A stepwise control strategy for glutathione synthesis in Saccharomyces cerevisiae based on oxidative stress and energy metabolism.

A stepwise control strategy for enhancing glutathione (GSH) synthesis in yeast based on oxidative stress and energy metabolism was investigated. First, molasses and corn steep liquor were selected and fed as carbon source mixture at a flow rate of 1.5 g/L/h and 0.4 g/L/h, respectively, for increasing cell density in a 10 L fermenter. When the biomass reached 90 g/L, the KMnO4 sustained-release particles, composed of 1.5% KMnO4, 3% stearic acid, 2% polyethylene glycol and 3% agar powder, were prepared and added to the fermentation broth for maintaining the oxidative stress. The results showed that the maximum GSH accumulation of the group fed KMnO4 sustained-release particles was 39.0% higher than that of KMnO4-fed group. In addition to the improved average GSH productivity and average specific production rate, the activities of GSH peroxidase, γ-glutamylcysteine synthetase and GSH reductase, enzymes taking part in GSH metabolism, were also significantly enhanced by KMnO4 sustained-release particles feeding. Finally, 6 g/L sodium citrate fed as an energy adjuvant elevated the intracellular ATP level for further enhancing GSH production. Through the above stepwise strategy, the GSH accumulation reached 5.76 g/L, which was 2.84-fold higher than that of the control group. The stepwise control strategy based on oxidative stress and energy metabolism significantly improved GSH accumulation in yeast.

A high throughput method for total alcohol determination in fermentation broths.

BACKGROUND: The potassium dichromate oxidation method used in determination of alcohols in fermentation has two major disadvantages. This method cannot be used to determine alcohols in raw fermentation broth samples, which often contain various reducing sugars. The method is not environment friendly due to the carcinogenicity of Cr (VI) used. RESULTS: A new method for determination of reducing sugars and total alcohols in raw fermentation broths was developed. The fermentation broth was pretreated to remove proteins, polysaccharides, glycerol and organic acids. The colorimetric change from both total alcohols and reducing sugars by potassium permanganate oxidation was measured. The portion of colorimetric change from oxidation of reducing sugars was determined by DNS test and subtracted. The remaining portion of colorimetric change was then used to calculate the total alcohol concentration in the sample. CONCLUSIONS: Using this method, total alcohol concentration can be easily and accurately determined in both distilled samples and raw fermentation broth samples. It is fast and environmental friendly.

Experimental investigation and mathematical modeling for microbial removal using potassium permanganate as an oxidant-case study: water treatment plant No. 1, Mashhad, Iran.

Preoxidation is an important unit process which can partially remove organic and microbial contaminations. Due to the high concentrations of organic matter entering the water treatment plant, originating from surface water resources, preoxidation by using chlorinated compounds may increase the possibility of trihalomethane (THM) formation. Therefore, in order to reduce the concentration of THMs, different alternatives such as injection of potassium permanganate are utilized. The present study attempts to investigate the efficiency of the microbial removal from raw water entering the water treatment plant No. 1 in Mashhad, Iran, through various doses of potassium permanganate. Then, an examination of the predictive models is done in order to indicate the residual Escherichia coli and total coliform resulted from injecting the potassium permanganate. Finally, the coefficients of the proposed models were optimized using the genetic algorithm. The results of the study show that 0.5 mg L-1 of potassium permanganate would remove 50% of total coliform as well as 80% of Escherichia coli in the studied water treatment plant. Also, assessing the performance of different models in predicting the residual microbial concentration after injection of potassium permanganate suggests the Gaussian model as the one resulting the highest conformity. Moreover, it can be concluded that employing smart models leads to an optimization of the injected potassium permanganate at the levels of 27% and 73.5%, for minimum and maximum states during different seasons of a year, respectively.

A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non-eDNA networks in Pseudomonas aeruginosa biofilms.

Biofilms are etiologically important in the development of chronic medical and dental infections. The biofilm extracellular polymeric substance (EPS) determines biofilm structure and allows bacteria in biofilms to adapt to changes in mechanical loads such as fluid shear. However, EPS components are difficult to visualize microscopically because of their low density and molecular complexity. Here, we tested potassium permanganate, KMnO4, for use as a non-specific EPS contrast-enhancing stain using confocal laser scanning microscopy in reflectance mode. We demonstrate that KMnO4 reacted with EPS components of various strains of Pseudomonas, Staphylococcus and Streptococcus, yielding brown MnO2 precipitate deposition on the EPS, which was quantifiable using data from the laser reflection detector. Furthermore, the MnO2 signal could be quantified in combination with fluorescent nucleic acid staining. COMSTAT image analysis indicated that KMnO4 staining increased the estimated biovolume over that determined by nucleic acid staining alone for all strains tested, and revealed non-eDNA EPS networks in Pseudomonas aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but did not react strongly with DNA or alginate. KMnO4 staining may have application as a research tool and for diagnostic potential for biofilms in clinical samples.

Comparative study of MnO2 nanoparticle synthesis by marine bacterium Saccharophagus degradans and yeast Saccharomyces cerevisiae.

Microorganisms are one of the most attractive and simple sources for the synthesis of different types of metal nanoparticles. The synthesis of manganese dioxide nanoparticles (MnO2 NPs) by microorganisms from reducing potassium permanganate was investigated for the first time in the present study. The microbial supernatants of the bacterium Saccharophagus degradans ATCC 43961 (Sde 2-40) and of the yeast Saccharomyces cerevisiae showed positive reactions to the synthesis of MnO2 NPs by displaying a change of color in the permanganate solution from purple to yellow. KMnO4-specific peaks also disappeared and MnO2-specific peaks emerged at an absorption maximum of 365 nm in UV-visible spectrophotometry. The washed Sde 2-40 cells did not show any ability to synthesize MnO2 NPs. The medium and medium constituents of Sde 2-40 showed similar positive reactions as supernatants, which indicate the role of the Sde 2-40 medium constituents in the synthesis of MnO2 NPs. This suggests that microorganisms without nanoparticle synthesis ability can be misreported for their abilities to synthesize nanoparticles. S. cerevisiae washed cells showed an ability to synthesize MnO2 NPs. The strategies of keeping yeast cells in tea bags and dialysis membranes showed positive tests for the synthesis of MnO2 NPs. A Fourier transform-infrared spectroscopy study suggested roles for the proteins, alcoholic compounds, and cell walls of S. cerevisiae cells in the synthesis of MnO2 NPs. Electron-dispersive X-ray spectroscopy analyses confirmed the presence of Mn and O in the sample. X-ray photoelectron spectroscopy revealed characteristic binding energies for MnO2 NPs. Transmission electron microscopy micrographs revealed the presence of uniformly dispersed hexagonal- and spherical-shaped particles with an average size of 34.4 nm. The synthesis approach using yeast is possible by a simple reaction at low temperature without any need for catalysts, templates, or expensive and precise equipment. Therefore, this study will be useful for the easy, cost-effective, reliable, and eco-friendly production of nanomaterials.

Impact of potassium permanganate on cyanobacterial cell integrity and toxin release and degradation.

Potassium permanganate (KMnO4) is commonly used as a pre-treatment oxidant to remove soluble manganese (Mn) and iron (Fe) which can contribute to dirty water in drinking water supplies. Because Mn and Fe problems are commonly associated with thermal stratification in summer and autumn, they frequently coincide with the presence of cyanobacteria. The use of KMnO4 as an oxidant for Mn and Fe control therefore needs to consider the potential impacts on cyanobacterial cell integrity and toxin release. This study aims to assess the effect of KMnO4 on cyanobacteria cell integrity, toxin release and toxin oxidation. A toxic strain of Microcystis aeruginosa was exposed to various concentrations of KMnO4 and the cell integrity of cyanobacteria was measured with flow cytometry. Further the intra- and extra-cellular toxin concentrations were quantified and it was apparent that KMnO4 reduced both the intra- and extra-cellular toxins at low initial concentrations of 1 and 3 mg L(-1) without complete cell lysis. However, the cell integrity of cyanobacteria was compromised at KMnO4 concentrations of 5 mg L(-1) and 10 mg L(-1) and led to intracellular toxin release. In the 10 mg L(-1) KMnO4 treatment, the total toxin was oxidised after 7h contact time. A model describing the two step process of release and degradation was developed and may provide a tool to assess the risk water quality posed by toxin release. Consequently, it may be possible to use KMnO4 as a pre-treatment for Mn and Fe at concentrations<3 mg L(-1) and short contact time when cyanobacteria are also present.

A novel and simple method of screening compounds for interaction with DNA: A validation study.

We report the development of a simple, cost-effective assay for detecting compounds that have the ability to interact with and modify DNA. Potential uses for the assay lie in the areas of early genotoxicity testing of drug candidates, anticancer and antibiotic drug discovery, environmental monitoring and testing in the food, beverage and cosmetics industries. At present the assay has been used to assess direct-acting compounds only and it is yet to be established whether the assay is compatible with bio-activation. The methodology is based on the oxidative reaction of potassium permanganate with pyrimidine bases, which have become perturbed and more reactive by the agent under test. Results are recorded by use of UV/vis spectroscopy. The adaptation to a multi-well plate format provides the capacity for high throughput utilizing small amounts of compounds. Over 100 compounds, comprising different classes of DNA-binding chemicals as well as non-binding controls, have been put through the assay and the results compared with existing genotoxicity testing data from other methods. The assay has shown to be predictive of the results of other genotoxicity testing methods. We have found that the method is overall predictive of 71% of Ames bacterial reverse-mutation test results (where data are given) encompassing both negative and positive results.

Neurotoxicity following chronic intravenous use of "Russian cocktail".

INTRODUCTION: Recently, neurological abnormalities in methcathinone users have been attributed to manganese. We report similar toxicity in three patients following the use of a mixture similar to methcathinone: potassium permanganate, ephedrine, and aspirin. CASE REPORTS: Three teenagers (15 to 19 years old) presented with extrapyramidal abnormalities and movement disorders following chronic intravenous use of a mixture known as "Russian Cocktail". All three patients had multiple movement disorders. One patient had normal blood manganese concentration (<19 microg/L) and MRI. The other two had elevated blood manganese (2100 microg/L and 3176 microg/L) and MRIs showing bilateral symmetric hyper-intensities on T1-weighted-images in the dentate nucleus, subcortical white substance of cerebellar hemisphere, globus pallidus, and putamen. Abstinence and treatment with EDTA, levodopa, and para-aminosalicylic acid was associated with decreasing blood manganese concentrations and subjective improvement, but no change in objective findings. DISCUSSION: The "Russian Cocktail" likely contains manganese as a result of the oxidation of ephedrine by potassium permanganate in water acidified by acetylsalicylic acid. We believe that manganese with the possible contribution of methcathinone caused the neurological impairments. CONCLUSIONS: Three toxic substances have been made into a mixture administered intravenously, similar to methcathinone. Our patients learned of this mixture, called "Russian Cocktail", from their friends. The toxicity from repeated use of this mixture is one of extrapyramidal abnormalities and movement disorders. Standard therapies were unsuccessful in reversing the clinical toxicity.

Removal of arsenic from drinking water by chemical precipitation--a modeling and simulation study of the physical-chemical processes.

A dynamic mathematical model was developed for removal of arsenic from drinking water by chemical coagulation-precipitation and was validated experimentally in a bench-scale set-up. While examining arsenic removal efficiency of the scheme under different operating conditions, coagulant dose, pH and degree of oxidation were found to have pronounced impact. Removal efficiency of 91-92% was achieved for synthetic feed water spiked with 1 mg/L arsenic and pre-oxidized by potassium permanganate at optimum pH and coagulant dose. Model predictions corroborated well with the experimental findings (the overall correlation coefficient being 0.9895) indicating the capability of the model in predicting performance of such a treatment plant under different operating conditions. Menu-driven, user-friendly Visual Basic software developed in the study will be very handy in quick performance analysis. The simulation is expected to be very useful in full-scale design and operation of the treatment plants for removal of arsenic from drinking water.

The role of the lid element in transcription by E. coli RNA polymerase.

The recently described crystal structures of multi-subunit RNA polymerases (RNAPs) reveal a conserved loop-like feature called the lid. The lid projects from the clamp domain and contacts the flap, thereby enclosing the RNA transcript in RNAP's RNA-exit channel and forming the junction between the exit channel and the main channel, which holds the RNA:DNA hybrid. In the initiating form of bacterial RNAP (holoenzyme containing sigma), the lid interacts with sigma region 3 and encloses an extended linker between sigma region 3 and sigma region 4 in place of the RNA in the exit channel. During initiation, the lid may be important for holding open the transcription bubble and may help displace the RNA from the template DNA strand. To test these ideas, we constructed and characterized a mutant RNAP from which the lid element was deleted. Deltalid RNAP exhibited dramatically reduced activity during initiation from -35-dependent and -35-independent promoters, verifying that the lid is important for stabilizing the open promoter complex during initiation. However, transcript elongation, RNA displacement, and, surprisingly, transcriptional termination all occurred normally in Deltalid RNAP. Importantly, Deltalid RNAP behaved differently from wild-type RNAP when transcribing single-stranded DNA templates where it synthesized long, persistent RNA:DNA hybrids, in contrast to efficient transcriptional arrest by wild-type RNAP.

The role of the largest RNA polymerase subunit lid element in preventing the formation of extended RNA-DNA hybrid.

Analysis of multi-subunit RNA polymerase (RNAP) structures revealed several distinct elements that may perform partial functions of the enzyme. One such element, the "lid", is formed by an evolutionarily conserved segment of the RNAP largest subunit (beta' in bacterial RNAP). The beta' lid contacts the nascent RNA at the upstream edge of the RNA-DNA hybrid, where the RNA gets separated from the DNA template-strand and double-stranded upstream DNA is formed. To test the beta' lid functions, we generated bacterial RNAP lacking the lid and studied the mutant enzyme's properties in vitro. Our results demonstrate that removal of the lid has minimal consequences on transcription elongation from double-stranded DNA. On single-stranded DNA, the mutant RNAP generates full-sized transcripts that remain annealed to the DNA throughout their length. In contrast, the wild-type enzyme produces short, 18-22 nucleotide transcripts that remain part of the transcription complex but cannot be further elongated. The cessation of transcription is apparently triggered by a clash between the lid and the nascent RNA 5' end. The results show that the lid's function is redundant in the presence of the non-template DNA strand, which alone can control the proper geometry of nucleic acids at the upstream edge of the transcription complex. Structural considerations suggest that in the absence of the non-template strand and the lid, a new channel opens within the RNAP molecule that allows continuous DNA-RNA hybrid to exit RNAP.

Mg2+-modulated KMnO4 reactivity of thymines in the open transcription complex reflects variation in the negative electrostatic potential along the separated DNA strands. Footprinting of Escherichia coli RNA polymerase complex at the lambdaP(R) promoter revisited.

There is still a controversy over the mechanism of promoter DNA strand separation upon open transcription complex (RPo) formation by Escherichia coli RNA polymerase: is it a single or a stepwise process controlled by Mg2+ ions and temperature? To resolve this question, the kinetics of pseudo-first-order oxidation of thymine residues by KMnO4 in the -11 ... +2 DNA region of RPo at the lambdaP(R) promoter was examined under single-hit conditions as a function of temperature (13-37 degrees C) in the absence or presence of 10 mm MgCl2. The reaction was also studied with respect to thymidine and its nucleotides (TMP, TTP and TpT) as a function of temperature and [MgCl2]. The kinetic parameters, (ox)k and (ox)E(a), and Mg-induced enhancement of (ox)k proved to be of the same order of magnitude for RPo-lambdaP(R) and the nucleotides. Unlike the complex, (ox)E(a) for the nucleotides was found to be Mg-independent. The isothermal increase in (ox)k with increasing [Mg2+] was thus interpreted in terms of a simple model of screening of the negative charges on phosphate groups by Mg2+ ions, lowering the electrostatic barrier to the diffusion of MnO4- anions to the reactive double bond of thymine. Similar screening isotherms were determined for the oxidation of two groups of thymines in RPo at a consensus-like Pa promoter, differing in the magnitude of the Mg effect. Together, the findings show that: (a) the two DNA strands in the -11...+2 region of RPo-lambdaP(R) are completely separated over the whole range of temperatures investigated (13-37 degrees C) in the absence of Mg2+ (b) Mg2+ ions induce an increase in the rate of the oxidation reaction by screening negatively charged phosphate and carboxylate groups; and (c) the observed thymine reactivity and the magnitude of the Mg effect reflect variation in the strength of the electrostatic potential along the separated DNA strands, in agreement with the current structural model of RPo.

Succession of aquatic microbial communities as a result of the water quality variations in continuous water.

The changes of structural and functional parameters of aquatic microbial communities in continuous water on campus of Tsinghua University, China are investigated, by polyurethane foam unit (PFU) method. The measured compositions of the communities include alga, protozoa, and some metazoa (such as rotifers). The measured indicators of water quality include water temperature, pH value, dissolved oxygen (DO), potassium permanganate index (COD(Mn)), total nitrogen (TN), total phosphorus (TP) and chlorophyll-a (Chla). The trophic level, expressed by the trophic level indices (TLIc), is assessed with analytic hierarchy process and principal component analysis (AHP-PCA) method. The changing trends of the structural and functional parameters of aquatic microbial communities, such as Margalef index of diversity (D), Shannon-weaver index of diversity (H), Heterotropy index (HI), number of species when the colonization gets equilibrium (S(eq)), colonizing speed constant (G) and time spent when 90 percent of S(eq) colonized in PFU (T(80%)), are also analyzed. The experimental results showed the succession of aquatic microbial communities along the water flow is consistent with the water quality changes, so the parameters of microbial community can reflect the changes of water quality from the ecological view.

A mutant spacer sequence between -35 and -10 elements makes the Plac promoter hyperactive and cAMP receptor protein-independent.

To determine whether the spacer region between the -35 and -10 elements plays any sequence-specific role, we randomized the GC-rich sequence ((-20)CCGGCTCG(-13)) within the spacer region of the cAMP-dependent lac promoter and selected an activator-independent mutant, which showed extraordinarily high intrinsic activity. The hyperactive promoter is obtained by incorporation of a specific 10-bp-long AT-rich DNA sequence within the spacer, referred to as the -15 sequence, which must be juxtaposed to the upstream end of the -10 sequence for the hyperactivity. The transcription enhancement functions only in the presence of a -35 element. The spacer sequence enhanced both RNA polymerase binding and open complex formation. Isolated in the lac promoter, it also enhanced transcription when placed at two other unrelated promoters. Sequence analysis shows a low GC content and an abundance of stereochemically flexible TG:CA and TA:TA dimeric steps in the -18/-9 region and a strong correlation between the presence of flexible dimeric steps in this region and the intrinsic strength of the promoter.

Potassium permanganate as a probe to map DNA-protein interactions in vivo.

Potassium permanganate (KMnO4) has widely been used in genomic footprinting assays to map unusual gene structures, including the melting DNA block in transcriptional elongation that results from promoter-proximal pausing of RNA polymerase (Pol) II complexes. Although it has been assumed that DNA-bound proteins do not protect underlying nucleic acids from KMnO4 modifications, we provide evidence herein that this chemical can readily be used to detect nuclear factor loading at a promoter when using optimized conditions. Moreover, by comparing parallel KMnO4 and dimethylsulfate (DMS) in vivo footprintings, we show that the utilization of KMnO4 in combination with another chemical probe maximizes the detection of factor occupancy at a DNA regulatory region, thus providing a better opportunity to define the actual profiles of DNA-protein contacts at given genomic sites in living cells.

UV-visible spectral identification of the solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid.

Solution-phase and solid-phase permanganate oxidation reactions of thymine acetic acid were investigated by spectroscopy. The spectral data showed the formation of a stable organomanganese intermediate, which was responsible for the rise in the absorbance at 420 nm. This result enables unambiguous interpretation of the absorbance change at 420 nm, as the intermediate permanganate ions could be isolated on the solid supports.

Mechanism of transcriptional activation by FIS: role of core promoter structure and DNA topology.

The Escherichia coli DNA architectural protein FIS activates transcription from stable RNA promoters on entry into exponential growth and also reduces the level of negative supercoiling. Here we show that such a reduction decreases the activity of the tyrT promoter but that activation by FIS rescues tyrT transcription at non-optimal superhelical densities. Additionally we show that three different "up" mutations in the tyrT core promoter either abolish or reduce the dependence of tyrT transcription on both high negative superhelicity and FIS in vivo and infer that the specific sequence organisation of the core promoter couples the control of transcription initiation by negative superhelicity and FIS. In vitro all the mutations potentiate FIS-independent untwisting of the -10 region while at the wild-type promoter FIS facilitates this step. We propose that this untwisting is a crucial limiting step in the initiation of tyrT RNA synthesis. The tyrT core promoter structure is thus optimised to combine high transcriptional activity with acute sensitivity to at least three major independent regulatory inputs: negative superhelicity, FIS and ppGpp.

The TRTGn motif stabilizes the transcription initiation open complex.

The effect on transcription initiation by the extended -10 motif (5'-TRTG(n)-3'), positioned upstream of the -10 region, was investigated using a series of base substitution mutations in the alpha-amylase promoter (amyP). The extended -10 motif, previously referred to as the -16 region, is found frequently in Gram-positive bacterial promoters and several extended -10 promoters from Escherichia coli. The inhibitory effects of the non-productive promoter site (amyP2), which overlaps the upstream region of amyP, were eliminated by mutagenesis of the -35 region and the TRTG motif of amyP2. Removal by mutagenesis of the competitive effects of amyP2 resulted in a reduced dependence of amyP on the TRTG motif. In the absence of the second promoter, mutations in the TRTG motif of amyP destabilized the open complex and prevented the maintenance of open complexes at low temperatures. The open complex half-life was up to 26-fold shorter in the mutant TRTG motif promoters than in the wild-type promoter. We demonstrate that the amyP TRTG motif dramatically stabilizes the open complex intermediate during transcription initiation. Even though the open complex is less stable in the mutant promoters, the region of melted DNA is the same in the wild-type and mutant promoters. However, upon addition of the first three nucleotides, which trap RNAP (RNA polymerase) in a stable initiating complex, the melted DNA region contracts at the 5'-end in a TRTG motif promoter mutant but not at the wild-type promoter, indicating that the motif contributes to maintaining DNA-strand separation.