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Bacterial biofilms are a common cause of chronic medical implant infections. Treatment and eradication of biofilms by conventional antibiotic therapy has major drawbacks including toxicity and side effects associated with high-dosage antibiotics. Additionally, administration of high doses of antibiotics may facilitate the emergence of antibiotic resistant bacteria. Thus, there is an urgent need for the development of treatments that are not based on conventional antibiotic therapies. Presented herein is a novel bacterial biofilm combination treatment independent of traditional antibiotics, by using low electric fields in combination with small molecule inhibitors of bacterial quorum sensing - autoinducer-2 analogs. We investigate the effect of this treatment on mature Escherichia coli biofilms by application of an alternating and offset electric potential in combination with the small molecule inhibitor for 24 h using both macro and micro-scale devices. Crystal violet staining of the macro-scale biofilms shows a 46 % decrease in biomass compared to the untreated control. We demonstrate enhanced treatment efficacy of the combination therapy using a high-throughput polydimethylsiloxane-based microfluidic biofilm analysis platform. This microfluidic flow cell is designed to reduce the growth variance of in vitro biofilms while providing an integrated control, and thus allows for a more reliable comparison and evaluation of new biofilm treatments on a single device. We utilize linear array charge-coupled devices to perform real-time tracking of biomass by monitoring changes in optical density. End-point confocal microscopy measurements of biofilms treated with the autoinducer analog and electric fields in the microfluidic device show a 78 % decrease in average biofilm thickness in comparison to the negative controls and demonstrate good correlation with real-time optical density measurements. Additionally, the combination treatment showed 76 % better treatment efficacy compared to conventional antibiotic therapy. Taken together these results suggest that the antibiotic-free combination treatment described here may provide an effective alternative to traditional antibiotic therapies against bacterial biofilm infections. Use of this combination treatment in the medical and environmental fields would alleviate side effects associated with high-dosage antibiotic therapies, and reduce the rise of antibiotic-resistant bacteria.
Assuntos
Biofilmes/efeitos dos fármacos , Eletricidade , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Homosserina/análogos & derivados , Lactonas/química , Lactonas/farmacologia , Escherichia coli/citologia , Homosserina/química , Homosserina/farmacologia , Dispositivos Lab-On-A-Chip , Microscopia Confocal , Percepção de Quorum/efeitos dos fármacosRESUMO
Reduced forms of the C56S and C60S variants of the thioredoxin-like Clostridium pasteurianum [Fe2S2] ferredoxin (CpFd) provide the only known examples of valence-delocalized [Fe2S2](+) clusters, which constitute a fundamental building block of all higher nuclearity Fe-S clusters. In this work, we have revisited earlier work on the CpFd variants and carried out redox and spectroscopic studies on the [Fe2S2](2+,+) centers in wild-type and equivalent variants of the highly homologous and structurally characterized Aquifex aeolicus ferredoxin 4 (AaeFd4) using EPR, UV-visible-NIR absorption, CD and variable-temperature MCD, and protein-film electrochemistry. The results indicate that the [Fe2S2](+) centers in the equivalent AaeFd4 and CpFd variants reversibly interconvert between similar valence-localized S = 1/2 and valence-delocalized S = 9/2 forms as a function of pH, with pKa values in the range 8.3-9.0, because of protonation of the coordinated serinate residue. However, freezing high-pH samples results in partial or full conversion from valence-delocalized S = 9/2 to valence-localized S = 1/2 [Fe2S2](+) clusters. MCD saturation magnetization data for valence-delocalized S = 9/2 [Fe2S2](+) centers facilitated determination of transition polarizations and thereby assignments of low-energy MCD bands associated with the Fe-Fe interaction. The assignments provide experimental assessment of the double exchange parameter, B, for valence-delocalized [Fe2S2](+) centers and demonstrate that variable-temperature MCD spectroscopy provides a means of detecting and investigating the properties of valence-delocalized S = 9/2 [Fe2S2](+) fragments in higher nuclearity Fe-S clusters. The origin of valence delocalization in thioredoxin-like ferredoxin Cys-to-Ser variants and Fe-S clusters in general is discussed in light of these results.
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Ferredoxinas/química , Ferro/química , Enxofre/química , Tiorredoxinas/química , Aquifoliaceae , Clostridium , Ferredoxinas/metabolismo , Ferro/metabolismo , Oxirredução , Análise Espectral , Enxofre/metabolismoRESUMO
Fumarate and nitrate reduction (FNR) regulatory proteins are O(2)-sensing bacterial transcription factors that control the switch between aerobic and anaerobic metabolism. Under anaerobic conditions [4Fe-4S](2+)-FNR exists as a DNA-binding homodimer. In response to elevated oxygen levels, the [4Fe-4S](2+) cluster undergoes a rapid conversion to a [2Fe-2S](2+) cluster, resulting in a dimer-to-monomer transition and loss of site-specific DNA binding. In this work, resonance Raman and UV-visible absorption/CD spectroscopies and MS were used to characterize the interconversion between [4Fe-4S](2+) and [2Fe-2S](2+) clusters in Escherichia coli FNR. Selective (34)S labeling of the bridging sulfides in the [4Fe-4S](2+) cluster-bound form of FNR facilitated identification of resonantly enhanced Cys(32)S-(34)S stretching modes in the resonance Raman spectrum of the O(2)-exposed [2Fe-2S](2+) cluster-bound form of FNR. This result indicates O(2)-induced oxidation and retention of bridging sulfides in the form of [2Fe-2S](2+) cluster-bound cysteine persulfides. MS also demonstrates that multiple cysteine persulfides are formed on O(2) exposure of [4Fe-4S](2+)-FNR. The [4Fe-4S](2+) cluster in FNR can also be regenerated from the cysteine persulfide-coordinated [2Fe-2S](2+) cluster by anaerobic incubation with DTT and Fe(2+) ion in the absence of exogenous sulfide. Resonance Raman data indicate that this type of cluster conversion involving sulfide oxidation is not unique to FNR, because it also occurs in O(2)-exposed forms of O(2)-sensitive [4Fe-4S] clusters in radical S-adenosylmethionine enzymes. The results provide fresh insight into the molecular mechanism of O(2) sensing by FNR and iron-sulfur cluster conversion reactions in general, and suggest unique mechanisms for the assembly or repair of biological [4Fe-4S] clusters.
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Cisteína/química , Proteínas de Escherichia coli/química , Compostos Ferrosos/química , Proteínas Ferro-Enxofre/química , Modelos Químicos , Oxigênio/química , Cisteína/metabolismo , Proteínas de Escherichia coli/metabolismo , Compostos Ferrosos/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Oxigênio/metabolismo , Análise EspectralRESUMO
The biosynthesis of the organometallic H cluster of [Fe-Fe] hydrogenase requires three accessory proteins, two of which (HydE and HydG) belong to the radical S-adenosylmethionine enzyme superfamily. The third, HydF, is an Fe-S protein with GTPase activity. The [4Fe-4S] cluster of HydF is bound to the polypeptide chain through only the three, conserved, cysteine residues present in the binding sequence motif CysXHisX(46-53)HisCysXXCys. However, the involvement of the two highly conserved histidines as a fourth ligand for the cluster coordination is controversial. In this study, we set out to characterize further the [4Fe-4S] cluster of HydF using Mössbauer, EPR, hyperfine sublevel correlation (HYSCORE), and resonance Raman spectroscopy in order to investigate the influence of nitrogen ligands on the spectroscopic properties of [4Fe-4S](2+/+) clusters. Our results show that Mössbauer, resonance Raman, and EPR spectroscopy are not able to readily discriminate between the imidazole-coordinated [4Fe-4S] cluster and the non-imidazole-bound [4Fe-4S] cluster with an exchangeable fourth ligand that is present in wild-type HydF. HYSCORE spectroscopy, on the other hand, detects the presence of an imidazole/histidine ligand on the cluster on the basis of the appearance of a specific spectral pattern in the strongly coupled region, with a coupling constant of approximately 6 MHz. We also discovered that a His-tagged version of HydF, with a hexahistidine tag at the N-terminus, has a [4Fe-4S] cluster coordinated by one histidine from the tag. This observation strongly indicates that care has to be taken in the analysis of data obtained on tagged forms of metalloproteins.
Assuntos
Proteínas Ferro-Enxofre/química , Thermotoga maritima/enzimologia , Espectroscopia de Ressonância de Spin Eletrônica , Histidina/química , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Espectroscopia de Mossbauer , Análise Espectral Raman , Thermotoga maritima/química , Thermotoga maritima/metabolismoRESUMO
Nfu-type proteins are essential in the biogenesis of iron-sulfur (Fe-S) clusters in numerous organisms. A number of phenotypes including low levels of Fe-S cluster incorporation are associated with the deletion of the gene encoding a chloroplast-specific Nfu-type protein, Nfu2 from Arabidopsis thaliana (AtNfu2). Here, we report that recombinant AtNfu2 is able to assemble both [2Fe-2S] and [4Fe-4S] clusters. Analytical data and gel filtration studies support cluster/protein stoichiometries of one [2Fe-2S] cluster/homotetramer and one [4Fe-4S] cluster/homodimer. The combination of UV-visible absorption and circular dichroism and resonance Raman and Mössbauer spectroscopies has been employed to investigate the nature, properties, and transfer of the clusters assembled on Nfu2. The results are consistent with subunit-bridging [2Fe-2S](2+) and [4Fe-4S](2+) clusters coordinated by the cysteines in the conserved CXXC motif. The results also provided insight into the specificity of Nfu2 for the maturation of chloroplastic Fe-S proteins via intact, rapid, and quantitative cluster transfer. [2Fe-2S] cluster-bound Nfu2 is shown to be an effective [2Fe-2S](2+) cluster donor for glutaredoxin S16 but not glutaredoxin S14. Moreover, [4Fe-4S] cluster-bound Nfu2 is shown to be a very rapid and efficient [4Fe-4S](2+) cluster donor for adenosine 5'-phosphosulfate reductase (APR1), and yeast two-hybrid studies indicate that APR1 forms a complex with Nfu2 but not with Nfu1 and Nfu3, the two other chloroplastic Nfu proteins. This cluster transfer is likely to be physiologically relevant and is particularly significant for plant metabolism as APR1 catalyzes the second step in reductive sulfur assimilation, which ultimately results in the biosynthesis of cysteine, methionine, glutathione, and Fe-S clusters.
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Proteínas de Arabidopsis/química , Cloroplastos/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Endonucleases/metabolismo , Glutarredoxinas/metabolismo , Proteínas Ferro-Enxofre/química , Espectrofotometria Ultravioleta , Análise Espectral RamanRESUMO
The roles of four conserved basic amino acids in the reaction catalyzed by the ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 have been investigated using site-directed mutagenesis in combination with measurements of steady-state kinetics, substrate-binding affinities, and spectroscopic properties of the enzyme's two prosthetic groups. Replacement of either Lys58 or Arg70 by glutamine leads to a complete loss of activity, both with the physiological electron donor, reduced ferredoxin, and with a nonphysiological electron donor, reduced methyl viologen. More conservative, charge-maintaining K58R and R70K variants were also completely inactive. Replacement of Lys130 by glutamine produced a variant that retained 26% of the wild-type activity with methyl viologen as the electron donor and 22% of the wild-type activity with ferredoxin as the electron donor, while replacement by arginine produces a variant that retains a significantly higher percentage of the wild-type activity with both electron donors. In contrast, replacement of Arg146 by glutamine had minimal effect on the activity of the enzyme. These results, along with substrate-binding and spectroscopic measurements, are discussed in terms of an in silico structural model for the enzyme.
Assuntos
Aminoácidos Básicos/química , Ferredoxinas/química , Nitrato Redutase/química , Synechococcus/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sequência Conservada , Glutamina/química , Glutamina/genética , Dados de Sequência Molecular , Nitrato Redutase/genética , Especificidade por Substrato/genética , Synechococcus/genéticaRESUMO
We report stable and heterogeneous graphene oxide (GO)-intercalated copper as an efficient catalyst for the organic transformations in green solvents. The GO-intercalated copper(II) complex of bis(1,4,7,10-tetraazacyclododecane) [Cu(II)-bis-cyclen] was prepared by a facile synthetic approach with a high dilution technique. The as-prepared GO-Cu(II)-bis-cyclen nanocomposite was used as a click catalyst for the 1,3 dipolar Huisgen cycloaddition reaction of terminal alkyne and azide substrates. On directing a great deal of attention toward the feasibility of the rapid electron transfer rate of the catalyst in proliferating the yield of 1,2,3-triazole products, the click catalyst GO-Cu(II)-bis-cyclen nanocomposite was designed and synthesized via non-covalent functionalization. The presence of a higher coordination site in an efficient 2D nanocomposite promotes the stabilization of Cu(I) L-acetylide intermediate during the catalytic cycle initiated by the addition of reductants. From the XRD analysis, the enhancement in the d-interlayer spacing of 1.04 nm was observed due to the intercalation of the Cu(II)-bis-cyclen complex in between the GO basal planes. It was also characterized by XPS, FT-IR, RAMAN, UV, SEM, AFM, and TGA techniques. The recyclability of the heterogeneous catalyst [GO-Cu(II)-cyclen] with the solvent effect has also been studied. This class of GO-Cu(II)-bis-cyclen nanocomposite paves the way for bioconjugation of macromolecules through the click chemistry approach.
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Biofilms are the primary cause of clinical bacterial infections and are impervious to typical amounts of antibiotics, necessitating very high doses for elimination. Therefore, it is imperative to have suitable methods for characterization to develop novel methods of treatment that can complement or replace existing approaches using significantly lower doses of antibiotics. This review presents some of the current developments in microsystems for characterization and sensing of bacterial biofilms. Initially, we review current standards for studying biofilms that are based on invasive and destructive end-point biofilm characterization. Additionally, biofilm formation and growth is extremely sensitive to various growth and environmental parameters that cause large variability in biofilms between repeated experiments, making it very difficult to compare experimental repeats and characterize the temporal characteristics of these organisms. To address these challenges, recent developments in the field have moved toward systems and miniature devices that can aid in the non-invasive characterization of bacterial biofilms. Our review focuses on several types of microsystems for biofilm evaluation including optical, electrochemical, and mechanical systems. This review will show how these devices can lead to better understanding of the physiology and function of these communities of bacteria, which can eventually lead to the development of novel treatments that do not rely on high-dosage antibiotics.
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WhiD, a member of the WhiB-like (Wbl) family of iron-sulfur proteins found exclusively within the actinomycetes, is required for the late stages of sporulation in Streptomyces coelicolor. Like all other Wbl proteins, WhiD has not so far been purified in a soluble form that contains a significant amount of cluster, and characterization has relied on cluster-reconstituted protein. Thus, a major goal in Wbl research is to obtain and characterize native protein containing iron-sulfur clusters. Here we report the analysis of S. coelicolor WhiD purified anaerobically from Escherichia coli as a soluble protein containing a single [4Fe-4S](2+) cluster ligated by four cysteines. Upon exposure to oxygen, spectral features associated with the [4Fe-4S] cluster were lost in a slow reaction that unusually yielded apo-WhiD directly without significant concentrations of cluster intermediates. This process was found to be highly pH dependent with an optimal stability observed between pH 7.0 and pH 8.0. Low molecular weight thiols, including a mycothiol analogue and thioredoxin, exerted a small but significant protective effect against WhiD cluster loss, an activity that could be of physiological importance. [4Fe-4S](2+) WhiD was found to react much more rapidly with superoxide than with either oxygen or hydrogen peroxide, which may also be of physiological significance. Loss of the [4Fe-4S] cluster to form apoprotein destabilized the protein fold significantly but did not lead to complete unfolding. Finally, apo-WhiD exhibited negligible activity in an insulin-based disulfide reductase assay, demonstrating that it does not function as a general protein disulfide reductase.
Assuntos
Proteínas Ferro-Enxofre/química , Streptomyces coelicolor/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/isolamento & purificação , Proteínas Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Estabilidade Proteica , Solubilidade , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
The S-adenosylmethionine-dependent enzyme MoaA, in concert with MoaC, catalyzes the first step of molybdenum cofactor biosynthesis, the conversion of guanosine 5'-triphosphate (5'-GTP) into precursor Z. A published X-ray crystal structure of MoaA with the substrate 5'-GTP revealed that the substrate might be bound to the unique iron of one of two 4Fe-4S clusters through either or both the amino and N1 nitrogen nuclei. Use of 35 GHz continuous-wave ENDOR spectroscopy of MoaA with unlabeled and (15)N-labeled substrate and a reduced [4Fe-4S](+) cluster now demonstrates that only one nitrogen nucleus is bound to the cluster. Experiments with the substrate analogue inosine 5'-triphosphate further demonstrate that it is the N1 nitrogen that binds. Two of the more distant nitrogen nuclei have also been detected by 35 GHz pulsed ENDOR spectroscopy, allowing a rough approximation of their distances from the cluster to be calculated. Combining this information with the crystal structure, we propose that the guanine base adopts the enol tautomer as N1 binds to Fe4 and the O6-H hydroxyl group forms a hydrogen bond with S4 of the 4Fe-4S cluster, and that this binding-induced tautomerization may have important mechanistic ramifications.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , Guanina/química , Guanosina Trifosfato/metabolismo , Hidrolases/química , Hidrolases/metabolismo , Staphylococcus aureus/enzimologia , Sítios de Ligação , Cristalografia por Raios X , Guanina/metabolismo , Guanosina Trifosfato/química , Hidrolases/genética , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação ProteicaRESUMO
GOAL: This paper reports a platform for real-time monitoring and treatment of biofilm formation on three-dimensional biomedical device surfaces. METHODS: We utilize a flexible platform consisting of gold interdigitated electrodes patterned on a polyimide substrate. The device was integrated onto the interior of a urinary catheter and characterization was performed in a custom-developed flow system. Biofilm growth was monitored via impedance change at 100 Hz ac with a 50 mV signal amplitude. RESULTS: A 30% impedance decrease over 24 h corresponded to Escherichia coli biofilm formation. The platform also enabled removal of the biofilm through the bioelectric effect; a low concentration of antibiotic combined with the applied ac voltage signal led to a synergistic reduction in biofilm resulting in a 12% increase in impedance. Biomass characterization via crystal violet staining confirmed that the impedance detection results correlate with changes in the amount of biofilm biomass on the sensor. We also demonstrated integration with a chip-based impedance converter to enable miniaturization and allow in situ wireless implementation. A 5% impedance decrease measured with the impedance converter corresponded to biofilm growth, replicating the trend measured with the potentiostat. CONCLUSION: This platform represents a promising solution for biofilm infection management in diverse vulnerable environments. SIGNIFICANCE: Biofilms are the dominant mode of growth for microorganisms, where bacterial cells colonize hydrated surfaces and lead to recurring infections. Due to the inaccessible nature of the environments where biofilms grow and their increased tolerance of antimicrobials, identification, and removal on medical devices poses a challenge.
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Biofilmes , Técnicas Biossensoriais/métodos , Impedância Elétrica , Escherichia coli , Biofilmes/crescimento & desenvolvimento , Biofilmes/efeitos da radiação , Biomassa , Desenho de Equipamento , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/efeitos da radiação , Ouro , Microeletrodos , Maleabilidade , Análise Espectral , Cateteres Urinários/microbiologiaRESUMO
Bacterial biofilms are the primary cause of infections in medical implants and catheters. Delayed detection of biofilm infections contributes to the widespread use of high doses of antibiotics, leading to the emergence of antibiotic-resistant bacterial strains. Accordingly, there is an urgent need for systems that can rapidly detect and treat biofilm infections in situ. As a step toward this goal, in this work we have developed for the first time a threshold-activated feedback-based impedance sensor-treatment system for combined real-time detection and treatment of biofilms. Specifically, we demonstrate the use of impedimetric sensing to accurately monitor the growth of Escherichia coli biofilms in microfluidic flow cells by measuring the fractional relative change (FRC) in absolute impedance. Furthermore, we demonstrate the use of growth measurements as a threshold-activated trigger mechanism to initiate successful treatment of biofilms using bioelectric effect (BE), applied through the same sensing electrode array. This was made possible through a custom program that (a) monitored the growth and removal of biofilms within the microfluidic channels in real-time and (b) enabled the threshold-based activation of BE treatment. Such BE treatment resulted in a â¼74.8 % reduction in average biofilm surface coverage as compared to the untreated negative control. We believe that this smart microsystem for integrated biofilm sensing and treatment will enable future development of autonomous biosensors optimized for accurate real-time detection of the onset of biofilms and their in situ treatment, directly on the surfaces of medical implants.
Assuntos
Biofilmes , Técnicas Biossensoriais , Impedância Elétrica , Escherichia coli , MicrofluídicaRESUMO
BACKGROUND/OBJECTIVES: The use of electric fields in combination with small doses of antibiotics for enhanced treatment of biofilms is termed the 'bioelectric effect' (BE). Different mechanisms of action for the AC and DC fields have been reported in the literature over the last two decades. In this work, we conduct the first study on the correlation between the electrical energy and the treatment efficacy of the bioelectric effect on Escherichia coli K-12 W3110 biofilms. METHODS: A thorough study was performed through the application of alternating (AC), direct (DC) and superimposed (SP) potentials of different amplitudes on mature E. coli biofilms. The electric fields were applied in combination with the antibiotic gentamicin (10 µg/ml) over a course of 24 h, after the biofilms had matured for 24 h. The biofilms were analysed using the crystal violet assay, the colony-forming unit method and fluorescence microscopy. RESULTS: Results show that there is no statistical difference in treatment efficacy between the DC-, AC- and SP-based BE treatment of equivalent energies (analysis of variance (ANOVA) P>0.05) for voltages <1 V. We also demonstrate that the efficacy of the BE treatment as measured by the crystal violet staining method and colony-forming unit assay is proportional to the electrical energy applied (ANOVA P<0.05). We further verify that the treatment efficacy varies linearly with the energy of the BE treatment (r2 =0.984). Our results thus suggest that the energy of the electrical signal is the primary factor in determining the efficacy of the BE treatment, at potentials less than the media electrolysis voltage. CONCLUSIONS: Our results demonstrate that the energy of the electrical signal, and not the type of electrical signal (AC or DC or SP), is the key to determine the efficacy of the BE treatment. We anticipate that this observation will pave the way for further understanding of the mechanism of action of the BE treatment method and may open new doors to the use of electric fields in the treatment of bacterial biofilms.
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Loop mediated isothermal amplification (LAMP) is a highly efficient, selective and rapid DNA amplification technique for genetic screening of pathogens. However, despite its popularity, there is yet no mathematical model to quantify the outcome and no well-defined metric for comparing results that are available. LAMP is intrinsically complex and involves multiple pathways for gene replication, making fundamental modelling nearly intractable. To circumvent this difficulty, an alternate, empirical model is introduced that will allow one to extract a set of parameters from the concentration versus time curves. A simple recipe to deduce the time to positive, Tp--a parameter analogous to the threshold cycling time in polymerase chain reaction (PCR), is also provided. These parameters can be regarded as objective and unambiguous indicators of LAMP amplification. The model is exemplified on Escherichia coli strains by using the two gene fragments responsible for vero-toxin (VT) production and tested against VT-producing (O157 and O45) and non-VT producing (DH5 alpha) strains. Selective amplification of appropriate target sequences was made using well established LAMP primers and protocols, and the concentrations of the amplicons were measured using a Qubit 2.0 fluorometer at specific intervals of time. The data is fitted to a generalized logistic function. Apart from providing precise screening indicators, representing the data with a small set of numbers offers significant advantages. It facilitates comparisons of LAMP reactions independently of the sampling technique. It also eliminates subjectivity in interpretation, simplifies data analysis, and allows easy data archival, retrieval and statistical analysis for large sample populations. To our knowledge this work represents a first attempt to quantitatively model LAMP and offer a standard method that could pave the way towards high throughput automated screening.
Assuntos
DNA Bacteriano/genética , Escherichia coli O157/genética , Escherichia coli/genética , Modelos Estatísticos , Técnicas de Amplificação de Ácido Nucleico/métodos , Toxinas Shiga/genética , Calibragem , Primers do DNA/química , Escherichia coli/metabolismo , Escherichia coli O157/metabolismo , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Toxinas Shiga/biossínteseRESUMO
Due to the growing need to provide alternatives to fossil fuels as efficiently, economically, and sustainably as possible there has been growing interest in improved biofuel production systems. Biofuels produced from microalgae are a particularly attractive option since microalgae have production potentials that exceed the best terrestrial crops by 2 to 10-fold. In addition, autotrophically grown microalgae can capture CO2 from point sources reducing direct atmospheric greenhouse gas emissions. The enhanced biomass production potential of algae is attributed in part to the fact that every cell is photosynthetic. Regardless, overall biological energy capture, conversion, and storage in microalgae are inefficient with less than 8% conversion of solar into chemical energy achieved. In this review, we examine the thermodynamic and kinetic constraints associated with the autotrophic conversion of inorganic carbon into storage carbohydrate and oil, the dominant energy storage products in Chlorophytic microalgae. We discuss how thermodynamic restrictions including the loss of fixed carbon during acetyl CoA synthesis reduce the efficiency of carbon accumulation in lipids. In addition, kinetic limitations, such as the coupling of proton to electron transfer during plastoquinone reduction and oxidation and the slow rates of CO2 fixation by Rubisco reduce photosynthetic efficiency. In some cases, these kinetic limitations have been overcome by massive increases in the numbers of effective catalytic sites, e.g. the high Rubisco levels (mM) in chloroplasts. But in other cases, including the slow rate of plastoquinol oxidation, there has been no compensatory increase in the abundance of catalytically limiting protein complexes. Significantly, we show that the energetic requirements for producing oil and starch relative to the recoverable energy stored in these molecules are very similar on a per carbon basis. Presently, the overall rates of starch and lipid synthesis in microalgae are very poorly characterized. Increased understanding of the kinetic constraints of lipid and starch synthesis, accumulation and turnover would facilitate the design of improved biomass production systems.
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An electronic platform to detect very small amounts of genomic DNA from bacteria without the need for PCR amplification and molecular labeling is described. The system uses carbon nanotube field-effect transistor (FET) arrays whose electrical properties are affected by minute electrical charges localized on their active regions. Two pathogenic strains of E. coli are used to evaluate the detection properties of the transistor arrays. Described herein are the results for detection of synthetic oligomers, unpurified and highly purified genomic DNA at various concentrations and their comparison against non-specific binding. In particular, the capture of genomic DNA of E. coli O157:H7 by a specific oligonucleotide probe coated onto the transistor array results in a significant shift in the threshold (gate-source) voltage (V(th)). By contrast the signal under the same procedure using a different strain, E. coli O45 that is non-complementary to the probe remained nearly constant. This work highlights the detection sensitivity and efficacy of this biosensor without stringent requirement for DNA sample preparation.
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Técnicas Biossensoriais/instrumentação , DNA Bacteriano/genética , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Nanotubos de Carbono/química , Sequência de Bases , Técnicas Biossensoriais/economia , Técnicas Biossensoriais/métodos , DNA Bacteriano/análise , Desenho de Equipamento , Fatores de Tempo , Transistores EletrônicosRESUMO
Sulfatases form a major group of enzymes present in prokaryotes and eukaryotes. This class of hydrolases is unique in requiring essential post-translational modification of a critical active-site cysteinyl or seryl residue to C(alpha)-formylglycine (FGly). Herein, we report mechanistic investigations of a unique class of radical-S-adenosyl-L-methionine (AdoMet) enzymes, namely anaerobic sulfatase-maturating enzymes (anSMEs), which catalyze the oxidation of Cys-type and Ser-type sulfatases and possess three [4Fe-4S](2+,+) clusters. We were able to develop a reliable quantitative enzymatic assay that allowed the direct measurement of FGly production and AdoMet cleavage. The results demonstrate stoichiometric coupling of AdoMet cleavage and FGly formation using peptide substrates with cysteinyl or seryl active-site residues. Analytical and EPR studies of the reconstituted wild-type enzyme and cysteinyl cluster mutants indicate the presence of three almost isopotential [4Fe-4S](2+,+) clusters, each of which is required for the generation of FGly in vitro. More surprisingly, our data indicate that the two additional [4Fe-4S](2+,+) clusters are required to obtain efficient reductive cleavage of AdoMet, suggesting their involvement in the reduction of the radical AdoMet [4Fe-4S](2+,+) center. These results, in addition to the recent demonstration of direct abstraction by anSMEs of the C(beta) H-atom from the sulfatase active-site cysteinyl or seryl residue using a 5'-deoxyadenosyl radical, provide new insights into the mechanism of this new class of radical-AdoMet enzymes.
Assuntos
Alanina/análogos & derivados , Glicina/análogos & derivados , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Sulfatases/metabolismo , Alanina/metabolismo , Anaerobiose , Sítios de Ligação , Catálise , Cisteína/química , Espectroscopia de Ressonância de Spin Eletrônica , Eucariotos , Glicina/metabolismo , Oxirredução , S-Adenosilmetionina/classificação , S-Adenosilmetionina/metabolismo , Serina/química , Especificidade por Substrato , Sulfatases/químicaRESUMO
Sulfatases are a major group of enzymes involved in many critical physiological processes as reflected by their broad distribution in all three domains of life. This class of hydrolases is unique in requiring an essential post-translational modification of a critical active-site cysteine or serine residue to C(alpha)-formylglycine. This modification is catalyzed by at least three nonhomologous enzymatic systems in bacteria. Each enzymatic system is currently considered to be dedicated to the modification of either cysteine or serine residues encoded in the sulfatase-active site and has been accordingly categorized as Cys-type and Ser-type sulfatase-maturating enzymes. We report here the first detailed characterization of two bacterial anaerobic sulfatase-maturating enzymes (anSMEs) that are physiologically responsible for either Cys-type or Ser-type sulfatase maturation. The activity of both enzymes was investigated in vivo and in vitro using synthetic substrates and the successful purification of both enzymes facilitated the first biochemical and spectroscopic characterization of this class of enzyme. We demonstrate that reconstituted anSMEs are radical S-adenosyl-l-methionine enzymes containing a redox active [4Fe-4S](2+,+) cluster that initiates the radical reaction by binding and reductively cleaving S-adenosyl-l-methionine to yield 5 '-deoxyadenosine and methionine. Surprisingly, our results show that anSMEs are dual substrate enzymes able to oxidize both cysteine and serine residues to C(alpha)-formylglycine. Taken together, the results support a radical modification mechanism that is initiated by hydrogen abstraction from a serine or cysteine residue located in an appropriate target sequence.