in the adsorption and resistance of Ni2+ in the model organism Cuprividus pauculus 1490 (C. pauculus). Proteinase K treatment reduced Ni2+ resistance of C. pauculus underlining the essential role of e-PNs. Further three-dimension excitation-emission matrix fluorescence spectroscopy analysis demonstrated that tryptophan proteins as part of the e-PNs increased significantly with Ni2+ treatment. Proteomic and quantitative real-time polymerase chain reaction data indicated that major changes were induced in the metabolism of C. pauculus in response to Ni2+. Among those lipopolysaccharide biosynthesis, general secretion pathways, Ni2+-affiliated transporters and multidrug efflux play an essential role in Ni2+ resistance. Altogether the results provide a conceptual model for comprehending how e-PNs contribute to bacterial resistance and adsorption of Ni2+.

Combining microbiome and pseudotargeted metabolomics revealed the alleviative mechanism of Cupriavidus sp. WS2 on the cadmium toxicity in Vicia unijuga A.Br.

Cadmium (Cd) pollution is one of the most severe toxic metals pollution in grassland. Vicia unijuga (V. unijuga) A.Br. planted nearby the grassland farming are facing the risk of high Cd contamination. Here, we investigated the beneficial effects of a highly Cd tolerant rhizosphere bacterium, Cupriavidus sp. WS2, on Cd contaminated V. unijuga. Through plot experiments, we set up four groups of treatments: the control group (without WS2 or Cd), the Cd group (with only Cd addition), the WS2 group (with only WS2 addition), and the WS2/Cd group (with WS2 and Cd addition), and analyzed the changes in physiological indicators, rhizosphere microorganisms, and stem and leaf metabolites of V. unijuga. Results of physiological indicators indicated that Cupriavidus sp. WS2 had strong absorption and accumulation capacity of Cd, exogenous addition of strain WS2 remarkably decreased the Cd concentrations, and increased the plant heights, the biomass, the total protein concentrations, the chlorophyll contents and the photosynthetic rate in stems and leaves of V. unijuga under Cd stress. Cd treatment increased the abundance of Cd tolerant bacterial genera in rhizosphere microbiome, but these genera were down-regulated in the WS2/Cd group. Pseudotargeted metabolomic results showed that six common differential metabolites associated with antioxidant stress were increased after co-culture with WS2. In addition, WS2 activated the antioxidant system including glutathione (GSH) and catalase (CAT), reduced the contents of oxidative stress markers including malondialdehyde (MDA) and hydrogen peroxide (H2O2) in V. unijuga under Cd stress. Taken together, this study revealed that Cupriavidus sp.WS2 alleviated the toxicity of V. unijuga under Cd exposure by activating the antioxidant system, increasing the antioxidant metabolites, and reducing the oxidative stress markers.

Enhancing polyhydroxyalkanoate production in Cupriavidus sp. L7L through wcaJ gene deletion.

Cupriavidus sp. L7L synthesizes a high content of ductile polyhydroxyalkanoate. However, during fermentation, the medium's viscosity gradually increases, eventually reaching a level similar to 93 % glycerol, leading to fermentation termination and difficulties in cell harvest. A non-mucoid variant was isolated from a mini-Tn5 mutant library with the transposon inserted at the promoter sequence upstream of the wcaJ gene. Deletion of wcaJ eliminated the mucoid-colony appearance. The complementation experiment confirmed the association between wcaJ gene expression and mucoid-colony formation. Additionally, the wild-type strain exhibited a faster specific growth rate than the deletion strain using levulinate (Lev) as a carbon source. In fed-batch fermentation, Cupriavidus sp. L7L∆wcaJ showed similar PHA content and monomer composition to the wild-type strain. However, the extended fermentation time resulted in a 42 % increase in PHA concentration. After fed-batch fermentation, the deletion strain's medium had only 8.75 % of the wild-type strain's extracellular polymeric substance content. Moreover, the deletion strain's medium had a much lower viscosity (1.04 mPa·s) than the wild-type strain (194.7 mPa·s), making bacterial cell collection easier through centrifugation. In summary, Cupriavidus sp. L7L∆wcaJ effectively addressed difficulties in cell harvest, increased PHA production, and Lev-to-PHA conversion efficiency, making these characteristics advantageous for industrial-scale PHA production.

Characterization of newly isolated thermotolerant bacterium Cupriavidus sp. CB15 from composting and its ability to produce polyhydroxyalkanoate from glycerol.

BACKGROUND: This study aimed to isolate a novel thermotolerant bacterium that is capable of synthesizing polyhydroxyalkanoate from glycerol under high temperature conditions. RESULTS: A newly thermotolerant polyhydroxyalkanoate (PHA) producing bacterium, Cupriavidus sp. strain CB15, was isolated from corncob compost. The potential ability to synthesize PHA was confirmed by detection of PHA synthase (phaC) gene in the genome. This strain could produce poly(3-hydroxybutyrate) [P(3HB)] with 0.95 g/L (PHA content 75.3 wt% of dry cell weight 1.24 g/L) using glycerol as a carbon source. The concentration of PHA was enhanced and optimized based on one-factor-at-a-time (OFAT) experiments and response surface methodology (RSM). The optimum conditions for growth and PHA biosynthesis were 10 g/L glycerol, 0.78 g/L NH4Cl, shaking speed at 175 rpm, temperature at 45 °C, and cultivation time at 72 h. Under the optimized conditions, PHA production was enhanced to 2.09 g/L (PHA content of 74.4 wt% and dry cell weight of 2.81 g/L), which is 2.12-fold compared with non-optimized conditions. Nuclear magnetic resonance (NMR) analysis confirmed that the extracted PHA was a homopolyester of 3-hydyoxybutyrate. CONCLUSION: Cupriavidus sp. strain CB15 exhibited potential for cost-effective production of PHA from glycerol.

Comparative Genomic Analysis and BTEX Degradation Pathways of a Thermotolerant Cupriavidus cauae PHS1.

Volatile organic compounds such as benzene, toluene, ethylbenzene, and isomers of xylenes (BTEX) constitute a group of monoaromatic compounds that are found in petroleum and have been classified as priority pollutants. In this study, based on its newly sequenced genome, we reclassified the previously identified BTEX-degrading thermotolerant strain Ralstonia sp. PHS1 as Cupriavidus cauae PHS1. Also presented are the complete genome sequence of C. cauae PHS1, its annotation, species delineation, and a comparative analysis of the BTEX-degrading gene cluster. Moreover, we cloned and characterized the BTEX-degrading pathway genes in C. cauae PHS1, the BTEX-degrading gene cluster of which consists of two monooxygenases and meta-cleavage genes. A genome-wide investigation of the PHS1 coding sequence and the experimentally confirmed regioselectivity of the toluene monooxygenases and catechol 2,3-dioxygenase allowed us to reconstruct the BTEX degradation pathway. The degradation of BTEX begins with aromatic ring hydroxylation, followed by ring cleavage, and eventually enters the core carbon metabolism. The information provided here on the genome and BTEX-degrading pathway of the thermotolerant strain C. cauae PHS1 could be useful in constructing an efficient production host.

Efficient Knocking Out of the Organophosphorus Insecticides Degradation Gene opdB in Cupriavidus nantongensis X1T via CRISPR/Cas9 with Red System.

Cupriavidus nantongensis X1T is a type strain of the genus Cupriavidus, that can degrade eight kinds of organophosphorus insecticides (OPs). Conventional genetic manipulations in Cupriavidus species are time-consuming, difficult, and hard to control. The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) system has emerged as a powerful tool for genome editing applied in prokaryotes and eukaryotes due to its simplicity, efficiency, and accuracy. Here, we combined CRISPR/Cas9 with the Red system to perform seamless genetic manipulation in the X1T strain. Two plasmids, pACasN and pDCRH were constructed. The pACasN plasmid contained Cas9 nuclease and Red recombinase, and the pDCRH plasmid contained the dual single-guide RNA (sgRNA) of organophosphorus hydrolase (OpdB) in the X1T strain. For gene editing, two plasmids were transferred to the X1T strain and a mutant strain in which genetic recombination had taken place, resulting in the targeted deletion of opdB. The incidence of homologous recombination was over 30%. Biodegradation experiments suggested that the opdB gene was responsible for the catabolism of organophosphorus insecticides. This study was the first to use the CRISPR/Cas9 system for gene targeting in the genus Cupriavidus, and it furthered our understanding of the process of degradation of organophosphorus insecticides in the X1T strain.

Interplay between Two-Component Regulatory Systems Is Involved in Control of Cupriavidus metallidurans Metal Resistance Genes.

Metal resistance of Cupriavidus metallidurans is based on determinants that were acquired in the past by horizontal gene transfer during evolution. Some of these determinants encode transmembrane metal efflux systems. Expression of most of the respective genes is controlled by two-component regulatory systems composed of a membrane-bound sensor/sensory histidine kinase (HK) and a cytoplasmic, DNA-binding response regulator (RR). Here, we investigated the interplay between the three closely related two-component regulatory systems CzcRS, CzcR2S2, and AgrRS. All three systems regulate the response regulator CzcR, while the RRs AgrR and CzcR2 were not involved in czc regulation. Target promoters were czcNp and czcPp for genes upstream and downstream of the central czc gene region. The two systems together repressed CzcRS-dependent upregulation of czcP-lacZ at low zinc concentrations in the presence of CzcS but activated this signal transmission at higher zinc concentrations. AgrRS and CzcR2S2 interacted to quench CzcRS-mediated expression of czcNp-lacZ and czcPp-lacZ. Together, cross talk between the three two-component regulatory systems enhanced the capabilities of the Czc systems by controlling expression of the additional genes czcN and czcP. IMPORTANCE Bacteria are able to acquire genes encoding resistance to metals and antibiotics by horizontal gene transfer. To bestow an evolutionary advantage on their host cell, new genes must be expressed, and their expression should be regulated so that resistance-mediating proteins are produced only when needed. Newly acquired regulators may interfere with those already present in a host cell. Such an event was studied here in the metal-resistant bacterium Cupriavidus metallidurans. The results demonstrate how regulation by the acquired genes interacts with the host's extant regulatory network. This leads to emergence of a new system level of complexity that optimizes the response of the cell to periplasmic signals.

Production of polyhydroxyalkanoates by the thermophile Cupriavidus cauae PHS1.

Thermophilic production of polyhydroxyalkanoate is considered a very promising way to overcome the problems that may arise when using mesophilic strains. This study reports the first thermophilic polyhydroxybutyrate-producing Cupriavidus species, which are known as the best polyhydroxybutyrate-producing microorganisms. Cupriavidus cauae PHS1 harbors a phbCABR cluster with high similarity to the corresponding proteins of C. necator H16 (80, 93, 96, and 97 %). This strain can produce polyhydroxybutyrate from a range of substrates, including acetate (5 g/L) and phenol (1 g/L), yielding 7.6 % and 18.9 % polyhydroxybutyrate, respectively. Moreover, the strain produced polyhydroxybutyrate at temperatures ranging from 25 to 50 °C, with the highest polyhydroxybutyrate content (47 °C) observed at 45 °C from gluconate. Additionally, the strain could incorporate 3-hydroxyvalerate (12.5 mol. %) into the polyhydroxybutyrate polymer using levulinic acid as a precursor. Thus, Cupriavidus cauae PHS1 may be a promising polyhydroxybutyrate producer as alternative for mesophilic polyhydroxybutyrate-producing Cupriavidus species.

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.

Biological denitrification potential of cellulase-producing Cupriavidus sp. ZY7 and denitrifying Aquabacterium sp. XL4 at low carbon-to-nitrogen ratio: Performance and synergistic properties.

This study emphasizes on the cellulase production characteristics of strain ZY7 and its collaboration with nitrate-dependent ferrous oxidizing (NFO) strain XL4 to achieve efficient denitrification at low carbon-to-nitrogen (C/N) ratio. Results indicated that the denitrification efficiency increased from 65.47 to 97.99% at 24 h after co-culture at C/N of 1.0. Three-dimensional fluorescence excitation-emission matrix (3D-EEM) showed significant changes in the intensity of soluble microbial products (SMP), fulvic-like materials, and aromatic proteins after co-culture. Bio-precipitates were characterized by Scanning electron microscope (SEM), Fourier transform infrared spectrometer (FTIR), and X-ray diffraction (XRD), which showed that cellulose structure was disrupted and the metabolites were potential carbon source for denitrification. In addition, cellulase activity suggested that the hydrolysis of ß-1,4-glycosidic bonds and oligosaccharides may be the rate-limiting steps in cellulose degradation. This work promoted the understanding of denitrification characteristics of co-culture and expanded the application of cellulose degrading bacteria in sewage treatment.

Metal-free catalysis for organic micropollutant degradation in waste activated sludge via poly(3-hydroxybutyrate) biopolymers using Cupriavidus sp. L7L coupled with peroxymonosulfate.

This study employed a novel and environment-friendly biopolymer/oxidant catalytic system, viz., poly(3-hydroxybutyrate)/peroxymonosulfate (PHB/PMS), for pretreating wastewater sludge for the first time. Under optimal conditions, i.e., 3.1 × 10-4 M of PMS and 3.3 g/L of PHB at pH = 6.0, the PAHs in the sludge matrix was decreased by 79 % in 12 h. Increase in salinity (75 % synthetic seawater) achieved 83 % of PAHs degradation. Functional groups (CO) of the biopolymer matrix were active centers for biopolymer-mediated electron transfer that produced reactive oxygen species (SO4-, HO, and 1O2) for adsorption and catalytic oxidation of PAHs in the sludge. Functional metagenomic analysis revealed the main genus, Conexibacter (phylum, Actinobacteria) exhibited PAH-degrading function with high efficiency in the biodegradation of PAHs from sludge pretreated with PHB/PMS. Coupling chemical oxidation and biostimulation using bacterial polymer-based biomaterials is effective and beneficial for pretreating wastewater sludge toward circular bioeconomy.

Biodegradation of technical hexachlorocyclohexane by Cupriavidus malaysiensis.

Lindane (γ-Hexachlorocyclohexane) has been used extensively as a pesticide all over the world. The production of Lindane entails the formation of four major Hexachlorocyclohexane (HCH) isomers, that is, alpha, beta, gamma, and delta as muck. These have been used as Technical HCH in developing countries as an inexpensive alternate source. However, HCH isomers pose a severe environmental hazard due to their highly persistent nature and toxicity. In this study, the effect of HCH application on the soil microbial diversity was studied. The species which could persist even after prolonged exposure at high HCH concentration, was isolated, screened, and enriched as potential t-HCH degraders. The selected isolate could degrade 88.05%, 92.19%, 91.54%, and 82.85% of the alpha, gamma, beta, and delta isomers, respectively at 100 mg/L HCH concentration. Identification of the isolate by 16s rRNA sequencing was similar to Cupriavidus malaysiensis. To the best of the authors' knowledge, this is the first study to observe this particular strain's ability to simultaneously degrade the four isomers, especially the most recalcitrant beta isomer. Therefore, the degradative capability of this strain, as a sole carbon source at higher HCH concentration (100 mg/l), can be exploited for bioremediation of HCH contaminated sites.

Resistance properties and adaptation mechanism of cadmium in an enriched strain, Cupriavidus nantongensis X1T.

Bacterial adaption to heavy metal stress is a complex and comprehensive process of multi-response regulation. However, the mechanism is largely unexplored. In this study, cadmium (Cd) resistance and adaptation mechanism in Cupriavidus nantongensis X1T were investigated. Strain X1T could resist the stress of 307 mg/L Cd2+ and remove 70% Cd2+ in 48 h. Spectroscopic analyses suggested interactions between Cd2+ with C-N, -COOH, and -NH ligands of extracellular polymeric substances. Whole-genome sequencing found that the resistance of Cd2+ in strain X1T was caused by the joint action of Czc and Cad systems. Cd2+ at 20 mg/L elicited differential expression of 1157 genes in strain X1T. In addition to the reported effects of uptake, adsorption, effluxion, and accumulation system, the oxidative stress system, Type-VI secretory protein system, Fe-S protein synthesis, and cysteine synthesis system in strain X1T were involved in the Cd2+ resistance and accumulation. The intracellular accumulation content of Cd2+ in strain X1T was higher than the extracellular adsorption content made strain X1T to be an important resource strain in the bioremediation of Cd-contaminated sewage. The results provide a theoretical network for understanding the complex regulatory system of bacterial resistance and adaptation of Cd against stressful environments.