The Transcriptomic Landscape of Cupriavidus metallidurans CH34 Acutely Exposed to Copper.

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

Cupriavidus sp. strain Cd02-mediated pH increase favoring bioprecipitation of Cd2+ in medium and reduction of cadmium bioavailability in paddy soil.

This study investigated the effects of Cupriavidus sp. strain Cd02-mediated increase on biosorption and bioprecipitation of Cd2+ during the 144-h cultivation time as well as evaluated effectivenesses of changing soil pH and bioavailability of cadmium after bioaugmentation of strain Cd02 into Cd-contaminated paddy soil for 15 days. Results showed that strain Cd02-induced pH increase of the culture medium (from 7.40 to 8.68) facilitated biosorption of Cd2+ on Cd02 cell surface (4.82 mg/mg) and extracellular bioprecipitation in form of cadmium carbonate (3.07 mg/mg). Also, the pH values of Cd-contaminated paddy soil increased by 1.41 units after strain Cd02 was applied for 15 days, which thereby promoted the decrease of exchangeable fraction of Cd2+ by 6.5% in the tested paddy soil. Meanwhile, strain Cd02 could prosperously live in paddy soils after bioaugmentation. These results suggest that strain Cd02 may be applicable for bioremediation of the heavy metal-contaminated soils by bioaugmentation.

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).

Synergistic gold-copper detoxification at the core of gold biomineralisation in Cupriavidus metallidurans.

The bacterium Cupriavidus metallidurans is capable of reducing toxic Au(i/iii)-complexes into metallic gold (Au) nano-particles, thereby mediating the (trans)formation of Au nuggets in Earth surface environments. In this study we describe a novel detoxification pathway, which prevents synergistic copper (Cu)/Au-toxicity. Gold-complexes and Cu-ions exert cooperative toxicity, because cellular uptake of Au(i/iii)-complexes blocks Cu(i) export from the cytoplasm by the Cu-efflux pump CupA. Using a combination of micro-analytical and biochemical methods we show that inducible resistance to these Cu/Au mixtures is mediated by the periplasmic Cu(i)-oxidase CopA, which functions as an oxygen-consuming Au(i)-oxidase. With high Au-complex loads the enzymatic activity of CopA detoxifies the reduction pathway of Au(iii)-complexes via Au(i)-intermediates to Au(0) nanoparticles in the periplasm. Thereby the concentration of highly toxic Au(i) in the cytoplasm is diminished, while allowing direct reduction of Au(iii) to Au nanoparticles in the periplasm. This permits C. metallidurans to thrive in Au-rich environments and biomineralise metallic Au.

Spatio-temporal control of mutualism in legumes helps spread symbiotic nitrogen fixation.

Mutualism is of fundamental importance in ecosystems. Which factors help to keep the relationship mutually beneficial and evolutionarily successful is a central question. We addressed this issue for one of the most significant mutualistic interactions on Earth, which associates plants of the leguminosae family and hundreds of nitrogen (N2)-fixing bacterial species. Here we analyze the spatio-temporal dynamics of fixers and non-fixers along the symbiotic process in the Cupriavidus taiwanensis-Mimosa pudica system. N2-fixing symbionts progressively outcompete isogenic non-fixers within root nodules, where N2-fixation occurs, even when they share the same nodule. Numerical simulations, supported by experimental validation, predict that rare fixers will invade a population dominated by non-fixing bacteria during serial nodulation cycles with a probability that is function of initial inoculum, plant population size and nodulation cycle length. Our findings provide insights into the selective forces and ecological factors that may have driven the spread of the N2-fixation mutualistic trait.

Biotransformation and reduction of estrogenicity of bisphenol A by the biphenyl-degrading Cupriavidus basilensis.

The biphenyl-degrading Gram-negative bacterium Cupriavidus basilensis (formerly Ralstonia sp.) SBUG 290 uses various aromatic compounds as carbon and energy sources and has a high capacity to transform bisphenol A (BPA), which is a hormonally active substance structurally related to biphenyl. Biphenyl-grown cells initially hydroxylated BPA and converted it to four additional products by using three different transformation pathways: (a) formation of multiple hydroxylated BPA, (b) ring fission, and (c) transamination followed by acetylation or dimerization. Products of the ring fission pathway were non-toxic and all five products exhibited a significantly reduced estrogenic activity compared to BPA. Cell cultivation with phenol and especially in nutrient broth (NB) resulted in a reduced biotransformation rate and lower product quantities, and NB-grown cells did not produce all five products in detectable amounts. Thus, the question arose whether enzymes of the biphenyl degradation pathway are involved in the transformation of BPA and was addressed by proteomic analyses.

Biodegradation and detoxification of chloronitroaromatic pollutant by Cupriavidus.

Current study reports isolation of Cupriavidus strain a3 which can utilize 2-chloro-4-nitrophenol (C4NP) as sole source of carbon and nitrogen, leading to its detoxification. Degradation process was initiated by release of nitrite ion resulting in the formation of 2-chlorohydroquinone as intermediate. The nitrite releasing activity was also evident in the cell free protein extract. Different parameters for 2C4NP biodegradation were optimized. The degradation pattern followed Haldane substrate inhibition model with maximum specific degradation rate (qmax) of 0.13/h, half saturation constant (Ks) of 0.05mM, and 2C4NP inhibition constant (Ki) of 0.64mM. The isolate was successfully applied to remediation of 2C4NP-contaminated soil in microcosm study. 2-Dimensional protein electrophoresis analysis showed that growth of the isolate in the presence of 2C4NP resulted in modification of membrane permeability and induction of signal transduction protein. In our knowledge, this is the first study reporting degradation and detoxification of 2C4NP by Cupriavidus.

Proteomic responses to gold(iii)-toxicity in the bacterium Cupriavidus metallidurans CH34.

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

Use of Cupriavidus basilensis-aided bioabatement to enhance fermentation of acid-pretreated biomass hydrolysates by Clostridium beijerinckii.

Lignocellulose-derived microbial inhibitors (LDMICs) prevent efficient fermentation of Miscanthus giganteus (MG) hydrolysates to fuels and chemicals. To address this problem, we explored detoxification of pretreated MG biomass by Cupriavidus basilensis ATCC(®)BAA-699 prior to enzymatic saccharification. We document three key findings from our test of this strategy to alleviate LDMIC-mediated toxicity on Clostridium beijerinckii NCIMB 8052 during fermentation of MG hydrolysates. First, we demonstrate that growth of C. basilensis is possible on furfural, 5-hydroxymethyfurfural, cinnamaldehyde, 4-hydroxybenzaldehyde, syringaldehyde, vanillin, and ferulic, p-coumaric, syringic and vanillic acid, as sole carbon sources. Second, we report that C. basilensis detoxified and metabolized ~98 % LDMICs present in dilute acid-pretreated MG hydrolysates. Last, this bioabatement resulted in significant payoffs during acetone-butanol-ethanol (ABE) fermentation by C. beijerinckii: 70, 50 and 73 % improvement in ABE concentration, yield and productivity, respectively. Together, our results show that biological detoxification of acid-pretreated MG hydrolysates prior to fermentation is feasible and beneficial.

A study of synthesis and properties of poly-3-hydroxybutyrate/diethylene glycol copolymers.

This study investigates synthesis of poly(3-hydroxybutyrate)/diethylene glycol copolymers (P3HB/DEG) by Cupriavidus eutrophus B-10646 cells as related to DEG concentration in the medium and the time when it is added to the culture of cells synthesizing P3HB. The study determines the limits of physiological effect of DEG on C. eutrophus cells, showing that at DEG concentrations above 30 g/L, it inhibits cell growth, decreasing cell concentration and total P3HB/DEG yield and inducing an increase in the degree of saturation of fatty acids in lipids of cell cytoplasmic membrane. A series of copolymers containing different molar fractions of DEG (between 0.13 and 3.0 mol%) have been synthesized and their physicochemical, physical/mechanical, and biological properties have been investigated as related to the chemical composition and proportions of DEG monomers of the polymers. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1017-1028, 2016.

Bacterial biofilms on gold grains-implications for geomicrobial transformations of gold.

The biogeochemical cycling of gold (Au), i.e. its solubilization, transport and re-precipitation, leading to the (trans)formation of Au grains and nuggets has been demonstrated under a range of environmental conditions. Biogenic (trans)formations of Au grains are driven by (geo)biochemical processes mediated by distinct biofilm consortia living on these grains. This review summarizes the current knowledge concerning the composition and functional capabilities of Au-grain communities, and identifies contributions of key-species involved in Au-cycling. To date, community data are available from grains collected at 10 sites in Australia, New Zealand and South America. The majority of detected operational taxonomic units detected belong to the α-, ß- and γ-Proteobacteria and the Actinobacteria. A range of organisms appears to contribute predominantly to biofilm establishment and nutrient cycling, some affect the mobilization of Au via excretion of Au-complexing ligands, e.g. organic acids, thiosulfate and cyanide, while a range of resident Proteobacteria, especially Cupriavidus metallidurans and Delftia acidovorans, have developed Au-specific biochemical responses to deal with Au-toxicity and reductively precipitate mobile Au-complexes. This leads to the biomineralization of secondary Au and drives the environmental cycle of Au.

Interplay between seven secondary metal uptake systems is required for full metal resistance of Cupriavidus metallidurans.

The beta-proteobacterium Cupriavidus metallidurans is able to grow in metal-contaminated environments due to having sophisticated metal efflux systems. Here, the contribution of all seven known secondary metal uptake systems (ZupT, PitA, CorA1, CorA2, CorA3, ZntB, HoxN) to metal resistance is characterized. In a strategic deletion approach, all ten double deletion mutants, a variety of triple and quadruple mutants, and from the Δ4 mutant (ΔzupT ΔcorA1 ΔcorA2 ΔcorA3) the mutants Δ5 (=Δ4 ΔpitA), Δ6 (=Δ4 ΔpitA ΔzntB), and finally Δ7 (ΔzupT ΔcorA1 ΔcorA2 ΔcorA3 ΔpitA ΔzntB ΔhoxN) were constructed. Metal resistance, metal content, and regulation of expression of these genes were characterized in these mutants. The ΔzupT single deletion strain exhibited an extended lag phase in Tris-buffered liquid mineral salts medium (TMM) compared to its parent strain AE104, indicating a decreased fitness level. Further deletions up to Δ6 did not influence growth in TMM without added metals but fitness of the Δ7 strain dropped to a lower level compared to Δ6, Δ5 and ΔzupT. The cells of the Δ7 multiple deletion strain still contained all essential metals, demonstrating that additional metal import systems must exist in C. metallidurans. PitA was an important contributor of metal:phosphate complexes to C. metallidurans. Up to Δ5 no evidence was found for increased expression of the transporter genes to recruit substitutes for the deleted importers. Only the hoxN-lacZ reporter gene fusion displayed a changed expression pattern in the Δ6 strain, indicating recruitment of HoxN. Metal resistance of the deletion strains decreased along the deletion series although all strains still contained metal efflux systems: up to the Δ6 mutant the overall fitness was kept at the ΔzupT mutant strain level at the cost of a diminished competence to handle µM concentrations of transition metals. Together, these data demonstrated an important contribution of the seven secondary metal import systems to metal homeostasis in this bacterium.

Bioaccumulation and biosorption of Cd(2+) and Zn(2+) by bacteria isolated from a zinc mine in Thailand.

The three bacteria, Tsukamurella paurometabola A155, Pseudomonas aeruginosa B237, and Cupriavidus taiwanensis E324, were isolated from soils collected from a zinc mine in Tak Province, Thailand. Among these bacteria, P. aeruginosa B237 and C. taiwanensis E324 were tolerant of both cadmium and zinc, while T. paurometabola A155 was highly tolerant of zinc only. Bioaccumulation experiment revealed that Cd(2+) and Zn(2+) were mainly adsorbed on the cell walls of these bacteria rather than accumulated inside the cells. During Cd(2+) and Zn(2+) biosorption, P. aeruginosa B237 and T. paurometabola A155 showed the highest removal efficiencies for Cd(2+) and Zn(2+), respectively. The maximum biosorption capacities of P. aeruginosa B237 and T. paurometabola A155 biomasses for Cd(2+) and Zn(2+) biosorptions were 16.89 and 16.75 mg g(-1), respectively, under optimal conditions. The experimental data of Cd(2+) and Zn(2+) biosorptions fitted well with Langmuir isotherm model, suggesting that Cd(2+) and Zn(2+) adsorptions occurred in a monolayer pattern on a homogeneous surface. Furthermore, the pseudo-second order and pseudo-first order kinetic models best described the biosorption kinetics of Cd(2+) and Zn(2+) adsorptions, respectively, suggesting that the Cd(2+) and Zn(2+) adsorptions took place mainly by chemisorption (Cd(2+)) and physisorption (Zn(2+)).

Characterization of the molecular degradation mechanism of diphenyl ethers by Cupriavidus sp. WS.

Commonly used flame retardants, such as polybrominated diphenyl ethers, are extremely persistent in the environment, causing serious environmental risks. Certain strains of bacteria are able to degrade several low brominated congeners of PBDEs aerobically. However, the aerobic degradation pathway is not yet well understood, particularly at the genetic level. In this study, we isolated Cupriavidus sp. WS from the environment that could degrade diphenyl ether (DE), 4-bromodiphenyl ether, and 4,4'-bromodiphenyl ether. DE was completely degraded in 6 days without any detectable end-product. Using transposon mutagenesis, several DE degradation-deficient mutants were obtained. Knocking out bphA1, bphA2, and bphA3 eliminated the ability of the Cupriavidus sp. WS bacterium to degrade DE, indicating that the bph genes play a crucial role in DE degradation by this strain. The specific roles of bphA, bphB, and bphC were identified by systematically expressing these genes in Escherichia coli. The dihydrodiol product of BphA was dehydrogenated into 2,3-dihydroxydiphenyl ether by BphB. 2,3-Dihydroxydiphenyl ether was then decomposed into phenol and 2-pyrone-6-carboxylic acid by BphC. Thus, BphA, BphB, and BphC act sequentially in the aerobic degradation of DE, 4-bromodiphenyl ether, and 4,4'-dibromodiphenyl ether by the Cupriavidus sp. WS bacterium.

Hierarchy of Carbon Source Utilization in Soil Bacteria: Hegemonic Preference for Benzoate in Complex Aromatic Compound Mixtures Degraded by Cupriavidus pinatubonensis Strain JMP134.

Cupriavidus pinatubonensis JMP134, like many other environmental bacteria, uses a range of aromatic compounds as carbon sources. Previous reports have shown a preference for benzoate when this bacterium grows on binary mixtures composed of this aromatic compound and 4-hydroxybenzoate or phenol. However, this observation has not been extended to other aromatic mixtures resembling a more archetypal context. We carried out a systematic study on the substrate preference of C. pinatubonensis JMP134 growing on representative aromatic compounds channeled through different catabolic pathways described in aerobic bacteria. Growth tests of nearly the entire set of binary combinations and in mixtures composed of 5 or 6 aromatic components showed that benzoate and phenol were always the preferred and deferred growth substrates, respectively. This pattern was supported by kinetic analyses that showed shorter times to initiate consumption of benzoate in aromatic compound mixtures. Gene expression analysis by real-time reverse transcription-PCR (RT-PCR) showed that, in all mixtures, the repression by benzoate over other catabolic pathways was exerted mainly at the transcriptional level. Additionally, inhibition of benzoate catabolism suggests that its multiple repressive actions are not mediated by a sole mechanism, as suggested by dissimilar requirements of benzoate degradation for effective repression in different aromatic compound mixtures. The hegemonic preference for benzoate over multiple aromatic carbon sources is not explained on the basis of growth rate and/or biomass yield on each single substrate or by obvious chemical or metabolic properties of these aromatic compounds.

Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil.

The newly developed droplet digital PCR (DD-PCR) has shown promise as a DNA quantification technology in medical diagnostic fields. This study evaluated the applicability of DD-PCR as a quantitative tool for soil DNA using quantitative real-time PCR (qRT-PCR) as a reference technology. Cupriavidus sp. MBT14 and Sphingopyxis sp. MD2 were used, and a primer/TaqMan probe set was designed for each (CupMBT and SphMD2, respectively). Standard curve analyses on tenfold dilution series showed that both qRT-PCR and DD-PCR exhibited excellent linearity (R (2) = 1.00) and PCR efficiency (≥92 %) across their detectable ranges. However, DD-PCR showed a tenfold greater sensitivity than qRT-PCR. MBT14 and MD2 were added to non-sterile soil at 0 ~ 5 × 10(8) and 0 ~ 5 × 10(7) cells per gram of soil, respectively (n = 5). This bacterial load test indicated that DD-PCR was more sensitive and discriminating than qRT-PCR. For instance, DD-PCR showed a gradual DNA increase from 14 to 141,160 MBT14 rDNA copies µL DNA extract(-1) as the bacterial load increased, while qRT-PCR could quantify the DNA (6,432 copies µL DNA(-1)) at ≥5 × 10(5) MBT14 per gram of soil. When temporal DNA changes were monitored for 3 weeks in the amended soils, the two technologies exhibited nearly identical changes over time. Linearity tests (y = a · x) revealed excellent quantitative agreement between the two technologies (a = 0.98, R (2) = 0.97 in the CupMBT set and a = 0.90, R (2) = 0.94 in the SphMD2 set). These results suggest that DD-PCR is a promising tool to examine temporal dynamics of microorganisms in complex environments.

Cupriavidus metallidurans biomineralization ability and its application as a bioconsolidation enhancer for ornamental marble stone.

Bacterially induced calcium carbonate precipitation of a Cupriavidus metallidurans isolate was investigated to develop an environmentally friendly method for restoration and preservation of ornamental stones. Biomineralization performance was carried out in a growth medium via a Design of Experiments (DoE) approach using, as design factors, the temperature, growth medium concentration, and inoculum concentration. The optimum conditions were determined with the aid of consecutive experiments based on response surface methodology (RSM) and were successfully validated thereafter. Statistical analysis can be utilized as a tool for screening bacterial bioprecipitation as it considerably reduced the experimental time and effort needed for bacterial evaluation. Analytical methods provided an insight to the biomineral characteristics, and sonication tests proved that our isolate could create a solid new layer of vaterite on marble substrate withstanding sonication forces. C. metallidurans ACA-DC 4073 provided a compact vaterite layer on the marble substrate with morphological characteristics that assisted in its differentiation. The latter proved valuable during spraying minimum amount of inoculated media on marble substrate under conditions close to an in situ application. A sufficient and clearly distinguishable layer was identified.

Poly-ß-hydroxybutyrate content and dose of the bacterial carrier for Artemia enrichment determine the performance of giant freshwater prawn larvae.

The beneficial effects of poly-ß-hydroxybutyrate (PHB) for aquaculture animals have been shown in several studies. The strategy of applying PHB contained in a bacterial carrier has, however, hardly been considered. The effect of administering PHB-accumulated Alcaligenes eutrophus H16 containing 10 or 80 % PHB on dry weight, named A10 and A80, respectively, through the live feed Artemia was investigated on the culture performance of larvae of the giant freshwater prawn (Macrobrachium rosenbergii). Feeding larvae with Artemia nauplii enriched in a medium containing 100 and 1,000 mg L(-1) A80 significantly increased the survival with about 15 % and the development of the larvae with a larval stage index of about 1 as compared to feeding non-enriched Artemia. The survival of the larvae also significantly increased with about 35 % in case of a challenge with Vibrio harveyi. The efficiency of these treatments was equal to a control treatment of Artemia enriched in an 800 mg L(-1) PHB powder suspension, while Artemia enriched in 10 mg L(-1) A80, 100 mg L(-1) A10, and 1,000 mg L(-1) A10 did not bring similar effects. From our results, it can be concluded that PHB supplemented in a bacterial carrier (i.e., amorphous PHB) can increase the larviculture efficiency of giant freshwater prawn similar to supplementation of PHB in powdered form (i.e., crystalline PHB). When the level of PHB in the bacterial carrier is high, similar beneficial effects can be achieved as crystalline PHB, but at a lower live food enrichment concentration expressed on PHB basis.

Changes in the proteome of the cadmium-tolerant bacteria Cupriavidus taiwanensis KKU2500-3 in response to cadmium toxicity.

Cupriavidus taiwanensis KKU2500-3 is a cadmium (Cd)-tolerant bacterial strain that was previously isolated from rice fields contaminated with high levels of Cd. In 500 µmol/L CdCl2, the KKU2500-3 strain grew slower and with a more prolonged lag-phase than when grown in the absence of Cd. A proteomic approach was used to characterize the protein expression in the Cd-tolerant bacteria C. taiwanensis KKU2500-3 during growth under Cd stress. When compared with the untreated cells, a total of 982 differentially expressed protein spots were observed in the CdCl2-treated cells, and 59 and 10 spots exhibited >2- and >4-fold changes, respectively. The level of up- and downregulation varied from 2.01- to 11.26-fold and from 2.01- to 5.34-fold, respectively. Of the 33 differentially expressed protein spots analyzed by MALDI TOF MS/MS, 19 spots were successfully identified, many of which were involved in stress responses. The most highly upregulated protein (+7.95-fold) identified was the chaperone GroEL, which indicated that this factor likely contributed to the bacterial survival and growth in response to Cd toxicity. Detection of the downregulated protein flagellin (-3.52-fold) was consistent with the less effective ATP-mediated and flagella-driven motility. The flagella-losing cells were also observed in the Cd-treated bacteria when analyzed by scanning electron microscopy. Thus, the Cd-stressed cells may downregulate pathways involving ATP utilization in favor of other mechanisms in response to Cd toxicity. When the KKU2500-3 strain was grown in the presence of Cd, H2S was not detected, suggesting a possible role of the sulfur in precipitation with Cd. Apart from a general response, no specific process could be determined using the present proteomic approach. However, the potential role of protein folding-mediated GroEL, flagella-mediated motility and CdS biotransformation in Cd toxicity response observed in this study as well as the extent of Cd-tolerant mechanisms using other methods could facilitate the future application of this strain in addressing Cd environmental contamination.

A glucose-utilizing strain, Cupriavidus euthrophus B-10646: growth kinetics, characterization and synthesis of multicomponent PHAs.

This study investigates kinetic and production parameters of a glucose-utilizing bacterial strain, C. eutrophus B-10646, and its ability to synthesize PHA terpolymers. Optimization of a number of parameters of bacterial culture (cell concentration in the inoculum, physiological activity of the inoculum, determined by the initial intracellular polymer content, and glucose concentration in the culture medium during cultivation) provided cell concentrations and PHA yields reaching 110 g/L and 80%, respectively, under two-stage batch culture conditions. Addition of precursor substrates (valerate, hexanoate, propionate, γ-butyrolactone) to the culture medium enabled synthesis of PHA terpolymers, P(3HB/3HV/4HB) and P(3HB/3HV/3HHx), with different composition and different molar fractions of 3HB, 3HV, 4HB, and 3HHx. Different types of PHA terpolymers synthesized by C. eutrophus B-10646 were used to prepare films, whose physicochemical and physical-mechanical properties were investigated. The properties of PHA terpolymers were significantly different from those of the P3HB homopolymer: they had much lower degrees of crystallinity and lower melting points and thermal decomposition temperatures, with the difference between these temperatures remaining practically unchanged. Films prepared from all PHA terpolymers had higher mechanical strength and elasticity than P3HB films. In spite of dissimilar surface structures, all films prepared from PHA terpolymers facilitated attachment and proliferation of mouse fibroblast NIH 3T3 cells more effectively than polystyrene and the highly crystalline P3HB.