Oxidative stress antagonizes fluoroquinolone drug sensitivity via the SoxR-SUF Fe-S cluster homeostatic axis.

The level of antibiotic resistance exhibited by bacteria can vary as a function of environmental conditions. Here, we report that phenazine-methosulfate (PMS), a redox-cycling compound (RCC) enhances resistance to fluoroquinolone (FQ) norfloxacin. Genetic analysis showed that E. coli adapts to PMS stress by making Fe-S clusters with the SUF machinery instead of the ISC one. Based upon phenotypic analysis of soxR, acrA, and micF mutants, we showed that PMS antagonizes fluoroquinolone toxicity by SoxR-mediated up-regulation of the AcrAB drug efflux pump. Subsequently, we showed that despite the fact that SoxR could receive its cluster from either ISC or SUF, only SUF is able to sustain efficient SoxR maturation under exposure to prolonged PMS period or high PMS concentrations. This study furthers the idea that Fe-S cluster homeostasis acts as a sensor of environmental conditions, and because its broad influence on cell metabolism, modifies the antibiotic resistance profile of E. coli.

Impact of small fractions of abnormal erythrocytes on blood rheology.

Red blood cell (RBC) populations are inherently heterogeneous, given mature RBC lack the transcriptional machinery to re-synthesize proteins affected during in vivo aging. Clearance of older, less functional cells thus aids in maintaining consistent hemorheological properties. Scenarios occur, however, where portions of mechanically impaired RBC are re-introduced into blood (e.g., damaged from circulatory support, blood transfusion) and may alter whole blood fluid behavior. Given such perturbations are associated with poor clinical outcomes, determining the tolerable level of abnormal RBC in blood is valuable. Thus, the current study aimed to define the critical threshold of blood fluid properties to re-infused physically-impaired RBC. Cell mechanics of RBC were impaired through membrane cross-linking (glutaraldehyde) or intracellular oxidation (phenazine methosulfate). Mechanically impaired RBC were progressively re-introduced into the native cell population. Negative alterations of cellular deformability and high shear blood viscosity were observed following additions of only 1-5% rigidified RBC. Low-shear blood viscosity was conversely decreased following addition of glutaraldehyde-treated cells; high-resolution microscopy of these mixed cell populations revealed decreased capacity to form reversible aggregates and decreased aggregate size. Mixed RBC populations, when exposed to supraphysiological shear, presented with compounded mechanical impairment. Collectively, key determinants of blood flow behavior are sensitive to mechanical perturbations in RBC, even when only 1-5% of the cell population is affected. Given this fraction is well-below the volume of rigidified RBC introduced during circulatory support or transfusion practice, it is plausible that some adverse events following surgery and/or transfusion may be related to impaired blood fluidity.

Ecstasy metabolites and monoamine neurotransmitters upshift the Na+/K+ ATPase activity in mouse brain synaptosomes.

3,4-Methylenedioximethamphetamine (MDMA; "ecstasy") is a psychotropic drug with well-known neurotoxic effects mediated by hitherto not fully understood mechanisms. The Na+- and K+-activated adenosine 5'-triphosphatase (Na+/K+ ATPase), by maintaining the ion gradient across the cell membrane, regulates neuronal excitability. Thus, a perturbation of its function strongly impacts cell homeostasis, ultimately leading to neuronal dysfunction and death. Nevertheless, whether MDMA affects the Na+/K+ ATPase remains unknown. In this study, we used synaptosomes obtained from whole mouse brain to test the effects of MDMA, three of its major metabolites [α-methyldopamine, N-methyl-α-methyldopamine and 5-(glutathion-S-yl)-α-methyldopamine], serotonin (5-HT), dopamine, 3,4-dihydroxy-L-phenylalanine (L-Dopa) and 3,4-dihydroxyphenylacetic acid (DOPAC) on the Na+/K+ ATPase function. A concentration-dependent increase of Na+/K+ ATPase activity was observed in synaptosomes exposed to the tested compounds (concentrations ranging from 0.0625 to 200 µM). These effects were independent of protein kinases A and C activities. Nevertheless, a rescue of the compounds' effects was observed in synaptosomes pre-incubated with the antioxidant N-acetylcysteine (1 mM), suggesting a role for reactive species-regulated pathways on the Na+/K+ ATPase effects. In agreement with this hypothesis, a similar increase in the pump activity was found in synaptosomes exposed to the chemical generator of superoxide radicals, phenazine methosulfate (1-250 µM). This study demonstrates the ability of MDMA metabolites, monoamine neurotransmitters, L-Dopa and DOPAC to alter the Na+/K+ ATPase function. This could represent a yet unknown mechanism of action of MDMA and its metabolites in the brain.

High-pressure tuning of primary photochemistry in bacterial photosynthesis: membrane-bound versus detergent-isolated reaction centers.

While photosynthesis thrives at close to normal pressures and temperatures, it is presently well known that life is similarly commonplace in the hostile environments of the deep seas as well as around hydrothermal vents. It is thus imperative to understand how key biological processes perform under extreme conditions of high pressures and temperatures. Herein, comparative steady-state and picosecond time-resolved spectroscopic studies were performed on membrane-bound and detergent-purified forms of a YM210W mutant reaction center (RC) from Rhodobacter sphaeroides under modulating conditions of high hydrostatic pressure applied at ambient temperature. A previously established breakage of the lone hydrogen bond formed between the RC primary donor and the protein scaffold was shown to take place in the membrane-bound RC at an almost 3 kbar higher pressure than in the purified RC, confirming the stabilizing role of the lipid environment for membrane proteins. The main change in the multi-exponential decay of excited primary donor emission across the experimental 10 kbar pressure range involved an over two-fold continuous acceleration, the kinetics becoming increasingly mono-exponential. The fastest component of the emission decay, thought to be largely governed by the rate of primary charge separation, was distinctly slower in the membrane-bound RC than in the purified RC. The change in character of the emission decay with pressure was explained by the contribution of charge recombination to emission decreasing with pressure as a result of an increasing free energy gap between the charge-separated and excited primary donor states. Finally, it was demonstrated that, in contrast to a long-term experimental paradigm, adding a combination of sodium ascorbate and phenazine methosulfate to the protein solution potentially distorts natural photochemistry in bacterial RCs.

Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster's Blue in methanol dehydrogenase assays.

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

Phenazine methosulphate-treated red blood cells activate NF-κB and upregulate endothelial ICAM-1 expression.

Although enhanced Red Blood Cell (RBC) - Endothelial Cell (EC) interaction, as well as RBC induced EC activation, have been extensively studied in several RBC-linked pathologies, the specific individual effects of oxidatively modified RBC on EC activation has not yet been documented. However, increasing evidence in both experimental and clinical studies suggests that oxidatively modified RBC could be considered potential pathogenic determinants in several acute and chronic diseases displaying systemic oxidative stress. Therefore, the present study aimed to explore the specific effects of oxidized RBC interaction with endothelial cells on intracellular signaling pathways that promote EC activation. RBC were exposed to oxidative stress induced by phenazine methosulphate (PMS). It is shown that the interaction of oxidatively modified RBC with cultured human umbilical vein endothelial cells (HUVEC) results in: a) EC activation as indicated by the increased surface expression of intercellular adhesion molecule -1 (ICAM-1); b) the activation of transcription factor NF-κB, an indicator of cellular oxidant stress. These results emphasize the specific contribution of oxidatively modified RBC interaction to EC activation and their possible pathological role in vascular diseases and oxidative stress.

A fast, sensitive, single-step colorimetric dipstick assay for quantifying ascorbic acid in urine.

The presence of ascorbate in human urine has been shown to be a useful dietary, fruit or vitamin C intake biomarker. More recently it has been discovered that ascorbate levels in urine can be used to facilitate the detection of precancerous colorectal polyps. While there are a number elaborate HPLC, MS or multi-step enzymatic "kit" methods to detect and quantify urinary ascorbate, these are time consuming and expensive. There are also a number of low-cost paper-based ascorbate detection dipsticks. However, the limits of detection and quantification accuracy for these dipsticks are not adequate for applications with human urine. To address these limitations, we have developed a fast, sensitive, single-step colorimetric assay that can be used to quantify ascorbate in urine and other biological fluids. The assay uses the tetrazolium salt, methylthiazolyldiphenyl-tetrazolium bromide (MTT), with the electron carrier phenazine methosulfate (PMS), in a chelated acidic phosphate-buffer to produce a vivid purple color in the presence of ascorbate. Confirmation of the performance of the assay and of its standard curve in human urine was also done using independent LC-MS/MS and NMR analyses. The lower limit of detection of the ascorbate dipstick assay described here was found to be 3.2 µM. The paper dipsticks are stable over a wide range of temperatures and can be stored for up to 150-days.

A novel histochemistry assay to assess and quantify focal cytochrome c oxidase deficiency.

Defects in the respiratory chain, interfering with energy production in the cell, are major underlying causes of mitochondrial diseases. In spite of this, the surprising variety of clinical symptoms, disparity between ages of onset, as well as the involvement of mitochondrial impairment in ageing and age-related diseases continue to challenge our understanding of the pathogenic processes. This complexity can be in part attributed to the unique metabolic needs of organs or of various cell types. In this view, it remains essential to investigate mitochondrial dysfunction at the cellular level. For this purpose, we developed a novel enzyme histochemical method that enables precise quantification in fresh-frozen tissues using competing redox reactions which ultimately lead to the reduction of tetrazolium salts and formazan deposition in cytochrome c oxidase-deficient mitochondria. We demonstrate that the loss of oxidative activity is detected at very low levels - this achievement is unequalled by previous techniques and opens up new opportunities for the study of early disease processes or comparative investigations. Moreover, human biopsy samples of mitochondrial disease patients of diverse genotypic origins were used and the successful detection of COX-deficient cells suggests a broad application for this new method. Lastly, the assay can be adapted to a wide range of tissues in the mouse and extends to other animal models, which we show here with the fruit fly, Drosophila melanogaster. Overall, the new assay provides the means to quantify and map, on a cell-by-cell basis, the full extent of COX deficiency in tissues, thereby expending new possibilities for future investigation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Menadione-mediated WST1 reduction assay for the determination of metabolic activity of cultured neural cells.

Cellular reduction of tetrazolium salts to their respective formazans is frequently used to determine the metabolic activity of cultured cells as an indicator of cell viability. For membrane-impermeable tetrazolium salts such as WST1 the application of a membrane-permeable electron cycler is usually required to mediate the transfer of intracellular electrons for extracellular WST1 reduction. Here we demonstrate that in addition to the commonly used electron cycler M-PMS, menadione can also serve as an efficient electron cycler for extracellular WST1 reduction in cultured neural cells. The increase in formazan absorbance in glial cell cultures for the WST1 reduction by menadione involves enzymatic menadione reduction and was twice that recorded for the cytosolic enzyme-independent WST1 reduction in the presence of M-PMS. The optimized WST1 reduction assay allowed within 30 min of incubation a highly reliable detection of compromised cell metabolism caused by 3-bromopyruvate and impaired membrane integrity caused by Triton X-100, with a sensitivity as good as that of spectrophotometric assays which determine cellular MTT reduction or lactate dehydrogenase release. The short incubation period of 30 min and the observed good sensitivity make this optimized menadione-mediated WST1 reduction assay a quick and reliable alternative to other viability and toxicity assays.

Antimicrobial Efficacy of Salvadora persica Extracts on a Monospecies Biofilm on Orthodontic Brackets In Vitro.

PURPOSE: The oral cavity is a rich ecosystem with a plethora of microorganisms, and different components of fixed orthodontic appliances may contribute to a shift in the balance of oral ecology. The purpose of this study was to investigate the antimicrobial potential of hexane and ethanol extracts of Salvadora persica on a monospecies biofilm model established on orthodontic brackets in vitro. MATERIALS AND METHODS: Streptococcus mutans biofilm was formed on mini diamond orthodontic brackets following three days of anaerobic incubation at 37˚C. The bacterial cell viability of this biofilm was measured after their exposure to saline, hexane extract of S. persica, ethanol extract of S. persica and 0.2% chlorhexidine using 3-(4, 5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assay. On half of the brackets, the colony forming units (CFU) were counted. Both experiments were performed in triplicate. RESULTS: The absorbance values obtained from the MTS reduction assay after exposure to the different test agents showed a decline in the bacterial cell viability of the S. mutans biofilm as follows: chlorhexidine (+)0.05). The CFU counts of S. mutans obtained from chlorhexidine exposure were lower than from hexane and ethanol extracts. CONCLUSION: S. persica extracts were found to have antimicrobial effects on S. mutans biofilm established in vitro on orthodontic brackets suggestive of its potential use as an oral antimicrobial agent for orthodontic patients.

Gene identification and enzymatic characterization of the initial enzyme in pyrimidine oxidative metabolism, uracil-thymine dehydrogenase.

Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP+ or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD+, NADP+, flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors.

A shared mechanism of SoxR activation by redox-cycling compounds.

In this issue of Molecular Microbiology, Gu and Imlay show that a class of compounds known as redox-cycling agents directly activate the transcription factor SoxR of Escherichia coli and cause cellular toxicity independent of the production of the reactive oxygen species superoxide. Despite the fact that redox-cycling agents increase formation of superoxide in E. coli, the results described in this new publication revise the long-held assumption that superoxide is responsible for the activation of SoxR and for all of the major toxic effects of redox-cycling drugs. This study also suggests that the critical function of the SoxRS regulon in E. coli is in protection against redox-cycling agents and not exclusively the defence against superoxide.

The SoxRS response of Escherichia coli is directly activated by redox-cycling drugs rather than by superoxide.

When Escherichia coli is exposed to redox-cycling drugs, its SoxR transcription factor is activated by oxidation of its [2Fe-2S] cluster. In aerobic cells these drugs generate superoxide, and because superoxide dismutase (SOD) is a member of the SoxRS regulon, superoxide was initially thought to be the activator of SoxR. Its many-gene regulon was therefore believed to comprise a defence against superoxide stress. However, we found that abundant superoxide did not effectively activate SoxR in an SOD⁻ mutant, that overproduced SOD could not suppress activation by redox-cycling drugs, and that redox-cycling drugs were able to activate SoxR in anaerobic cells as long as alternative respiratory acceptors were provided. Thus superoxide is not the signal that SoxR senses. Indeed, redox-cycling drugs directly oxidized the cluster of purified SoxR in vitro, while superoxide did not. Redox-cycling drugs are excreted by both bacteria and plants. Their toxicity does not require superoxide, as they poisoned E. coli under anaerobic conditions, in part by oxidizing dehydratase iron-sulfur clusters. Under these conditions SoxRS induction was protective. Thus it is physiologically appropriate that the SoxR protein directly senses redox-cycling drugs rather than superoxide.

PMS: photosystem I electron donor or fluorescence quencher.

Light energy harvested by the pigments in Photosystem I (PSI) is used for charge separation in the reaction center (RC), after which the positive charge resides on a special chlorophyll dimer called P700. In studies on the PSI trapping kinetics, P700(+) is usually chemically reduced to re-open the RCs. So far, the information available about the reduction rate and possible chlorophyll fluorescence quenching effects of these reducing agents is limited. This information is indispensible to estimate the fraction of open RCs under known experimental conditions. Moreover, it would be important to understand if these reagents have a chlorophyll fluorescence quenching effects to avoid the introduction of exogenous singlet excitation quenching in the measurements. In this study, we investigated the effect of the commonly used reducing agent phenazine methosulfate (PMS) on the RC and fluorescence emission of higher plant PSI-LHCI. We measured the P700(+) reduction rate for different PMS concentrations, and show that we can give a reliable estimation on the fraction of closed RCs based on these rates. The data show that PMS is quenching chlorophyll fluorescence emission. Finally, we determined that the fluorescence quantum yield of PSI with closed RCs is 4% higher than if the RCs are open.

Ultrasensitive detection of the reduced form of nicotinamide adenine dinucleotide based on carbon nanotube field effect transistor.

We developed a simple, ultrasensitive, and quantitative detection method for the reduced form of nicotinamide adenine dinucleotide (NADH), based on carbon nanotube field effect transistors (CNTFETs). Following the injection of NADH at different concentrations, we obtained different electrical signals from a semiconductor characterization system mimicking biological catalysis of NADH dehydrogenase (CoI). Here, FET was fabricated via photolithography, attaching silicon wells, as the detection chamber, on the channel area of the single wall carbon nanotube (SWCNT). SWCNTs were functionalized with phenazine derivant, a counterpart of the key functional prosthetic group of CoI enzyme. In the presence of NADH, electrons transferred to phenazine derivant through SWCNT, by analogous means of the electron transport chain formed by a series of iron-sulfur (FeS) clusters in CoI. Using this method, the limit of detection was as low as 1 pM, and the range of linear response was 10 pM to 500 nM. Significantly, this approach possesses great potential for applications in real-time detection of NADH at extremely low concentrations, and rigorous analysis for NADH in electrochemical fields.

Aerobic denitration of 2,4,6-trinitrotoluene in the presence of phenazine compounds and reduced pyridine nucleotides.

Phenazine-containing spent culture supernatants of Pseudomonas aeruginosa concentrated with a C18 solid-phase extraction cartridge initiate NAD(P)H-dependent denitration of 2,4,6-trinitrotoluene (TNT). In this study, TNT denitration was investigated under aerobic conditions using two phenazine secondary metabolites excreted by P. aeruginosa, pyocyanin (Py) and its precursor phenazine-1- carboxylic acid (PCA), and two chemically synthesized pyocyanin analogs, phenazine methosulfate (PMS+) and phenazine ethosulfate (PES+). The biomimetic Py/NAD(P)H/O2 system was characterized and found to extensively denitrate TNT in unbuffered aqueous solution with minor production of toxic amino aromatic derivatives. To a much lesser extent, TNT denitration was also observed with PMS+ and PES+ in the presence of NAD(P)H. No TNT denitration was detected with the biomimetic PCA/NAD(P)H/O2 system. Electron paramagnetic resonance (EPR) spectroscopy analysis of the biomimetic Py/NAD(P)H/O2 system revealed the generation of superoxide radical anions (O2 •−). In vitro TNT degradation experiments in the presence of specific inhibitors of reactive oxygen species suggest a nucleophilic attack of superoxide radical anion followed by TNT denitration through an as yet unknown mechanism. The results of this research confirm the high functional versatility of the redox-active metabolite pyocyanin and the susceptibility of aromatic compounds bearing electron withdrawing substituents, such as nitro groups, to superoxide-driven nucleophilic attack.

Phenazine methosulfate decreases HIF-1α accumulation during the exposure of cells to hypoxia.

In HEK293 cells, exposure to various NAD(P)H oxidants, including phenazine methosulfate (PMS), that non-enzymatically oxidize intracellular NAD(P)H to NAD(P), decreased hypoxia-induced hypoxia-inducible factor 1 (HIF-1α) accumulation. RT-PCR and cycloheximide inhibition experiments indicated that PMS-induced HIF-1α decrease is involved in post-translational degradation during hypoxia. The decrease in HIF-1α caused by PMS was not eliminated by proteasome inhibitor MG132. Moreover, the increase in HIF-1α induced by exposure to MG132 alone in normoxia was diminished by PMS. In contrast, calpastatin peptide, a calpain inhibitor, fully prevented PMS-induced reduction in HIF-1α in hypoxic cells. These data suggest that the decreased stability of HIF-1α induced by PMS is due to the activation by PMS of a protein degradation system that is independent of the ubiquitin-proteasome pathway.

Protective effects of small heat shock proteins in Daphnia magna against heavy metal exposure.

Daphnia magna is one of the most commonly used model organisms to assess toxicity of heavy metal and other xenobiotics. However, the lack of knowledge about important stress-resistant molecules limits our understanding of the alteration of phenotypic and physiological traits of D. magna upon stress exposures. In this study, we focused on a chaperone family of small heat shock protein (sHSP) that has been found in archaea, bacteria and eukaryotes and plays an important role in stress tolerance. A total of eleven sHSP genes (termed DmsHSP1 - DmsHSP11) were identified from the D. magna genome, whose expression profiles during exposure to heavy metal (Cd2+, Cu2+ and Zn2+) and a few other potential pollutants were evaluated via qRT-PCR and RNA-Seq analysis. The results highlighted the predominant role of DmsHSP1 with the highest basal expression level in adults and robust upregulation upon exposure to heavy metals (Cu2+ > Cd2+ > Zn2+). In vivo, recombinant protein rDmsHSP1-21 and rDmsHSP11-12.8 could not only prevent model substrates agglutination induced by heavy metals or reducer dithiotreitol (DTT), but also protect tissue proteins and enzymes from denaturation and inactivation caused by heavy metals or high temperature. Ectopically expression of DmsHSP1-21 or DmsHSP11-12.8 in E. coli conferred host enhanced resistance against various abiotic stresses including Cd2+, Cu2+ and phenazine methosulfate (PMS). Knockdown of DmsHSP1-21 by RNAi, but not for DmsHSP11-12.8, significantly increased the vulnerability of D. magna to heavy metal exposure. Our work provides systematic information on the evolution and function of sHSPs in D. magna and leads to important insights into the mechanisms by which D. magna survive in adverse environments.

Oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor.

Cyanobacterial aldehyde decarbonylase (cAD) is, structurally, a member of the di-iron carboxylate family of oxygenases. We previously reported that cAD from Prochlorococcus marinus catalyzes the unusual hydrolysis of aldehydes to produce alkanes and formate in a reaction that requires an external reducing system but does not require oxygen [Das et al. (2011) Angew. Chem. 50, 7148-7152]. Here we demonstrate that cADs from divergent cyanobacterial classes, including the enzyme from N. puntiformes that was reported to be oxygen dependent, catalyze aldehyde decarbonylation at a much faster rate under anaerobic conditions and that the oxygen in formate derives from water. The very low activity (<1 turnover/h) of cAD appears to result from inhibition by the ferredoxin reducing system used in the assay and the low solubility of the substrate. Replacing ferredoxin with the electron mediator phenazine methosulfate allowed the enzyme to function with various chemical reductants, with NADH giving the highest activity. NADH is not consumed during turnover, in accord with the proposed catalytic role for the reducing system in the reaction. With octadecanal, a burst phase of product formation, k(prod) = 3.4 ± 0.5 min(-1), is observed, indicating that chemistry is not rate-determining under the conditions of the assay. With the more soluble substrate, heptanal, k(cat) = 0.17 ± 0.01 min(-1) and no burst phase is observed, suggesting that a chemical step is limiting in the reaction of this substrate.

Antifungal mechanisms by which a novel Pseudomonas aeruginosa phenazine toxin kills Candida albicans in biofilms.

Pseudomonas aeruginosa produces several phenazines including the recently described 5-methyl-phenazine-1-carboxylic acid (5MPCA), which exhibits a novel antibiotic activity towards pathogenic fungi such as Candida albicans. Here we characterize the unique antifungal mechanisms of 5MPCA using its analogue phenazine methosulphate (PMS). Like 5MPCA, PMS induced fungal red pigmentation and killing. Mass spectrometry analyses demonstrated that PMS can be covalently modified by amino acids, a process that yields red derivatives. Furthermore, soluble proteins from C. albicans grown with either PMS or P. aeruginosa were also red and demonstrated absorbance and fluorescence spectra similar to that of PMS covalently linked to either amino acids or proteins in vitro, suggesting that 5MPCA modification by protein amine groups occurs in vivo. The red-pigmented C. albicans soluble proteins were reduced by NADH and spontaneously oxidized by oxygen, a reaction that likely generates reactive oxygen species (ROS). Additional evidence indicated that ROS generation precedes 5MPCA-induced fungal death. Reducing conditions greatly enhanced PMS uptake by C. albicans and killing. Since 5MPCA was more toxic than other phenazines that are not modified, such as pyocyanin, we propose that the covalent binding of 5MPCA promotes its accumulation in target cells and contributes to its antifungal activity in mixed-species biofilms.