Type II thioesterase improves heterologous biosynthesis of valinomycin in Escherichia coli.

Heterologous expression of secondary metabolite biosynthesis pathways in a surrogate host, e.g. Escherichia coli, has emerged in recent years as an effective way to produce complex natural products. The nonribosomal peptide (NRP) antibiotic valinomycin has been recombinantly produced in E. coli through reconstitution of its biosynthetic pathway from the native producer Streptomyces tsusimaensis. In this study, a discrete protein type II thioesterase (TEII) encoded in the valinomycin gene cluster was coexpressed in the valinomycin producing E. coli strain. Valinomycin titers were significantly improved from 0.5 (without TEII coexpression) to 3.3 mg L(-1), which demonstrates the reconstitutive function of TEII involved in NRP biosynthesis. Based on a flask scale fed-batch cultivation system, repeated feeding of the glucose polymer during the cultivation further increased cell density and valinomycin titer up to 55 (OD600) and 13 mg L(-1), respectively. This indicates scalable high cell density cultivation in a bioreactor for overproduction of valinomycin will be a potential and feasible approach. In this work we present an in vivo example to show that TEII plays a positive role in heterologous valinomycin production.

Reconstituted biosynthesis of the nonribosomal macrolactone antibiotic valinomycin in Escherichia coli.

The structural complexity of nonribosomal peptides (NRPs) impeding economic chemical synthesis and poor cultivability of source organisms limits the development of bioprocesses for novel bioactive compounds. Since nonribosomal peptide synthetases (NRPSs) assemble NRPs from simple amino acid building blocks, heterologous expression of NRPSs in a robust and easy to manipulate expression host is an attractive strategy to make pharmaceutically relevant NRPs more accessible and is also a basis for engineering of these enzymes to generate novel synthetic bioactive compounds. Here we show a systematic approach for the heterologous expression of the 654 kDa heterodimeric valinomycin synthetase (VlmSyn) from Streptomyces tsusimaensis in a soluble and active form in Escherichia coli. VlmSyn activity and precursor requirements were determined in vitro and provided evidence for a previously proposed model of valinomycin biosynthesis. In vivo production of recombinant valinomycin, a macrolactone antibiotic with reported antifungal, antibacterial, and antiviral activities, was achieved using an engineered E. coli strain growing in inexpensive media and independent of the supplementation with precursors and further optimization of the cultivation conditions. Tailoring of VlmSyn in E. coli paves the way to the production of novel valinomycin analogues in the future.

Fungal and bacterial metabolites in commercial poultry feed from Nigeria.

Metabolites of toxigenic fungi and bacteria occur as natural contaminants (e.g. mycotoxins) in feedstuffs making them unsafe to animals. The multi-toxin profiles in 58 commercial poultry feed samples collected from 19 districts in 17 states of Nigeria were determined by LC/ESI-MS/MS with a single extraction step and no clean-up. Sixty-three (56 fungal and seven bacterial) metabolites were detected with concentrations ranging up to 10,200 µg kg⁻¹ in the case of aurofusarin. Fusarium toxins were the most prevalent group of fungal metabolites, whereas valinomycin occurred in more than 50% of the samples. Twelve non-regulatory fungal and seven bacterial metabolites detected and quantified in this study have never been reported previously in naturally contaminated stored grains or finished feed. Among the regulatory toxins in poultry feed, aflatoxin concentrations in 62% of samples were above 20 µg kg⁻¹, demonstrating high prevalence of unsafe levels of aflatoxins in Nigeria. Deoxynivalenol concentrations exceeded 1000 µg kg⁻¹ in 10.3% of samples. Actions are required to reduce the consequences from regulatory mycotoxins and understand the risks of the single or co-occurrence of non-regulatory metabolites for the benefit of the poultry industry.

Single drop microextraction coupled with matrix-assisted laser desorption/ionization mass spectrometry for rapid and direct analysis of hydrophobic peptides from biological samples in high salt solution.

Single drop microextraction (SDME) coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been applied for direct analysis of hydrophobic peptides (valinomycin and gramicidin D) from biological samples (urine and plasma) in high salt solution. The optimal conditions such as selection of extraction solvent, stirring rate, extraction time, effect of salt concentration and matrix-to-analyte ratio were investigated. The limits of detection (LODs) were found to be 73 nM to 170 nM for valinomycin and 96 nM to 5.5 µM for gramicidin D in high salt solution (1.7 M of NaCl) in MALDI-MS. The current approach can enhance the LODs by 11-320-fold for gramicidin D analysis in water, urine and plasma in high salt solution. Furthermore, the current approach has been successfully demonstrated for real-world sample analysis (ß-carotene from carrots) by MALDI-MS. The current approach is a rapid, simple and efficient clean-up platform for direct analysis of hydrophobic molecules in biological samples from high salt solution.

The influence of CO(2) on ISFETs with polymer membranes and characterization of a carbonate ion sensor.

The influence of CO(2) and acetic acid on the response of ISFET sensors with PVC and photocured polyurethane polymer membranes with valinomycin as an ionophore was assessed. Experimental results show that the presence of these compounds has no effect on sensors parameters even after prolonged soaking in a water solution. Using a photocured polyurethane polymer as an ion-selective membrane matrix for an ISFET, a carbonate ion sensor was developed with hexyl-p-trifluoroacetylbenzoate (HE) as an ionophore. Effect of cationic and anionic lipophilic additives on the sensors response was studied. Sensors with the optimized membrane composition based on HE (7.9%, w/w) and tridodecylmethylammonium chloride (5.7%, w/w) show sensitivity of 27-30mV per decade of carbonate ion concentration, sufficient selectivity in front of chloride ions, and a lifetime of 3-5 months.

Capillary affinity electrophoresis and ab initio calculation studies of valinomycin complexation with Na+ ion.

In a combined experimental and theoretical approach, the interactions of valinomycin (Val), macrocyclic depsipeptide antibiotic ionophore, with sodium cation Na(+ )have been investigated. The strength of the Val-Na(+ )complex was evaluated experimentally by means of capillary affinity electrophoresis. From the dependence of valinomycin effective electrophoretic mobility on the sodium ion concentration in the BGE (methanolic solution of 20 mM chloroacetic acid, 10 mM Tris, 0-40 mM NaCl), the apparent binding (stability) constant (K(b)) of the Val-Na(+ )complex in methanol was evaluated as log K(b) = 1.71 +/- 0.16. Besides, using quantum mechanical density functional theory (DFT) calculations, the most probable structures of the nonhydrated Val-Na(+) as well as hydrated Val-Na(+).H(2)O complex species were proposed. Compared to Val-Na(+), the optimized structure of Val-Na(+).H(2)O complex appears to be more realistic as follows from the substantially higher binding energy (118.4 kcal/mol) of the hydrated complex than that of the nonhydrated complex (102.8 kcal/mol). In the hydrated complex, the central Na(+) cation is bound by strong bonds to one oxygen atom of the respective water molecule and to four oxygens of the corresponding C=O groups of the parent valinomycin ligand.

Do collisions inside the collision cell play a relevant role in CID-LIFT experiments?

Collision experiments are a valid approach to characterize the ionic species generated by matrix assisted laser desorption ionization (MALDI). When a time-of-flight analyzer is employed, three different approaches are available for these experiments: the postsource decay (PSD), the LIFT and the MALDI-TOF/TOF. The last two are of particular interest because of the overcoming of the PSD problems related to mass calibration of the product ion spectra. Experiments performed by LIFT on linear or cyclic peptides, in presence or in absence of collision gas in the collision cell, gave evidence of an unexpected behavior: the two spectra were practically superimposable, and in the former case only a few new fragmentation channels were activated with low yield. These results mean that the selected ion exhibits a large amount of internal energy, capable of promoting fragmentation processes in the time window corresponding to the flight time between ion source and the acceleration electrode placed after the collision cell. Experiments performed by varying the plume density show that this internal energy uptake occurs in the expanding plume, through multiple collisions. The LIFT data have been compared with those achieved by collisions of ESI-generated [MH](+) ions of angotensin II performed under 'in-source' conditions and by triple-quadrupole experiments. The obtained results show a strong similarity among the spectra, indicating that the internal energy uptake in a MALDI source is comparable with that of 40-eV ions colliding with Ar in a triple-quadrupole instrument.

MS(n) characterization of protonated cyclic peptides and metal complexes.

MS(n) experiments involving low energy collisionally activated dissociation (CAD) in a quadrupole ion trap were used to characterize the fragmentation of alkali, alkaline earth and transition metal complexes of five cyclic peptides, and the results were compared with those obtained for protonated cyclic peptides. Complexes with metal ions produced enhanced abundances of the most diagnostic fragments for elucidating the primary structures. For cyclosporin A, nickel and lithium complexes gave additional sequence information compared with the protonated peptide. For depsipeptides, sodium and lead complexes were superior to the protonated peptide or other metal complexes for sequencing residues, and CAD of the lead complexes led to preferential cleavage of two residues at a time. For cyclic lipopeptides, complexes with silver, nickel and strontium ions provided enhanced abundances of key fragment ions.

Increasing sensitivity and decreasing spot size using an inexpensive, removable hydrophobic coating for matrix-assisted laser desorption/ionisation plates.

Spot size reduction and increased detection sensitivity in matrix-assisted laser desorption/ionisation (MALDI) of small molecules are accomplished by using an inexpensive and removable hydrophobic coating for MALDI targets, based on 3M Scotch Gard surface treatment. Several variations in sample preparation were explored, such as surface coating technique, identity of the matrix, solvent composition, and the type of metal support plate used. These were investigated on both uncoated and coated surfaces and their impact on spot size, crystal coverage, and sensitivity is presented here. Additionally, crystallisation behaviour obtained on coated plates is compared with that on uncoated plates using scanning electron microscope analysis. To demonstrate the potential of the new coating technique, erythromycin A and valinomycin are studied to determine the increase in detection sensitivity of coated plates in comparison to uncoated plates, and to reveal the suitability of the plates for application in combined high-performance liquid chromatography/MALDI (HPLC/MALDI), where widely varying solvent compositions and droplet volumes are observed. It is shown that enhancements in detection sensitivities correlate very well with the achieved spot size reduction. The versatility of the coated plates is also exhibited by the ease of removing the surface layer, after which the plates can be rigorously cleaned without worry about damaging the hydrophobic surface, followed by a quick reapplication of new hydrophobic coating material. This makes the non-polar coating superior to more expensive commercial hydrophobic-coated targets, which are much more delicate to clean. Furthermore, cleaning and reapplication eliminate potential carry-over effects and the easy application procedure also makes the fabrication of inexpensive, disposable MALDI targets readily possible.

Evaluation of the separate equilibrium processes that dictate the upper detection limit of neutral ionophore-based potentiometric sensors.

The upper detection limit of polar ionophore-based ion-selective electrode membranes is predicted by utilizing the coextraction constant of dissociated electrolyte, the stability constant of the ionophore, and the membrane composition. The coextraction constant of dissociated electrolytes into the polar poly(vinyl chloride) membrane plasticized with o-nitrophenyl octyl ether (PVC-NPOE) is here measured by a novel approach. The sandwich membrane technique is utilized, with one membrane segment containing a lipophilic cation exchanger and the other containing an anion exchanger. This yields information about the coextraction constant and the free ion concentrations of the electrolyte in the two segments. Predictions correlate quantitatively with the upper detection limit observed for ion-selective electrodes based on the ionophores valinomycin, tert-butylcalix[4]arene tetraethyl ester, and calcimycin. The difficulties of the prediction of the upper detection limit for nonpolar poly(vinyl chloride) membranes plasticized with bis(2-ethylhexyl sebacate) (PVC-DOS) due to ion association are discussed in detail. A thermodynamic cycle experiment with a series of sandwich membranes shows that the principal processes governing the upper detection limit of PVC-DOS membranes are identical to those for the PVC-NPOE membranes. However, the stability of the ion pairs between the ionophore-metal ion complexes and the extracted anion are different from that of ion pairs formed between the same anion and the lipophilic anion exchanger. This makes it difficult to quantitatively predict the upper detection limit on the basis of simple apparent coextraction and complexation data alone. The approach reported herein is useful not only for mechanistic purposes but also to shed light onto the many cases where coextraction effects need to be understood but are not directly experimentally accessible.

Quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, produced under various conditions.

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 microg ml(-1). The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 microg of cereulide g(-1) (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 microg of cereulide ml(-1) in liquid medium at room temperature (21 +/- 1 degrees C) in 1 to 3 days, during the stationary growth phase when cell density was 2 x 10(8) to 6 x 10(8) CFU ml(-1). Cereulide production at temperatures at and below 8 degrees C or at 40 degrees C was minimal.

Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique.

BACKGROUND: Membrane potential (MP) plays a critical role in bacterial physiology. Existing methods for MP estimation by flow cytometry are neither accurate nor precise, due in part to the heterogeneity of size of the particles analyzed. The ratio of a size- and MP-sensitive measurement, and an MP-independent, size-sensitive measurement, should provide a better estimate of MP. METHODS: Flow cytometry and spectrofluorometry were used to detect red (488 --> 600 nm) fluorescence associated with aggregates of diethyloxacarbocyanine (DiOC2(3)), which, in the monomeric state, is normally green (488 --> 530 nm) fluorescent. RESULTS: In bacteria incubated with 30 microM dye, aggregate formation increases with the magnitude of the interior-negative membrane potential. Green fluorescence from stained bacteria predominantly reflects particle size, and is relatively independent of MP, whereas red fluorescence is highly dependent on both MP and size. The ratio of red to green fluorescence provides a measure of MP that is largely independent of cell size, with a low coefficient of variation (CV). Calibration with valinomycin and potassium demonstrates that the method is accurate over the range from -50 mV through -120 mV; it also accurately tracks reversible reductions in MP produced by incubation at 4 degrees C and washing in glucose-free medium. CONCLUSIONS: The ratiometric technique for MP estimation using DiOC2(3) is substantially more accurate and precise than those previously available, and may be useful in studies of bacterial physiology and in investigations of the effects of antibiotics and other agents on microorganisms.

[Regulators of differentiation in Streptomyces cyaneofuscatus]. / Reguliatory differentsiatsii Streptomyces cyaneofuscatus.

Streptomyces cyaneofuscatus PRL 1642 producing valinomycin was shown to synthesize bioregulators of differentiation similar to A factor. The regulators stimulate spore formation and streptomycin synthesis in a Streptomyces griseus 1439 mutant deficient in A factor. Some S. cyaneofuscatus mutants respond to A factor addition into the medium by an increase in valinomycin synthesis and a change in morphological properties. The regulators from S. cyaneofuscatus are more effective toward mutants of this species than toward S. griseus mutants.

[Determination of several antibiotics using voltamperometry at the interface of nitrobenzene and water]. / Opredelenie nekotorykh antibiotikov metodom vol'tampermetrii na granitse razdela faz nitrobenzol--Voda.

A new electroanalytical method of voltamperometry at the interface of two immiscible electrolyte solutions (ITIES) is based on electrochemical polarization of a liquid/liquid interface. The resulting current voltage characteristics completely resemble those obtained with metallic electrodes. The charge transfer processes are either the direct ion transfer across the ITIES or the transfer facilitated by macrocyclic ionophores. Determination of tetracycline antibiotics is based on the direct transfer of the cationic forms of these substances in acid media. Determination of valinomycin, nonactin and monensin acting as ion carriers is connected with the facilitated alkali metal ion transfer. In general, antibiotic concentrations higher than 0.02-0.05 mmol/l can be determined with this method. Monensin can also be determined in the extracts of Streptomyces cinnamonensis.