Ratiometric Fluorescent Safranin-O Staining Allows the Quantification of Lignin Contents In Muro.

In some specific vascular plant tissues, lignin can impregnate the entire cell wall to make it more rigid and hydrophobic. Different techniques have been developed in the past years to make possible the quantification of this polyphenolic polymer at the organ or tissue level, but difficulties of access to the cellular level remain. Here we describe an approach based on ratiometric emission measurements using safranin-O and the development of a macro adapted for the FIJI software, which makes it possible to quantify lignin in three different layers of the cell wall on images captured on a fluorescent confocal microscope.

Development and validation of a liquid chromatography-tandem mass spectrometry method for CPUL1, a novel antitumor candidate compound, and its application to pharmacokinetic studies.

An active substance of pyrano[3,2-a]phenazine, also called CPUL1, is a synthesized phenazine derivative and displays broad-spectrum anticancer activities. Quantitative assessment of CPUL1 in biological samples has not been well established, hindering pharmaceutical development and application. According to international guidelines, a sensitive and selective liquid chromatography-tandem mass spectrometry method in negative ion mode was developed and validated for quantification of CPUL1 in human plasma, colorectal cancer cell lines, and rat plasma, whereby linearity and accuracy were demonstrated for the range of 1-1000 ng/ml. The validated liquid chromatography-tandem mass spectrometry method was successfully employed in pharmacokinetic studies of CPUL1 in vitro and in vivo. Notably, the cellular pharmacokinetic behavior of CPUL1 varies in colorectal cancer cell lines. Regarding the pharmacokinetic processes in vivo, oral absorption was less effective than an injection, with a bioavailability of 23.66%. CPUL1 was linearly eliminated after a single administration; however, it could accumulate in tissues (heart, liver, spleen, lung, and kidney) after multiple injections. In summary, this study established a capable bioanalytical method for CPUL1 and provided exploratory pharmacokinetic data, paving the way for use of this promising derivative in disease models.

Characteristics of biological control and mechanisms of Pseudomonas chlororaphis zm-1 against peanut stem rot.

BACKGROUND: Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this plant disease. Biological control is one of the most promising plant disease control measures. In this study, Pseudomonas chlororaphis subsp. aurantiaca strain zm-1, a bacterial strain with potential biocontrol properties isolated by our team from the rhizosphere soil of Anemarrhena asphodeloides, was studied to control this plant disease. METHODS: We prepared extracts of Pseudomonas chloroaphis zm-1 extracellular antibacterial compounds (PECEs), determined their antifungal activities by confrontation assay, and identified their components by UPLC-MS/MS. The gene knockout strains were constructed by homologous recombination, and the biocontrol efficacy of P. chlororaphis zm-1 and its mutant strains were evaluated by pot experiments under greenhouse conditions and plot experiments, respectively. RESULTS: P. chlororaphis zm-1 could produce extracellular antifungal substances and inhibit the growth of Sclerotium rolfsii, the main pathogenic fungus causing peanut stem rot. The components of PECEs identified by UPLC-MS/MS showed that three kinds of phenazine compounds, i.e., 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and the core phenazine, were the principal components. In particular, 1-hydroxyphenazine produced by P. chlororaphis zm-1 showed antifungal activities against S. rolfsii, but 2-hydroxyphenazine did not. This is quite different with the previously reported. The extracellular compounds of two mutant strains, ΔphzH and ΔphzE, was analysed and showed that ΔphzE did not produce any phenazine compounds, and ΔphzH no longer produced 1-hydroxyphenazine but could still produce PCA and phenazine. Furthermore, the antagonistic ability of ΔphzH declined, and that of ΔphzE was almost completely abolished. According to the results of pot experiments under greenhouse conditions, the biocontrol efficacy of ΔphzH dramatically declined to 47.21% compared with that of wild-type P. chlororaphis zm-1 (75.63%). Moreover, ΔphzE almost completely lost its ability to inhibit S. rolfsii (its biocontrol efficacy was reduced to 6.19%). The results of the larger plot experiments were also consistent with these results. CONCLUSIONS: P. chlororaphis zm-1 has the potential to prevent and control peanut stem rot disease. Phenazines produced and secreted by P. chlororaphis zm-1 play a key role in the control of peanut stem rot caused by S. rolfsii. These findings provide a new idea for the effective prevention and treatment of peanut stem rot.

Degradation, adsorption and leaching of phenazine-1-carboxamide in agricultural soils.

Phenazines, a large group of nitrogen-containing heterocycles with promising bioactivities, can be widely used as medicines and pesticides. But phenazines also generate toxicity risks due to their non-selective DNA binding. The environmental fate of phenazines in soils is the key to assess their risks; however, hitherto, there have been very few related studies. Therefore in the present study, the degradation, adsorption and leaching behaviors of a typical natural phenazine-phenazine-1-carboxamide (PCN) in agricultural soils from three representative places in China with different physicochemical properties were, for the first time, systematically studied in laboratory simulation experiments. Our results indicated that the degradation of PCN in all the tested soils followed the first order kinetics, with half-lives ranging from 14.4 to 57.8 d under different conditions. Soil anaerobic microorganisms, organic matter content and pH conditions are important factors that regulating PCN degradation. The adsorption data of PCN were found to be well fitted using the Freundlich model, with the r2 values above 0.978. Freundlich adsorption coefficient Kf of PCN ranged from 5.75 to 12.8 [(mg/kg)/(mg/L)1/n] in soils. The retention factor Rf values ranged from 0.0833 to 0.354, which means that the mobility of PCN in the three types of soil is between immobile to moderately mobile. Our results demonstrate that PCN is easily degraded, has high adsorption affinity and low mobility in high organic matter content and clay soils, thus resulting in lower risks of contamination to groundwater systems. In contrast, it degraded slowly, has low adsorption affinity and moderately mobile in soils with low organic matter and clay content, therefore it has higher polluting potential to groundwater systems. Overall, these findings provide useful insights into the future evaluation of environmental as well as health risks of PCN.

LC-MS/MS method for quantitation of the CK2 inhibitor silmitasertib (CX-4945) in human plasma, CSF, and brain tissue, and application to a clinical pharmacokinetic study in children with brain tumors.

Silmitasertib (CX-4945) as a potent and selective inhibitor of CK2 exhibited promising in vitro and in vivo anti-cancer activity. An assay employing cation-exchange solid phase extraction (SPE) followed by LC-MS/MS analysis was successfully developed and validated for the quantitation of silmitasertib in human plasma, brain tissue, and human cerebrospinal fluid (CSF). Reverse phase chromatographic separation was achieved using Synergi™ hydro-RP column (4 µm, 75 × 2.0 mm) and gradient elution with 5 mM ammonium formate aqueous solution (pH 6.5) as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. Multiple reaction monitoring (MRM) transition of m/z 350.2 â†’ 223.2 and m/z 316.2 â†’ 223.2 were chosen for detection of silmitasertib and internal standard (CX-4786) respectively. Since silmitasertib concentration in patient plasma is expected to be in a wide range due to the study design, two calibration curves with range 0.2-125 ng/ml and 32-20,000 ng/ml were established. A different curve ranging from 2 to 40 ng/g was used for measurement of silmitasertib in brain tissue, while another calibration curve ranging from 0.2 to 20 ng/ml was established for CSF. All these calibration curves corresponding to different matrices showed good linearity (R2 > 0.99) over the concentration range. This assay demonstrated excellent precision below 15% and accuracies between 85% and 115% within-day and between-day for all the concentration levels in each matrix. This assay was also validated for each matrix for selectivity, sensitivity, matrix effects, recovery, and stability. We applied the validated method to the analysis of plasma silmitasertib for a clinical study.

How best to approach these acute hand infections.

Antimicrobial therapy is not straightforward with the 4 infectious conditions reviewed here. Combination therapy at the start must usually be tailored once an organism is known.

Electrochemical monitoring of the impact of polymicrobial infections on Pseudomonas aeruginosa and growth dependent medium.

The opportunistic human pathogen Pseudomonas aeruginosa (Pa) causes several infections acquired in a healthcare setting. During initial stages of infection, Pa produces redox-active phenazine metabolites, including pyocyanin (PYO), 5-methylphenazine-1-carboxylic acid (5-MCA), and 1-hydroxyphenazine (OHPHZ), which have toxic effects on surrounding host cells and/or other microbes. Rapid and sensitive detection of these metabolites provides important evidence about the onset of Pa infections. Herein, we investigate differences in Pa phenazine production and dynamics in polymicrobial communities. Specifically, Pa was co-cultured with two pathogens of clinical relevance, Staphylococcus aureus (Sa) and Escherichia coli (Ec), which typically populate infection sites with Pa. Phenazine production rates and biosynthesis dynamics were electrochemically monitored during a 48-h period using recently developed transparent carbon ultramicroelectrode arrays (T-CUAs). Moreover, the effect on phenazine production rates and dynamics was explored in two growth media, lysogeny broth (LB) and tryptic soy broth (TSB). The concentrations of PYO and highly reactive 5-MCA were determined in different polymicrobial culture samples in both media. The results demonstrate that other bacterial pathogens noticeably influence Pa phenazine production and dynamics. In particular, Sa caused a decrease in phenazine production in TSB. However, the presence of Ec in polymicrobial samples drastically inhibited phenazine production rates in both LB and TSB. Conclusively, the media type significantly influences phenazine product distribution, especially in polymicrobial co-cultures, signifying the need for analytical standardization of simulation media in the study of polymicrobial communities.

Degradation of safranin by heterogeneous Fenton processes using peanut shell ash based catalyst.

Today, dyes are one of the major problematic pollutants in the environment and are broadly used in several industrial sectors. In the current research work, decolorization of safranin (basic dye) from aqueous solution was investigated using iron-impregnated peanut shell ash (Fe-PSA) as a catalyst in the UV-assisted heterogeneous Fenton process (Fe-PSA/H2O2/UV). The effect of parameters such as H2O2 concentration, catalyst dose, pH, initial dye concentration, temperature, and agitation speed was studied. The maximum decolorization of safranin was achieved at optimum parametric values of reagent dose = 8 mM, catalyst dose = 0.5 g, pH = 3, initial concentration of safranin = 50 ppm, temperature = 25 °C, and agitation speed = 200 rpm. The results revealed the efficient performance of Fe-PSA as catalyst in the Fe-PSA/H2O2/UV process for safranin treatment.

Real-Time Electrochemical Detection of Pseudomonas aeruginosa Phenazine Metabolites Using Transparent Carbon Ultramicroelectrode Arrays.

Here, we use a recently developed electrochemical sensing platform of transparent carbon ultramicroelectrode arrays (T-CUAs) for the in vitro detection of phenazine metabolites from the opportunistic human pathogen Pseudomonas aeruginosa. Specifically, redox-active metabolites pyocyanin (PYO), 5-methylphenazine-1-carboxylic acid (5-MCA), and 1-hydroxyphenazine (OHPHZ) are produced by P. aeruginosa, which is commonly found in chronic wound infections and in the lungs of cystic fibrosis patients. As highly diffusible chemicals, PYO and other metabolites are extremely toxic to surrounding host cells and other competing microorganisms, thus their detection is of great importance as it could provide insights regarding P. aeruginosa virulence mechanisms. Phenazine metabolites are known to play important roles in cellular functions; however, very little is known about how their concentrations fluctuate and influence cellular behaviors over the course of infection and growth. Herein we report the use of easily assembled, low-cost electrochemical sensors that provide rapid response times, enhanced sensitivity, and high reproducibility. As such, these T-CUAs enable real-time electrochemical monitoring of PYO and another extremely reactive and distinct redox-active phenazine metabolite, 5-methylphenazine-1-carboxylic acid (5-MCA), from a highly virulent laboratory P. aeruginosa strain, PA14. In addition to quantifying phenazine metabolite concentrations, changes in phenazine dynamics are observed in the biosynthetic route for the production of PYO. Our quantitative results, over a 48-h period, show increasing PYO concentrations during the first 21 h of bacterial growth, after which PYO levels plateau and then slightly decrease. Additionally, we explore environmental effects on phenazine dynamics and PYO concentrations in two growth media, tryptic soy broth (TSB) and lysogeny broth (LB). The maximum concentrations of cellular PYO were determined to be 190 ± 5 µM and 150 ± 1 µM in TSB and LB, respectively. Finally, using desorption electrospray ionization (DESI) and nanoelectrospray ionization (nano-ESI) mass spectrometry we confirm the detection and identification of reactive phenazine metabolites.

Efficient photocatalytic removal of safarnin-O dye pollutants from water under sunlight using synthetic bentonite/polyaniline@Ni2O3 photocatalyst of enhanced properties.

This study involves a synthesis of bentonite/polyaniline composite (BE/PANI) of enhanced physicochemical properties as catalyst support for Ni2O3 photocatalyst. The change in the structural properties, morphological features, and optical behavior was addressed utilizing several analytic techniques. The characterization results reflected considerable enhancement in the specific surface area after the integration between bentonite and polyaniline (127 m2/g) and after loading of the campsite by Ni2O3 forming bentonite/polyaniline@Ni2O3 composite (BE/PANI@Ni2O3) (231 m2/g). Additionally, the band gap energy was reduced to 2.41 eV and 1.61 eV for BE/PANI and BE/PANI@Ni2O3, respectively, as compared to that of 3.4 eV for pure Ni2O3. The photocatalytic removal of safranin-O dye under sunlight exposure using BE/PANI@Ni2O3 as catalyst revealed great enhancement in the removal percentages by 63%, 75%, and 72.35% higher than bentonite, polyaniline, and Ni2O3, respectively. Five milligrams per liter of safranin-O dye can be completely removed from 100 ml water using 0.05 g of the composite after 90 min. The catalyst also was applied effectively in the removal of safranin-O dye from raw water samples as a realistic application of the synthetic composite. Synthetic BE/PANI@Ni2O3 as photocatalyst showed very high stability and can be used seven times as photocatalytic at amazing removal percentages.

Reciprocal enhancement of gene expression between the phz and prn operon in Pseudomonas chlororaphis G05.

In previous studies with Pseudomonas chlororaphis G05, two operons (phzABCDEFG and prnABCD) were confirmed to respectively encode enzymes for biosynthesis of phenazine-1-carboxylic acid and pyrrolnitrin that mainly contributed to suppression of some fungal phytopathogens. Although some regulators were identified to govern their expression, it is not known how two operons coordinately interact. By constructing the phz- or/and prn- deletion mutants, we found that in comparison with the wild-type strain G05, phenazine-1-carboxylic acid production in the mutant G05Δprn obviously decreased in GA broth in the absence of prn, and pyrrolnitrin production in the mutant G05Δphz remarkably declined in the absence of phz. By generating the phzA and prnA transcriptional and translational fusions with a truncated lacZ on shuttle vector or on the chromosome, we found that expression of the phz or prn operon was correspondingly increased in the presence of the prn or phz operon at the post-transcriptional level, not at the transcriptional level. These results indicated that the presence of one operon would promote the expression of the other one operon between the phz and prn. This reciprocal enhancement would keep the strain G05 producing more different antifungal compounds coordinately and living better with growth suppression of other microorganisms.

Establishment of LCMS Based Platform for Discovery of Quorum Sensing Inhibitors: Signal Detection in Pseudomonas aeruginosa PAO1.

Targeting the main three networking systems, viz. Las, RhI, and PQS, via natural quenchers is a new ray of hope for combating the persistent behavior of Pseudomonas aeruginosa. In the bacterial chemical vocabulary pyocyanin, N-AHLs and rhamnolipids are the main keywords, which are responsible for the social and nomadic behavior of P. aeruginosa. In the present work, LC-MS based real-time qualitative and quantitative analysis of pyocyanin, green phenazine, N-AHLs, and rhamnolipids were performed on P. aeruginosa PAO1. The quantitative analysis indicates that the production of pyocyanin and NHSLs increases with time while the production of rhamnolipids discontinued after 16 h. This indicates the emergence of persisters in the medium instead of planktonic cells. Rhamnolipids acting as a surfactant enhances the motility of the bacterial cells, whereas the pyocyanin is responsible for the biofilm formation. In a microtiter plate based assay, an effect of capsaicin and 6-gingerol was recorded. In the presence of capsaicin and 6-gingerol, a substantial decrease in the production of rhamnolipids, phenazine, quinolone, and N-AHLs was observed. Most interestingly, the 6-gingerol treatment led to a drastic decrease of rhamnolipids, phenazine, quinolone, and N-AHLs versus capsaicin. These studies demonstrate the effectiveness of the capsaicin and 6-gingerol on Las, PQS, and Rhl circuits in a bacterium in order to understand the persistent and social behavior. Here, we are reporting LC-MS/MS based qualitative and quantitative analysis of QS molecules by taking a low volume of culture (up to 200 µL). This method can be used as a platform to screen the new antivirulence agents for fighting the resistant behavior of P. aeruginosa during biofilm formation.

Impact of Gram stain results on initial treatment selection in patients with ventilator-associated pneumonia: a retrospective analysis of two treatment algorithms.

BACKGROUND: Ventilator-associated pneumonia (VAP) is a common and serious problem in intensive care units (ICUs). Several studies have suggested that the Gram stain of endotracheal aspirates is a useful method for accurately diagnosing VAP. However, the usefulness of the Gram stain in predicting which microorganisms cause VAP has not been established. The purpose of this study was to evaluate whether a Gram stain of endotracheal aspirates could be used to determine appropriate initial antimicrobial therapy for VAP. METHODS: Data on consecutive episodes of microbiologically confirmed VAP were collected from February 2013 to February 2016 in the ICU of a tertiary care hospital in Japan. We constructed two hypothetical empirical antimicrobial treatment algorithms for VAP: a guidelines-based algorithm (GLBA) based on the recommendations of the American Thoracic Society-Infectious Diseases Society of America (ATS-IDSA) guidelines and a Gram stain-based algorithm (GSBA) which limited the choice of initial antimicrobials according to the results of bedside Gram stains. The GLBA and the GSBA were retrospectively reviewed for each VAP episode. The initial coverage rates and the selection of broad-spectrum antimicrobial agents were compared between the two algorithms. RESULTS: During the study period, 219 suspected VAP episodes were observed and 131 episodes were assessed for analysis. Appropriate antimicrobial coverage rates were not significantly different between the two algorithms (GLBA 95.4% versus GSBA 92.4%; p = 0.134). The number of episodes for which antimethicillin-resistant Staphylococcus aureus agents were selected as an initial treatment was larger in the GLBA than in the GSBA (71.0% versus 31.3%; p < 0.001), as were the number of episodes for which antipseudomonal agents were recommended as an initial treatment (70.2% versus 51.9%; p < 0.001). CONCLUSIONS: Antimicrobial treatment based on Gram stain results may restrict the administration of broad-spectrum antimicrobial agents without increasing the risk of treatment failure. TRIAL REGISTRATION: UMIN-CTR, UMIN000026457 . Registered 8 March 2017 (retrospectively registered).

Immunochemical Determination of Pyocyanin and 1-Hydroxyphenazine as Potential Biomarkers of Pseudomonas aeruginosa Infections.

A novel immunochemical approach to diagnose Pseudomonas aeruginosa infections is reported, which is based on the quantification of relevant and specific virulence factors secreted by this microorganism. Specific antibodies have been raised using hapten PC1 (a 1:1 mixture of 9-hydroxy- and 6-hydroxy-phenazine-2-carobxylic acids), designed to recognize 1-hydroxyphenazine (1-OHphz), which is the main metabolite of pyocyanin (PYO). PYO is one of the most important virulence factors produced by nearly all P. aeruginosa strains, and other species do not produce this factor. With these antibodies, an immunochemical analytical procedure able to quantify both 1-OHphz and PYO in complex clinical samples has been developed. 1-OHphz can be directly measured in solubilized sputum samples diluted 20 times with the assay buffer. Quantification of PYO is accomplished after conversion to 1-OHphz in just 20 min under basic conditions. A LOD of 0.60 ± 0.01 nM (4.80 ± 0.08 nmol kg(-1) sputum) is reached for both biomarker targets under the conditions established, a value that is much below the reported concentrations on sputum samples obtained from infected patients (up to 100 µM). The assay is robust, reproducible, accurate, can be run in about 2 h, and many samples can be measured simultaneously. The present reported assay could represent a significant improvement in the diagnosis of infectious diseases caused by this pathogen.

Real-time monitoring of phenazines excretion in Pseudomonas aeruginosa microbial fuel cell anode using cavity microelectrodes.

Phenazines are a kind of metabolites that can mediate extracellular Pseudomonas aeruginosa (P. aeruginosa) cells in microbial fuel cells (MFCs). However, it is still not clear that whether and how the excretion profile of phenazines is affected by the operating MFC. Here, we report a real time analysis approach based on a cavity microelectrode electrochemical sensor to investigate the phenazines excretion behavior of P. aeruginosa during MFC operation. The phenazine concentration increases at first 72 h, reaches a plateau and decreases after 120 h and also shows local dependent variation. It is dependent on the MFC current generation profile but also affect by the biofilm formation. Accordingly, a mechanism about phenazines excretion in MFC anode and the phenazines mediated extracellular electron transfer of the P. aeruginosa anode is proposed. This work provides a novel strategy for self-mediated extracellular electron transfer analysis in the operating MFCs.

Gram's Stain Does Not Cross the Bacterial Cytoplasmic Membrane.

For well over a century, Hans Christian Gram's famous staining protocol has been the standard go-to diagnostic for characterizing unknown bacteria. Despite continuous and ubiquitous use, we now demonstrate that the current understanding of the molecular mechanism for this differential stain is largely incorrect. Using the fully complementary time-resolved methods: second-harmonic light-scattering and bright-field transmission microscopy, we present a real-time and membrane specific quantitative characterization of the bacterial uptake of crystal-violet (CV), the dye used in Gram's protocol. Our observations contradict the currently accepted mechanism which depicts that, for both Gram-negative and Gram-positive bacteria, CV readily traverses the peptidoglycan mesh (PM) and cytoplasmic membrane (CM) before equilibrating within the cytosol. We find that not only is CV unable to traverse the CM but, on the time-scale of the Gram-stain procedure, CV is kinetically trapped within the PM. Our results indicate that CV, rather than dyes which rapidly traverse the PM, is uniquely suited as the Gram stain.

The structural and compositional transition of the meniscal roots into the fibrocartilage of the menisci.

The meniscal roots, or insertional ligaments, firmly attach the menisci to tibial plateau. These strong attachments anchor the menisci and allow for the generation of hoop stress in the tissue. The meniscal roots have a ligament-like structure that transitions into the fibrocartilagenous structure of the meniscal body. The purpose of this study was to carry out a complete analysis of the structure and tissue organization from the body of the meniscus through the transition region and into the insertional roots. Serial sections were obtained from the meniscal roots into the meniscal body in fixed juvenile bovine menisci. Sections were stained for collagen and proteoglycans (PG) using fast green and safranin-o staining protocols. Unstained sections were imaged used a backlit stereo microscope. Optical projection tomography (OPT) was employed to evaluate the three-dimensional collagen architecture of the root-meniscus transition in lapine menisci. Tie-fibres were observed in the sections of the ligaments furthest from the bovine meniscal body. Blood vessels were observed to be surrounded by these tie-fibres and a PG-rich region within the ligaments. Near the tibial insertion, the roots contained large ligament-like collagen fascicles. In sections approaching the meniscus, there was an increase in tie-fibre size and density. Small tie-fibres extended into the ligament from the epiligamentous structure in the outermost sections of the meniscal roots, while large tie-fibre bundles were apparent at the meniscus transition. The staining pattern indicates that the root may continue into the outer portion of the meniscus where it then blends with the more fibrocartilage-like inner portions of the tissue. In unstained sections it was observed that the femoral side of the epiligamentous structure surrounding the root becomes more fibrous and thickens in the inferior inner portion of the posterior medial root. This thickening changes the shape of the root to more closely resemble the meniscus wedge shape. These observations support the concept of root continuity with the outer portion of the meniscus, thereby connecting with the hoop-like structure of the peripheral meniscus. OPT identified continuous collagen organization from the root into the meniscal body in longitudinal sections. In the radial direction, the morphology of the root continues into the meniscal body consistent with the serially sectioned bovine menisci. Blood vessels were prevalent on the periphery of the root. These blood vessels then arborized to cover the anterior femoral surface of the meniscus. This is the first study of the structural transition between the insertional ligaments (roots) and the fibrocartilagenous body of the menisci. These new structural details are important to understanding the meniscal load-bearing mechanism in the knee.

An UV-vis spectroelectrochemical approach for rapid detection of phenazines and exploration of their redox characteristics.

Phenazines are widely distributed in the environment and play an important role in various biological processes to facilitate microbial metabolism and electron transfer. In this work, an efficient and reliable spectroelectrochemical method is developed to quantitatively detect 1-hydroxyphenazine (1-OHPZ), a representative phenazine, and explore its redox characteristics. This approach is based on the sensitive absorption change of 1-OHPZ in response to its changes under redox state in rapid electrochemical reduction. The redox reaction of 1-OHPZ in aqueous solution is a proton-coupled electron transfer process, with a reversible one-step 2e(-)/2H(+) transfer reaction. This spectroelectrochemical approach exhibits good linear response covering two magnitudes to 1-OHPZ with a detection limit of 0.48µM, and is successfully applied to detect 1-OHPZ from a mixture of phenazines produced by Pseudomonas aeruginosa cultures. This method might also be applicable in exploring the abundance and redox processes of a wide range of other redox-active molecules in natural and engineered environments.

Overexpression of afsR and Optimization of Metal Chloride to Improve Lomofungin Production in Streptomyces lomondensis S015.

As a global regulatory gene in Streptomyces, afsR can activate the biosynthesis of many secondary metabolites. The effect of afsR on the biosynthesis of a phenazine metabolite, lomofungin, was studied in Streptomyces lomondensis S015. There was a 2.5-fold increase of lomofungin production in the afsR-overexpressing strain of S. lomondensis S015 N1 compared with the wild-type strain. Meanwhile, the transcription levels of afsR and two important genes involved in the biosynthesis of lomofungin (i.e., phzC and phzE) were significantly upregulated in S. lomondensis S015 N1. The optimization of metal chlorides was investigated to further increase the production of lomofungin in the afsR-overexpressing strain. The addition of different metal chlorides to S. lomondensis S015 N1 cultivations showed that CaCl2, FeCl2, and MnCl2 led to an increase in lomofungin biosynthesis. The optimum concentrations of these metal chlorides were obtained using response surface methodology. CaCl2 (0.04 mM), FeCl2 (0.33 mM), and MnCl2 (0.38 mM) gave a maximum lomofungin production titer of 318.0 ± 10.7 mg/l, which was a 4.1-fold increase compared with that of S. lomondensis S015 N1 without the addition of a metal chloride. This work demonstrates that the biosynthesis of phenazine metabolites can be induced by afsR. The results also indicate that metal chlorides addition might be a simple and useful strategy for improving the production of other phenazine metabolites in Streptomyces.