Metabolic network and recovery mechanism of Escherichia coli associated with triclocarban stress.

Although the toxicity of triclocarban at molecular level has been investigated, the metabolic networks involved in regulating the stress processes are not clear. Whether the cells would maintain specific phenotypic characteristics after triclocarban stress is also needed to be clarified. In this study, Escherichia coli was selected as a model to elucidate the cellular metabolism response associated with triclocarban stress and the recovery metabolic network of the triclocarban-treated cells using the proteomics and metabolomics approaches. Results showed that triclocarban caused systematic metabolic remodeling. The adaptive pathways, glyoxylate shunt and acetate-switch were activated. These arrangements allowed cells to use more acetyl-CoA and to reduce carbon atom loss. The upregulation of NH3-dependent NAD+ synthetase complemented the NAD+ consumption by catabolism, maintaining the redox balance. The synthesis of 1-deoxy-D-xylulose-5-phosphate was suppressed, which would affect the accumulation of end products of its downstream pathway of isoprenoid synthesis. After recovery culture for 12 h, the state of cells returned to stability and the main impacts on metabolic network triggered by triclocarban have disappeared. However, drug resistance caused by long-term exposure to environmentally relevant concentration of triclocarban is still worthy of attention. The present study revealed the molecular events under triclocarban stress and clarified how triclocarban influence the metabolic networks.

Cellular metabolism network of Bacillus thuringiensis related to erythromycin stress and degradation.

Erythromycin is one of the most widely used macrolide antibiotics. To present a system-level understanding of erythromycin stress and degradation, proteome, phospholipids and membrane potentials were investigated after the erythromycin degradation. Bacillus thuringiensis could effectively remove 77% and degrade 53% of 1 µM erythromycin within 24 h. The 36 up-regulated and 22 down-regulated proteins were mainly involved in spore germination, chaperone and nucleic acid binding. Up-regulated ribose-phosphate pyrophosphokinase and ribosomal proteins confirmed that the synthesis of protein, DNA and RNA were enhanced after the erythromycin degradation. The reaction network of glycolysis/gluconeogenesis was activated, whereas, the activity of spore germination was decreased. The increased synthesis of phospholipids, especially, palmitoleic acid and oleic acid, altered the membrane permeability for erythromycin transport. Ribose-phosphate pyrophosphokinase and palmitoleic acid could be biomarkers to reflect erythromycin exposure. Lipids, disease, pyruvate metabolism and citrate cycle in human cells could be the target pathways influenced by erythromycin. The findings presented novel insights to the interaction among erythromycin stress, protein interaction and metabolism network, and provided a useful protocol for investigating cellular metabolism responses under pollutant stress.

Metabolic network and proteomic expression perturbed by cyclosporine A to model microbe Escherichia coli.

Cyclosporine A (CsA) is a model drug that has caused great concern due to its widespread use and abuse in the environment. However, the potential harm of CsA to organisms also remains largely unknown, and this issue is exceptionally important for the health risk assessment of antibiotics. To address this concern, the crosstalk between CsA stress and cellular metabolism at the proteomic level in Escherichia coli was investigated and dissected in this study. The results showed that CsA inhibited E. coli growth in a time-dependent manner. CsA induced reactive oxygen species (ROS) overproduction in a dose- and time-dependent manner, leading to membrane depolarization followed by cell apoptosis. In addition, translation, the citric acid cycle, amino acid biosynthesis, glycolysis and responses to oxidative stress and heat were the central metabolic pathways induced by CsA stress. The upregulated proteins, including PotD, PotF and PotG, controlled cell growth. The downregulated proteins, including SspA, SspB, CstA and DpS, were regulators of self-feedback during the starvation process. And the up- and downregulated proteins, including AtpD, Adk, GroS, GroL and DnaK, controlled energy production. These results provide an important reference for the environmental health risk assessment of CsA.

The synergy of Fe(III) and NO2- drives the anaerobic oxidation of methane.

The anaerobic oxidation of methane (AOM) driven by NO2- or Fe(III) alone was limited by slow electron delivery and ineffective enrichment of microbes. The flexible coupling between Fe(III) and NO2- potentially cooperated to accelerate AOM. One negative control was fed CH4 and NO2-, and four treatment reactors were supplemented with CH4, NO2- and ferric citrate (FC)/ferric chloride (FCH)/ chelate iron (FCI)/ferric hydroxide (FH) and were anaerobically operated for 1200 days to verify the synergy and promicrobial roles of Fe(III) and NO2- in improving AOM. The changes in gas and ion profiles were observed in the reactors, and microbial development was studied using 16S rRNA gene sequencing with the Illumina platform. The results indicated that the combined Fe(III) and NO2- treatment improved AOM, and their synergy followed the order of FC > FCI > FCH > FH. The biochemical reaction of Fe3+ with NO2- and its secondary process accelerated electron transfer to microbial cells and subsequently enhanced AOM in the reactors. The total organic carbon (TOC) content, NH4+ content, NO3- content, and pH value altered the dominant bacteria the most in the FC reactor, FCI, FCH, and FH groups, respectively. Several dominant bacterial species were enriched, whereas only two archaea were highly concentrated in the FC and FCI groups. Only bacteria were detected in the FCH group, and archaea contributed substantially to the FH group. These findings contribute to an improved understanding of the interactions among nitrogen, iron and CH4 that are paramount to accelerating the process of AOM for engineering applications.

The molecular effects of ultrasound on the expression of cellular proteome.

High frequency and low intensity, diagnostic ultrasound methods are recognized to be safe in epidemiology and pathology but the bioeffects of these methods on molecular and proteomic levels are unknown. As a representative organism that can directly reflect the molecular response to stresses, Escherichia coli was selected for exposure to ultrasound probes C1-5, M5s and 9 L for 10 min and 20 min. ITRAQ was used to measure the expression of the cellular proteome. The results showed that both the frequency and time of exposure to ultrasound affected the proteome expression. Fifty biological processes were affected and nineteen metabolic processes, including carbohydrate metabolism, asparagine metabolism and phosphate import were differentially regulated. Lower frequency ultrasound caused copper export and iron­sulfur cluster biosynthesis upregulation. Nine proteins (GlpD, AsnB, TdcB, CopA, IscR, IscU, IscS, IscA, RecA) were key for the adaption to ultrasound. Accordingly, the results of the potential risks based on the calculation of the orthologous genome clarified that relevant pathways and potentially sensitive individuals were worthy of further study. These findings offer insights into reveal the bioeffects of ultrasound at the metabolic network and proteomic levels.

Bisphenol A degradation pathway and associated metabolic networks in Escherichia coli harboring the gene encoding CYP450.

Although bisphenol A (BPA) can be transformed by CYP450, the metabolic networks involved in regulating the transformation processes are not clear. In this study, Escherichia coli harboring the gene encoding CYP450 was used as a model to elucidate the BPA degradation pathway and the associated metabolic network using a proteomic approach. The results showed that CYP450 promotes the transformation of BPA, generating 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol, with hydroquinone and 4-(2-hydroxypropan-2-yl)phenol formed in another pathway. The DNA adducts formed by 1,4-benzoquinone were reduced, and CYP450 played a positive role in cellular homeostasis by promoting the transformation of BPA and mismatch repair. An increase in the synthesis of cell membrane lipids was observed after dislodging BPA. BPA disturbed folate metabolism by decreasing the abundance of dihydrofolate reductase, which inhibited microbial metabolism in the absence of CYP450. The findings of this study revealed the molecular mechanism associated with the metabolic network responsible for pollutant tolerance and degradation.

Molecular response mechanism in Escherichia coli under hexabromocyclododecane stress.

The effects of hexabromocyclododecane (HBCD) on the relationship between physiological responses and metabolic networks remains unclear. To this end, cellular growth, apoptosis, reactive oxygen species, exometabolites and the proteome of Escherichia coli were investigated following exposure to 0.1 and 1 µM HBCD. The results showed that although there were no significant changes in the pH value, apoptosis and reactive oxygen species under HBCD stress, cell growth was inhibited. The metabolic network formed by glycolysis, oxidative phosphorylation, amino acids biosynthesis, membrane proteins biosynthesis, ABC transporters, glycogen storage, cell recognition, compound transport and nucleotide excision repair was disrupted. Cell chemotaxis and DNA damage repair were the effective approaches to alleviate HBCD stress. This work improves our understanding of HBCD toxicity and provides insight into the toxicological mechanism of HBCD at the molecular and network levels.

Global proteomic responses of Escherichia coli and evolution of biomarkers under tetracycline stress at acid and alkaline conditions.

The global proteomic regulation and the mechanism of biomolecule evolution in acid and alkaline ecosystems triggered by tetracycline, a representative of antibiotics, are not clear. To reveal the related mechanisms, the global responses of Escherichia (E.) coli to tetracycline in acid and alkaline conditions were analyzed using a proteomic approach. The specific phospholipid C16:1ω9c showed a significant decrease between the treatment and control groups. The 77 and 111 upregulated proteins in E. coli in acid and alkaline groups were mainly involved in carbohydrate transport and metabolism and energy metabolism, whereas, the 78 downregulated proteins were related to ribosome and bacterial chemotaxis in the acid group. The 110 downregulated proteins involved in carbon, glycine, serine, threonine, glyoxylate, and dicarboxylate metabolism, biosynthesis of antibiotics, fatty acids, and secondary metabolites in the alkaline group. Protein sequence analysis showed that the respective distribution of phosphorylation, glycosylation, and methylation sites among stable-expressed, upregulated, and downregulated proteins all showed a significant difference. TolC and phosphoenolpyruvate carboxykinase (Pck) in E. coli could be biomarkers to reflect tetracycline stress under extreme conditions with high sequence homology in Homo sapiens, implying the potential impact of tetracycline on humans at the network level. Generally, E. coli in the acid group accelerated the highly efficient protection mechanism to defend against tetracycline stress, while E. coli in the alkaline group strongly impaired the protection mechanism. These findings provide important clues to reveal the microbial antibiotic resistance mechanism in E. coli under extreme conditions and perfect the antibiotic usage.

Toxicity of lanthanum oxide nanoparticles to the fungus Moniliella wahieum Y12T isolated from biodiesel.

Moniliella wahieum Y12T, isolated from biodiesel was used as a model organism to assess the use of lanthanum oxide (La2O3) (60-80 nm) and silver oxide (AgO) (10-40 nm) nanoparticles as potential fungal inhibitors. This is the first study to investigate the use of nanoscale La2O3 as a eukaryotic bio-inhibitor. The AgO nanoparticles were relatively effective at inhibiting the growth of M. wahieum Y12T. The half maximal effective concentration (EC50) for AgO was 0.012 mg/mL as compared with 4.63 mg/mL of La2O3. Fluorescein diacetate analysis showed that AgO nanoparticles significantly reduced metabolic activity in M. wahieum Y12T. The results of this study indicated that AgO nanoparticles can be a nonspecific inhibitor for the treatment of M. wahieum Y12T, a eukaryotic biodiesel contaminant.

Proteome and phospholipid alteration reveal metabolic network of Bacillus thuringiensis under triclosan stress.

Triclosan is a common antibacterial agent widely applied in various household and personal care products. The molecule, cell, organ and organism-level understanding of its toxicity pose to some target organisms has been investigated, whereas, the alteration of a single metabolic reaction, gene or protein cannot reflect the impact of triclosan on metabolic network. To clarify the interaction between triclosan stress and metabolism at network and system levels, phospholipid synthesis, and cellular proteome and metabolism of Bacillus thuringiensis under 1µM of triclosan stress were investigated through omics approaches. The results showed that C14:0, C16:1ω7, C16:0 and C18:2ω6 were significantly up-produced, and 19 proteins were differentially expressed. Whereas, energy supply, protein repair and the synthesis of DNA, RNA and protein were down-regulated. PyrH and Eno could be biomarkers to reflect triclosan stress. At network level, the target proteins ACOX1, AHR, CAR, CYP1A, CYP1B1, DNMT1, ENO, HSP60, HSP70, SLC5A5, TPO and UGT expressed in different species shared high sequence homology with the same function proteins found in Homo sapiens not only validated their role as biomarkers but also implied the potential impact of triclosan on the metabolic pathways and network of humans. These findings provided novel insights into the metabolic influence of triclosan at network levels, and developed an omics approach to evaluate the safety of target compound.

Metabolic and proteomic mechanism of bisphenol A degradation by Bacillus thuringiensis.

Bisphenol A (BPA) is a worldwide, widespread pollutant with estrogen mimicking and hormone-like properties. To date, some target biomolecules associated with BPA toxicity have been confirmed. The limited information has not clarified the related metabolism at the pathway and network levels. To this end, metabolic and proteomic approaches were performed to reveal the synthesis of phospholipids and proteins and the metabolic network during the BPA degradation process. The results showed that the degradation efficiency of 1 µM of BPA by 1 g L-1 of Bacillus thuringiensis was up to 85% after 24 h. During this process, BPA significantly changed the membrane permeability; altered sporulation, amino acid and protein expression, and carbon, purine, pyrimidine and fatty acid metabolism; enhanced C14:0, C16:1ω7, C18:2ω6, C18:1ω9t and C18:0 synthesis; and increased the trans/cis ratio of C18:1ω9t/C18:1ω9c. It also depressed the spore DNA stability of B. thuringiensis. Among the 14 upregulated and 7 down-regulated proteins, SasP-1 could be a biomarker to reflect BPA-triggered spore DNA impairment. TpiA, RpoA, GlnA and InfA could be phosphorylated at the active sites of serine and tyrosine. The findings presented novel insights into the interaction among BPA stress, BPA degradation, phospholipid synthesis and protein expression at the network and phylogenetic levels.

[Effect of PFOA on Oxidative Stress and Membrane Damage of Escherichia coli].

Perfluorooctanoic acid (PFOA) is widely used in industrial production because of its strong chemical stabilities and good hydrophobic and oleophobic properties. It was considered to be a widespread persistent organic pollutant in environment in recent years. The oxidative stress and membrane damage of Escherichia coli exposed to PFOA were measured by flow cytometry (FCM) and the toxic mechanism of PFOA was also preliminarily explored. The results showed that, under the stress of PFOA, the intracellular reactive oxygen species (ROS) content of E. coli increased, the unsaturation degree of fatty acid decreased, the malondialdehyde (MDA) content increased, the membrane permeability increased, the membrane potential decreased, and the activities of Na+K+-ATPase and Ca2+Mg2+-ATPase showed a compensatory increase first and then decreased. Therefore, owing to the stress of PFOA, the higher intracellular ROS in E. coli reacted with membrane unsaturated fatty acids by peroxidation,and then reduced cell membrane fatty acid saturation, accumulated MDA in cells, and further caused damage to cell membrane, reduced the ATPase activity, and eventually resulted in inactivation or apoptosis of E. coli. This study provided more evidence for the further study on environmental ecological toxicology of PFOA.

Triphenyltin recognition by primary structures of effector proteins and the protein network of Bacillus thuringiensis during the triphenyltin degradation process.

Herein, triphenyltin (TPT) biodegradation efficiency and its transformation pathway have been elucidated. To better understand the molecular mechanism of TPT degradation, the interactions between amino acids, primary structures, and quaternary conformations of effector proteins and TPT were studied. The results verified that TPT recognition and binding depended on amino acid sequences but not on secondary, tertiary or quaternary protein structure. During this process, TPT could change the molecular weight and isoelectric point of effector proteins, induce their methylation or demethylation, and alter their conformation. The effector proteins, alkyl hydroperoxide reductase and acetyl-CoA acetyltransferase, recognizing TPT were crucial to TPT degradation. Electron transfer flavoprotein subunit alpha, phosphoenolpyruvate carboxykinase, aconitate hydratase, branched-chain alpha-keto acid dehydrogenase E1 component, biotin carboxylase and superoxide dismutase were related to energy and carbon metabolism, which was consistent with the results in vivo. The current findings develop a new approach for investigating the interactions between proteins and target compounds.

Influence of perfluorooctanoic acid on proteomic expression and cell membrane fatty acid of Escherichia coli.

Perfluorooctanoic acid (PFOA) has received an increasing attention in the agricultural and food industries due to its risk to human health. To facilitate the development of novel biomarkers of Escherichia coli against PFOA through multi-omics technologies, and to reveal the resistance mechanism of E. coli against PFOA at protein levels, the interactions among pollutant stress, protein expression and cell metabolism was investigated by using iTRAQ-based quantitative proteomic analysis. The results revealed that the 63 up-regulated proteins mainly involved in tricarboxylic acid cycle, glyoxylate and dicarboxylate metabolism and fatty acid biosynthesis, whereas, the 69 down-regulated proteins related to oxidative phosphorylation, pyruvate metabolism and the cell cycle-caulobacter pathway, were also associated with the increase of membrane permeability, excessive expenditure of ATP, disruption of fatty acid biosynthesis under PFOA stress. The results provide novel insights into the influence mechanisms of PFOA on fatty acid and protein networks.

Interactions among triphenyltin degradation, phospholipid synthesis and membrane characteristics of Bacillus thuringiensis in the presence of d-malic acid.

Degradation pathway and surface biosorption of triphenyltin (TPT) by effective microbes have been investigated in the past. However, unclear interactions among membrane components and TPT binding and transport are still obstacles to understanding TPT biotransformation. To reveal the mechanism involved, the phospholipid expression, membrane potential, cellular mechanism and molecular dynamics between TPT and fatty acids (FAs) during the TPT degradation process in the presence of d-malic acid (DMA) were studied. The results show that the degradation efficiency of 1 mg L-1 TPT by Bacillus thuringiensis (1 g L-1) with 0.5 or 1 mg L-1 DMA reached values up to approximately 90% due to the promotion of element metabolism and cellular activity, and the depression of FA synthesis induced by DMA. The addition of DMA caused conversion of more linoleic acid into 10-oxo-12(Z)-octadecenoic acid, increased the membrane permeability, and alleviated the decrease in membrane potential, resulting in TPT transport and degradation. Fluorescence analysis reveals that the endospore of B. thuringiensis could act as an indicator for membrane potential and cellular activities. The current findings are advantageous for acceleration of biosorption, transport and removal of pollutants from natural environments.

Complete degradation of the endocrine disruptor di-(2-ethylhexyl) phthalate by a novel Agromyces sp. MT-O strain and its application to bioremediation of contaminated soil.

A newly isolated strain Agromyces sp. MT-O could utilize various phthalates and efficiently degraded di-(2-ethylhexyl) phthalate (DEHP). Response surface methodology was successfully employed for the optimization of culture conditions including pH (7.2), temperature (29.6), and inoculum size (OD600 of 0.2), resulting in almost complete degradation of DEHP (200mgL(-1)) within 7days. At different initial concentrations (50-1000mgL(-1)), DEHP degradation curves were fitted well with the first-order kinetic model, and the half-life of DEHP degradation ranged from 0.83 to 2.92days. Meanwhile, the substrate inhibition model was used to describe the special degradation rate with qmax, Ks, and Ki of 0.6298day(-1), 86.78mgL(-1), and 714.3mgL(-1), respectively. The GC-MS analysis indicated that DEHP was degraded into mono-ethylhexyl phthalate and phthalate acid before its complete mineralization. Bioaugmentation of DEHP-contaminated soils with strain MT-O has greatly enhanced DEHP disappearance rate in soils, providing great potential for efficiently remediating DEHP-contaminated environment.

[Characteristics and functional protein analysis of an effective decabromodiphenyl ether-degrading strain].

An effective decabromodiphenyl ether (BDE-209) degrading strain was isolated and identified as Enterococcus casseliflavus based on the 16S rRNA gene sequence analysis. The optimal conditions for strain growth were pH 7 and culture time of 48 h, respectively. E. casseliflavus has a good ability to degrade BDE-209. The biodegradation rate of 1 mg.L-1 BDE-209 by 1 g.L-1 E. casseliflavus reached the highest of 56. 7% after 4 days degradation with 5 mg.L-1 glucose as the additional carbon source. During the degradation process of BDE-209, SDS-PAGE demonstrated that some new extracellular proteins were induced under 2 mg.L-1 and 5 mg.L-1 BDE-209. As for the intracellular proteins, the quantity of protein expression varied, and some proteins even disappeared compared with the blank control. Two-dimensional electrophoresis steps for protein analysis detected 31 different protein points, demonstrating that during the degradation process, the conformation of some proteins which were related with degradation was changed, and resulted in the variation of type and content of the proteins.

Biodegradation of anthracene by Aspergillus fumigatus.

An anthracene-degrading strain, identified as Aspergillus fumigatus, showed a favorable ability in degradation of anthracene. The degradation efficiency could be maintained at about 60% after 5d with initial pH of the medium kept between 5 and 7.5, and the optimal temperature of 30 °C. The activity of this strain was not affected significantly by high salinity. Exploration on co-metabolism showed that the highest degradation efficiency was reached at equal concentration of lactose and anthracene. Excessive carbon source would actually hamper the degradation efficiency. Meanwhile, the strain could utilize some aromatic hydrocarbons such as benzene, toluene, phenol etc. as sole source of carbon and energy, indicating its degradation diversity. Experiments on enzymatic degradation indicated that extracellular enzymes secreted by A. fumigatus could metabolize anthracene effectively, in which the lignin peroxidase may be the most important constituent. Analysis of ion chromatography showed that the release of anions of A. fumigatus was not affected by addition of anthracene. GC-MS analysis revealed that the molecular structure of anthracene changed with the action of the microbe, generating a series of intermediate compounds such as phthalic anhydride, anthrone and anthraquinone by ring-cleavage reactions.

[Effect of heavy metal ions on triphenyltin enzymatic degradation].

The influence of different metal ions and different forms of addition on triphenyltin enzymatic degradation was investigated under conditions using enzyme obtained from Klebsiella pneumoniae. The objective of this study is to illuminate the mechanism of enzymatic degradation of triphenyltin (TPhT). The results demonstrated that the strain was able to tolerate K+, Mg2+, CU2+, Ca2+ and Fe3+ at high concentrations. High concentrations of Zn2+ and Fe2+ had some toxic effects on the strain, thus affecting its growth. The endoenzyme activity was enhanced by metal ions such as K+, Mg2+, Zn2+, Cu2+ and Fe2+ at certain concentrations. In the presence of 30 mg/L of Mg2+, the removal percentage of TPhT was up to 77.22%. Fe3+ restrained the enzyme activity at certain concentrations. Adding K+, Mg2+, Zn2+, Cu2+ into medium can promote the production of enzyme, among which Mg2+ demonstrated up to 85.66% of removal percentage of TPhT, suggesting some metal ions at the appropriate concentration range can be used as enzyme activator for the enzymatic degradation of triphenyltin. Metal ions showed no relevant impact on the cell growth and enzyme production. Certain metal ions can only serve as activators of endoenzyme and exhibited no similar effect towards exoenzyme.

[Cation exchanges during the process of Cd(2+) absorption by Alfalfa in aqueous solutions].

A hydroponic experiment was conducted to investigate the cation exchanges during the process of Cd2+ absorption by Alfalfa in aqueous solution. The absorption efficiency of Alfalfa plants with 0-10 mg x L(-1) Cd2+ treatments, changes of Na+, K+, Mg2+, Ca2+ and NH4(+) concentration, and the variation of pH values at different absorption time (0, 1, 2, 4, 8, 12, 24 and 72 h) were studied separately. The multiple linear regressions between Cd2+ absorption and cation variation were analyzed. The results indicated that when Cd2+ concentrations were 0.1, 1, 5, 10 mg x L(-1), the absorption efficiencies of Cd2+ by Alfalfa after 72 h were 85.86%, 52.14%, 15.97% and 7.81%. Cation exchange was involved in the removal of Cd2+ by Alfalfa in aqueous solution. Except for NH4(+), the concentrations of cationic metals Na+, K+, Mg2+ and Ca2+ in aqueous solution increased over time, which increased 11.30% - 61.72%, 21.44% - 98.73%, 24.09% - 8.90% and 37.04% - 191.96%, respectively. Kinetic studies illuminated that the release of Na+, K+, Mg2+ and Ca2+ by Alfalfa in Cd2+ solution with initial concentrations of 0, 0. 1, 1, 5, 10 mg x L(-1) best fitted pseudo-second-order equation,while the NH4(+) release fitted this model when Cd2+ concentrations were 1, 5, 10 mg x L(-1). The gradual decrease of pH during adsorption of Cd2+ by Alfalfa was observed. As the competition ion of Cd2+, H+ might affect the capacity of Alfalfa root system to absorb Cd2+. The ternary linear equation results demonstrated that the content of Cd2+ absorption by Alfalfa strongly related with the release of Ca2+, Mg2+, Na+. And this exchange mainly occurred among Cd2+ and divalent cations.