Rewiring the respiratory pathway of Lactococcus lactis to enhance extracellular electron transfer.

Lactococcus lactis, a lactic acid bacterium with a typical fermentative metabolism, can also use oxygen as an extracellular electron acceptor. Here we demonstrate, for the first time, that L. lactis blocked in NAD+ regeneration can use the alternative electron acceptor ferricyanide to support growth. By electrochemical analysis and characterization of strains carrying mutations in the respiratory chain, we pinpoint the essential role of the NADH dehydrogenase and 2-amino-3-carboxy-1,4-naphtoquinone in extracellular electron transfer (EET) and uncover the underlying pathway systematically. Ferricyanide respiration has unexpected effects on L. lactis, e.g., we find that morphology is altered from the normal coccoid to a more rod shaped appearance, and that acid resistance is increased. Using adaptive laboratory evolution (ALE), we successfully enhance the capacity for EET. Whole-genome sequencing reveals the underlying reason for the observed enhanced EET capacity to be a late-stage blocking of menaquinone biosynthesis. The perspectives of the study are numerous, especially within food fermentation and microbiome engineering, where EET can help relieve oxidative stress, promote growth of oxygen sensitive microorganisms and play critical roles in shaping microbial communities.

Fast fabrication of reusable polyethersulfone microbial biosensors through biocompatible phase separation.

In biosensors fabrication, entrapment in polymeric matrices allows efficient immobilization of the biorecognition elements without compromising their structure and activity. When considering living cells, the biocompatibility of both the matrix and the polymerization procedure are additional critical factors. Bio-polymeric gels (e.g. alginate) are biocompatible and polymerize under mild conditions, but they have poor stability. Most synthetic polymers (e.g. PVA), on the other hand, present improved stability at the expense of complex protocols involving chemical/physical treatments that decrease their biological compatibility. In an attempt to explore new solutions to this problem we have developed a procedure for the immobilization of bacterial cells in polyethersulfone (PES) using phase separation. The technology has been tested successfully in the construction of a bacterial biosensor for toxicity assessment. Biosensors were coated with a 300  µm bacteria-containing PES membrane, using non-solvent induced phase separation (membrane thickness ≈ 300 µm). With this method, up to 2.3 × 106 cells were immobilized in the electrode surface with an entrapment efficiency of 8.2%, without compromising cell integrity or viability. Biosensing was performed electrochemically through ferricyanide respirometry, with metabolically-active entrapped bacteria reducing ferricyanide in the presence of glucose. PES biosensors showed good stability and reusability during dry frozen storage for up to 1 month. The analytical performance of the sensors was assessed carrying out a toxicity assay in which 3,5-dichlorophenol (DCP) was used as a model toxic compound. The biosensor provided a concentration-dependent response to DCP with half-maximal effective concentration (EC50) of 9.2 ppm, well in agreement with reported values. This entrapment methodology is susceptible of mass production and allows easy and repetitive production of robust and sensitive bacterial biosensors.

Screen-Printed Carbon Electrodes Modified with Graphene Oxide for the Design of a Reagent-Free NAD+-Dependent Biosensor Array.

A facile approach for the construction of reagent-free electrochemical dehydrogenase-based biosensors is presented. Enzymes and cofactors (NAD+ and Fe(CN)63-) were immobilized by modification of screen-printed carbon electrodes with graphene oxide (GO) and an additional layer of cellulose acetate. The sensor system was exemplarily optimized for an l-lactate electrode in terms of GO concentration, working potential, and pH value. The biosensor exhibited best characteristics at pH 7.5 in 100 mM potassium phosphate buffer at an applied potential of +0.250 V versus an internal pseudo Ag reference electrode. Thereby, sensor performance was characterized by a linear working range from 0.25 to 4 mM and a sensitivity of 0.14 µA mM-1. The detection principle was additionally evaluated with three other dehydrogenases (d-lactate dehydrogenase, alcohol dehydrogenase, and formate dehydrogenase, respectively). The developed reagentless biosensor array enabled simultaneous and cross-talk free determination of l-lactate, d-lactate, ethanol, and formate.

Determination of Polyhexamethylene Biguanide Utilizing a Glucose Oxidase Enzymatic Reaction.

Polyhexamethylene biguanide (PHMB) is a cationic disinfectant widely used for personal-care products and for sanitizers in swimming pools. This paper describes a promotion effect of PHMB on a glucose oxidase (GOx) enzymatic reaction with ferricyanide ion and its analytical application. The promotion effect arose from a polyion complex formation between polycationic PHMB and polyanionic GOx. The promoted GOx reaction was analyzed by Michaelis-Menten equation, and the Michalis constant and catalytic constant were estimated to be 240 µM and 31 s-1, respectively. Utilizing the promotion effect, PHMB was successfully determined in the range of 0.05 to 0.40 ppm, and the detection limit was calculated to be 0.027 ppm. The visual detection and semi-determination of PHMB with the same concentration level were also possible. As an application, the method was applied to the determination of PHMB in a contact lens detergent and its model solution.

Regionally Impaired Redox Homeostasis in the Brain of Rats Subjected to Global Perinatal Asphyxia: Sustained Effect up to 14 Postnatal Days.

The present report evaluates the effect of global perinatal asphyxia on several parameters of oxidative stress and cell viability in rat brain tissue sampled at an extended neonatal period up to 14 days, a period characterised by intensive neuritogenesis, synaptogenesis, synaptic consolidation, pruning and delayed cell death. Perinatal asphyxia was induced by immersing foetus-containing uterine horns removed by a caesarean section from on term rat dams into a water bath at 37 °C for 21 min. Asphyxia-exposed and sibling caesarean-delivered foetuses were manually resucitated and nurtured by surrogate dams for 1 to 14 postnatal (P) days. Brain samples (mesencephalon, telencephalon and hippocampus) were assayed for glutathione (reduced and oxidated levels; spectrophotometry), tissue reducing capacity (potassium ferricyanide reducing assay, FRAP), catalase (the key enzyme protecting against oxidative stress and reactive oxygen species, Western blots and ELISA) and cleaved caspase-3 (the key executioner of apoptosis, Western blots) levels. It was found that global PA produced a regionally specific and sustained increase in GSSG/GSH ratio, a regionally specific decrease in tissue reducing capacity and a regionally and time specific decrease of catalase activity and increase of cleaved caspase-3 levels. The present study provides evidence for regionally impaired redox homeostasis in the brain of rats subjected to global PA, an effect observed up to P14, mainly affecting mesencephalon and hippocampus, suggesting a sustained oxidative stress after the posthypoxia period. The oxidative stress observed postnatally can in part be associated to a respiratory apneic-like deficit, since there was a statistically significant decrease in respiration frequency in AS compared to CS neonates, also up to P14, together with the signs of a decreased peripheral blood perfusion (pink-blue skin colour in AS, compared to the pink colour observed in all CS neonates). It is proposed that PA implies a long-term metabolic insult, triggered by the length of hypoxia, the resuscitation/reoxigenation manoevres, but also by the developmental stage of the affected brain regions, and the integrity of cardiovascular and respiratory physiological functions, which are fundamental for warrantying a proper development.

Anodic electro-fermentation: Anaerobic production of L-Lysine by recombinant Corynebacterium glutamicum.

Microbial electrochemical technologies (MET) are promising to drive metabolic processes for the production of chemicals of interest. They provide microorganisms with an electrode as an electron sink or an electron source to stabilize their redox and/or energy state. Here, we applied an anode as additional electron sink to enhance the anoxic metabolism of the industrial bacterium Corynebacterium glutamicum through an anodic electro-fermentation. In using ferricyanide as extracellular electron carrier, anaerobic growth was enabled and the feedback-deregulated mutant Corynebacterium glutamicum lysC further accumulated L-lysine. Under such oxidizing conditions we achieved L-lysine titers of 2.9 mM at rates of 0.2 mmol/L/hr. That titer is comparable to recently reported L-lysine concentrations achieved by anaerobic production under reductive conditions (cathodic electro-fermentation). However unlike other studies, our oxidative conditions allowed anaerobic cell growth, indicating an improved cellular energy supply during anodic electro-fermentation. In that light, we propose anodic electro-fermentation as the right choice to support C. glutamicum stabilizing its redox and energy state and empower a stable anaerobic production of L-lysine.

Electrochemical System for the Study of Trans-Plasma Membrane Electron Transport in Whole Eukaryotic Cells.

The study of trans-plasma membrane electron transport (tPMET) in oncogenic systems is paramount to the further understanding of cancer biology. The current literature provides methodology to study these systems that hinges upon mitochondrial knockout genotypes in conjunction with cell surface oxygen consumption, or the detection of an electron acceptor using colorimetric methods. However, when using an iron redox based system to probe tPMET, there is yet to be a method that allows for the simultaneous quantification of iron redox states while providing an exceptional level of sensitivity. Developing a method to simultaneously analyze the redox state of a reporter molecule would give advantages in probing the underlying biology. Herein, we present an electrochemical based method that allows for the quantification of both ferricyanide and ferrocyanide redox states to a highly sensitive degree. We have applied this system to a novel application of assessing oncogenic cell-driven iron reduction and have shown that it can effectively quantitate and identify differences in iron reduction capability of three lung epithelial cell lines.

The FNR modules contribute to control nitric oxide synthase catalysis revealed by chimera enzymes.

The reductase domains of neuronal NOS, endothelial NOS and two constitutive nitric oxide synthase (cNOS) share higher sequence similarity (>60%). In order to evaluate the role of ferredoxin­NADP+ reductase (FNR) module in adjusting NOS catalytic activities, chimeras were by interchanging the FNR­like module between endothelial NOS and neuronal NOS in the present study. The assays of steady­state enzymatic activities for cytochrome c and ferricyanide reduction, NO synthesis and NADPH oxidation were performed spectrophotometrically. The two NOS FNR modules transferred their ferricyanide reductase character to the chimera enzymes. Results showed that the FNR module was important in adjusting electrons flow through the reductase domain and out of the FMN module. Results indicated that the FNR module was critical in controlling the electron transfer capacities of the FMN module.

3-Acetyl-Chlorophyll Formation in Light-Harvesting Complexes of Purple Bacteria by Chemical Oxidation.

Dedicated to the memory of Yuriy Nikolayevich Kozlov Oxidation of bacteriochlorophyll (BChl) with potassium ferricyanide in membranes and LH2 complexes (carotenoid-less and control samples) from the purple bacteria Allochromatium minutissimum and Rhodobacter sphaeroides as well as BChl photobleaching in a model system have been studied. The oxidation of BChl depended on the type of bacteria. BChl850 was rapidly oxidized in samples from Alc. minutissimum, and BChl800 and BChl850 were slowly oxidized in samples from Rb. sphaeroides. The carotenoids were not involved in protecting BChl from chemical oxidation in the light-harvesting complexes. The appearance of BChl oxidation product was registered in the absorption spectra (absorption maximum about 700 nm) and by HPLC analysis. The oxidized BChl was identified as 3-acetyl-chlorophyll. It differed from BChl by the presence of a double bond in pyrrole ring II at the 7-8 position. The extinction coefficient of 3-acetyl-chlorophyll was about 10 times less than that of BChl850 in the LH2 complex from Alc. minutissimum. In the BChl → 3-acetyl-chlorophyll transition, the binding constant of the latter with LH2 complex as compared with that of BChl did not change dramatically, as indicated by: (i) preserved electrophoretic mobility of the complex; (ii) the presence of 3-acetyl-chlorophyll in the complex after separation; (iii) the presence of a 3-acetyl-chlorophyll CD signal that was proportional to its absorption spectrum.

Possible evidence for contribution of arbuscular mycorrhizal fungi (AMF) in phytoremediation of iron-cyanide (Fe-CN) complexes.

Arbuscular mycorrhizal fungi (AMF) are integral functioning parts of plant root systems and are widely recognized for enhancing contaminants uptake and metabolism on severely disturbed sites. However, the patterns of their influence on the phytoremediation of iron-cyanide (Fe-CN) complexes are unknown. Fe-CN complexes are of great common interest, as iron is one of the most abundant element in soil and water. Effect of ryegrass (Lolium perenne L.) roots inoculation, using mycorrhizal fungi (Rhizophagus irregularis and a mixture of R. irregularis, Funneliformis mosseae, Rhizophagus aggregatus, and Claroideoglomus etunicatum), on iron-cyanide sorption was studied. Results indicated significantly higher colonization of R. irregularis than the mixture of AMF species on ryegrass roots. Series of batch experiments using potassium hexacyanoferrate (II) solutions, in varying concentrations revealed significantly higher reduction of total CN and free CN content in the mycorrhizal roots, indicating greater cyanide decrease in the treatment inoculated with R. irregularis. Our study is a first indication of the possible positive contribution of AM fungi on the phytoremediation of iron-cyanide complexes.

A high effective NADH-ferricyanide dehydrogenase coupled with laccase for NAD(+) regeneration.

OBJECTIVES: To find an efficient and cheap system for NAD(+) regeneration RESULTS: A NADH-ferricyanide dehydrogenase was obtained from an isolate of Escherichia coli. Optimal activity of the NADH dehydrogenase was at 45 °C and pH 7.5, with a K m value for NADH of 10 µM. By combining the NADH dehydrogenase, potassium ferricyanide and laccase, a bi-enzyme system for NAD(+) regeneration was established. The system is attractive in that the O2 consumed by laccase is from air and the sole byproduct of the reaction is water. During the reaction process, 10 mM NAD(+) was transformed from NADH in less than 2 h under the condition of 0.5 U NADH dehydrogenase, 0.5 U laccase, 0.1 mM potassium ferricyanide at pH 5.6, 30 °C CONCLUSION: The bi-enzyme system employed the NADH-ferricyanide dehydrogenase and laccase as catalysts, and potassium ferricyanide as redox mediator, is a promising alternative for NAD(+) regeneration.

Ubiquinol and plastoquinol triphenylphosphonium conjugates can carry electrons through phospholipid membranes.

Many mitochondria-targeted antioxidants (MTAs) that comprise a quinol moiety covalently attached through an aliphatic carbon chain to the lipophilic triphenylphosphonium cation are widely used for evaluating the role of mitochondria in pathological processes involving oxidative stress. The potency of MTAs to carry electrons across biological membranes and thereby mediate transmembrane redox processes was unknown. To assess this, we measured the rate of ferricyanide reduction inside liposomes by external ascorbate. Here, we show that MTAs containing ubiquinone (MitoQ series) or plastoquinone (SkQ series) can carry electrons through lipid membranes, with the rate being inversely proportional to the length of the hydrocarbon linker group. Furthermore, this process was stimulated by the hydrophobic anion tetraphenylborate suggesting that permeation of the cationic MTA through the membrane was the rate-limiting step of the process. This conclusion was supported by the observation that the rate of MTA-induced electron transfer was insensitive to nigericin, in contrast to electron transfer mediated by neutral quinone derivatives. These findings indicate that MTAs can be utilized to transfer electrons across lipid membranes and this may be applicable to the study of the electron-transport chain in mitochondria and other natural membranes exhibiting redox processes.

A Surface-Confined Proton-Driven DNA Pump Using a Dynamic 3D DNA Scaffold.

A proton-driven molecular pump is devised using a surface-confined dynamic 3D DNA scaffold. A dynamic DNA tetrahedral nanostructure is designed by incorporating a pH-sensitive i-motif sequence in one edge, which serves as the scaffold to ensure highly ordered orientation and spatial isolation of this nanomachine on the macroscopic gold surface. It is found that the switching ability of this dynamic tetrahedron is fully maintained on the surface. Importantly, this proton-driven nanomachine can reversibly pump water and ferricynide in response to pH variation in solution.

Colorimetric method to detect ε-poly-l-lysine using glucose oxidase.

We describe a new colorimetric assay method using glucose oxidase (GOx) to detect ε-poly-l-lysine (εPL). This method uses εPL's remarkable effect of promoting the enzymatic reaction of GOx with ferricyanide ion. This reaction reduces ferricyanide ion to ferrocyanide ion, accompanied by a color change from yellow to colorless. In this colorimetric assay, the detection limit of εPL was estimated to be approximately 0.5 mg/L when purified εPL samples were used. εPL has usually been produced by a fermentation process using Streptomyces albulus species. The components of the culture broth showed interference effects against the assay method. However, due to the high sensitivity of the assay method for εPL, εPL could be detected in the culture broth without any pretreatment. The detectable concentration of εPL in the culture broth, cPL,ac, was estimated to be approximately 20 mg/L. By combining the Berlin blue reaction with this method, the cPL,ac was reduced to 10 mg/L. In light of the proposed method's simplicity and sensitivity, it could be useful for screening εPL synthetic enzymes and microorganisms.

Determination of the Michaelis-Menten kinetics and the genes expression involved in phyto-degradation of cyanide and ferri-cyanide.

Hydroponic experiments were conducted with different species of plants (rice, maize, soybean and willow) exposed to ferri-cyanide to investigate the half-saturation constant (K M ) and the maximal metabolic capacity (v max ) involved in phyto-assimilation. Three varieties for each testing species were collected from different origins. Measured concentrations show that the uptake rates responded biphasically to ferri-cyanide treatments by showing increases linearly at low and almost constant at high concentrations from all treatments, indicating that phyto-assimilation of ferri-cyanide followed the Michaelis-Menten kinetics. Using non-linear regression, the highest v max was by rice, followed by willows. The lowest v max was found for soybean. All plants, except maize (DY26) and rice (XJ12), had a similar K M value, suggesting the same enzyme was active in phyto-assimilation of ferri-cyanide. Transcript level, by real-time quantitative PCR, of enzymes involved in degradation of cyanides showed that the analyzed genes were differently expressed during different cyanides exposure. The expression of CAS and ST genes responded positively to KCN exposure, suggesting that ß-CAS and ST pathways were two possible pathways for cyanide detoxification in rice. The transcript level of NIT and ASPNASE genes also showed a remarkable up-regulation to KCN, implying the contribution to the pool of amino acid aspartate, which is an end product of CN metabolism. Up-regulation of GS genes suggests that acquisition of ammonium released from cyanide degradation may be an additional nitrogen source for plant nutrition. Results also revealed that the expressions of these genes, except for GS, were relatively constant during iron cyanide exposure, suggesting that they are likely metabolized by plants through a non-defined pathway rather than the ß-CAS pathway.

A rapid and sensitive colorimetric measurement of antibiotic efficacy against Escherichia coli in vitro.

A common dye of prussian blue (PB) as an indicator was used to develop a colorimetric method for detecting the efficacy of the antibiotics in vitro. Considering the electronic production capacity of microbial respiration, ferricyanide was employed in transferring electrons from target microorganism of Escherichia coli (E. coli) to produce ferrocyanide. Subsequently, ferrocyanide reacted with ferric ions to form PB. In view of relationship between the PB yield and the bacterial activity, the efficacy of the antibiotics on E. coli was directly detected at 700 nm of PB absorption. When the 5% activity of antibiotics on 20 isolates of E. coli was quantified as 5% efficacy, the applied concentrations of eight antibiotics, such as cefepime, ceftriaxone sodium, cefoperazone sodium, piperacillin sodium, amoxicillin, gentamicin, amikacin and levofloxacin were 2, 2, 4, 4, 10, 4, 8 and 8 µg mL⁻¹, respectively. To compare with minimum inhibitory concentration results obtained by Clinical and Laboratory Standards Institute broth macrodilution method, the results of PB methods showed good agreements except with gentamicin. Paired t-test result (P) also showed that difference between two methods was statistically significant (P = 0.006).

Synthesis of polypyrrole within the cell wall of yeast by redox-cycling of [Fe(CN)6](3-)/[Fe(CN)6](4-).

Yeast cells are often used as a model system in various experiments. Moreover, due to their high metabolic activity, yeast cells have a potential to be applied as elements in the design of biofuel cells and biosensors. However a wider application of yeast cells in electrochemical systems is limited due to high electric resistance of their cell wall. In order to reduce this problem we have polymerized conducting polymer polypyrrole (Ppy) directly in the cell wall and/or within periplasmic membrane. In this research the formation of Ppy was induced by [Fe(CN)6](3-)ions, which were generated from K4[Fe(CN)6], which was initially added to polymerization solution. The redox process was catalyzed by oxido-reductases, which are present in the plasma membrane of yeast cells. The formation of Ppy was confirmed by spectrophotometry and atomic force microscopy. It was confirmed that the conducting polymer polypyrrole was formed within periplasmic space and/or within the cell wall of yeast cells, which were incubated in solution containing pyrrole, glucose and [Fe(CN)6](4-). After 24h drying at room temperature we have observed that Ppy-modified yeast cell walls retained their initial spherical form. In contrast to Ppy-modified cells, the walls of unmodified yeast have wrinkled after 24h drying. The viability of yeast cells in the presence of different pyrrole concentrations has been evaluated.

Nanochannel-based electrochemical assay for transglutaminase activity.

A novel electrochemical assay to quantify transglutaminase activity is reported. The assay is based on the enzyme-controlled diffusion of Fe(CN)6(3-/4-) through amino-functionalized nanochannels of a mesoporous silica thin film on a Au surface in the presence of N-benzyloxycarbonyl-L-glutaminylglycine.

Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system.

We evaluated the intracellular NAD(P)H: quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione-ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 µM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 µM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system.

Electrochemical detection of intracellular and cell membrane redox systems in Saccharomyces cerevisiae.

Redox mediators can interact with eukaryote cells at a number of different cell locations. While cell membrane redox centres are easily accessible, the redox centres of catabolism are situated within the cytoplasm and mitochondria and can be difficult to access. We have systematically investigated the interaction of thirteen commonly used lipophilic and hydrophilic mediators with the yeast Saccharomyces cerevisiae. A double mediator system is used in which ferricyanide is the final electron acceptor (the reporter mediator). After incubation of cells with mediators, steady state voltammetry of the ferri/ferrocyanide redox couple allows quantitation of the amount of mediator reduced by the cells. The plateau current at 425 mV vs Ag/AgCl gives the analytical signal. The results show that five of the mediators interact with at least three different trans Plasma Membrane Electron Transport systems (tPMETs), and that four mediators cross the plasma membrane to interact with cytoplasmic and mitochondrial redox molecules. Four of the mediators inhibit electron transfer from S. cerevisiae. Catabolic inhibitors were used to locate the cellular source of electrons for three of the mediators.