Cezomycin Is Activated by CalC to Its Ester Form for Further Biosynthesis Steps in the Production of Calcimycin in Streptomyces chartreusis NRRL 3882.

Calcimycin, N-demethyl calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between calcimycin and cezomycin. In this study, we observed that when a Cal- (calB1 mutant) derivative of a calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, calcimycin production was restored. This suggested that calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal-S. chartreusis mutants lacking the ability to convert cezomycin to calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein's apparent Km and kcat for cezomycin were observed to be 190 µM and 3.98 min-1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form.IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both calcimycin's biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues', divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for calcimycin's production.

Recycling of Overactivated Acyls by a Type II Thioesterase during Calcimycin Biosynthesis in Streptomyces chartreusis NRRL 3882.

Type II thioesterases typically function as editing enzymes, removing acyl groups that have been misconjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic, and we have identified the presence of a putative type II thioesterase-like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic's synthesis. However, targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in calcimycin production but rather reduced the strain's production of its biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin coenzyme A (cezomycin-CoA), as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC; the mimic of the polyketide chain-releasing precursor). Further investigation of the enzyme's activity showed that it possessed a cezomycin-CoA hydrolysis Km of 0.67 mM and a kcat of 17.77 min-1 and was significantly inhibited by the presence of Mn2+ and Fe2+ divalent cations. Interestingly, when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO2, it was observed that the production of calcimycin rather than cezomycin was promoted. Also, supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca2+, Mg2+, Mn2+, and Fe2+ had a similar effect. Taken together, these observations suggest that CalG is not responsible for megasynthase polyketide precursor chain release during the synthesis of calcimycin or for retaining the catalytic efficiency of the megasynthase enzyme complex as is supposed to be the function for type II thioesterases. Rather, our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of type II thioesterases.IMPORTANCE Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production, thereby maintaining the efficiency of the megasynthase involved in this class of secondary metabolites' biosynthesis. Specifically, this class of enzyme is believed to be involved in editing misprimed precursors, controlling initial units, providing key intermediates, and releasing final synthetic products in the biosynthesis of this class of secondary metabolites. Our results indicate that the putative TEII CalG present in the calcimycin (A23187)-producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or for the release of the product compound from, the megasynthase involved in calcimycin biosynthesis. Rather, the enzyme is involved in regulating/controlling the pool size of the calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolites.

Surface engineering of nanoparticles in suspension for particle based bio-sensing.

Surface activation of nanoparticles in suspension using amino organosilane has been carried out via strict control of a particle surface ad-layer of water using a simple but efficient protocol 'Tri-phasic Reverse Emulsion' (TPRE). This approach produced thin and ordered layers of particle surface functional groups which allowed the efficient conjugation of biomolecules. When used in bio-sensing applications, the resultant conjugates were highly efficient in the hybrid capture of complementary oligonucleotides and the detection of food borne microorganism. TPRE overcomes a number of fundamental problems associated with the surface modification of particles in aqueous suspension viz. particle aggregation, density and organization of resultant surface functional groups by controlling surface condensation of the aminosilane. The approach has potential for application in areas as diverse as nanomedicine, to food technology and industrial catalysis.

Headgroup effects on the Krafft temperatures and self-assembly of ω-hydroxy and ω-carboxy hexadecyl quaternary ammonium bromide bolaform amphiphiles: micelles versus molecular clusters?

Eight bolaform amphiphiles were synthesised and characterised; 4 α,ω-hydroxy-alkane trialkyl (and pyridyl) ammonium bromides and 4 α,ω-carboxy-alkane trialkyl (and pyridyl) ammonium bromides where the alkyl groups were methyl, ethyl and propyl. Four of these represented new compounds. Overall the Krafft temperatures (T(K)) of the eight amphiphiles were high, with 6 of the eight possessing T(K)s greater than 45 °C. Thus most of the amphiphiles could only expect to find applications at raised temperatures limiting their potential utility. However in addition to the previously reported α,ω-hydroxy-hexadecyl triethylammonium bromide (2b) with a T(K) of 19.1 °C, another amphiphile, α,ω-carboxy-hexadecyl tripropylammonium bromide (2c) has been identified with a T(K) near ambient temperatures (T(K) of 22.1 °C). This provides an acid functional ammonium bolaform amphiphile that micellises at ambient temperatures to complement the hydroxyl derivative. A correlation between T(K) and the product of the enthalpies and T(m)s of the compounds was observed for 7 of the eight compounds. No correlation between the amphiphile critical micelle concentrations (cmc) and T(K)s was observed confirming previous reports that T(K) values are predominantly determined by crystalline stability rather than solubility. Considerable differences were observed between the various amphiphile T(K)s at different pHs but no clear trend was apparent for the various compounds (despite the degree to which the compounds' carboxylic acid and hydroxyl functionalities were likely to be ionised). The cmcs for the amphiphiles were an order of magnitude larger than those for analogous mono-ammonium amphiphiles with little difference in between the hydroxyl- and carboxy-functionalised compounds. The aggregation numbers (N(agg)) obtained for all compounds were very low (N(agg)<7) and the apparent micellar diameters for the hydroxyl-bolaforms were in the range 1.0-1.4 nm whereas those for the carboxy-compounds were in the range 2.1-2.4 nm. These results strongly suggest a difference in the packing of the two sets of amphiphiles with loose low density aggregates or 'molecular clusters' for the carboxy compounds and denser classical micellar type aggregates for the hydroxyl-compounds. In both cases however the sizes and the low aggregation numbers point suggest that these aggregates are more characteristic of the pre-micellar aggregates observed for many amphiphiles but in particular gemini surfactants.

QD-antibody conjugates via carbodiimide-mediated coupling: a detailed study of the variables involved and a possible new mechanism for the coupling reaction under basic aqueous conditions.

A detailed study into the optimization of carbodiimide-mediated coupling of antibodies (Ab) and quantum dots (QD) for use in cellular imaging has been undertaken. This involved the grafting of commercially available carboxyl-modified QDs (Evident Technologies "Lake Placid Blue" Evitag and eBioscience's eflour nanocrystals) with anti-Cdc8 Abs to produce conjugates with specific affinity for fission yeast tropomyosin Cdc8 protein. The water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to activate the QDs prior to their incubation with antibody, and a range of QD-carboxyl/EDC/Ab mole ratios were used in the experiments in attempts to optimize fluorescence and bioaffinity of the conjugate products (EDC to QD-carboxyl-600 nmol/15 pmol to 0.12 nmol/15 pmol and QD to Ab 120 pmol/24 pmol to 120 pmol/1.2 pmol). It was observed that a specific "optimum" ratio of the three reactants was required to produce the most fluorescent and biologically active product and that it was generated at alkaline pH 10.8. Increasing the ratio of Ab to QD produced conjugate which was less fluorescent while reducing the ratio of EDC to QD in the activation step led to increased fluorescence of product. Conjugates were tested for their possession of antibody by measurement of their absorption at OD(280 nm) and for their fluorescence by assay λ(max(em)) at 495 nm. A quantitative assay of the bioactivity of the conjugates was developed whereby a standardized amount of Cdc8 antigen was spotted onto nylon membranes and reacted with products from conjugation reactions in a sandwich-type colormetric assay The "best" conjugate was used in intracellular imaging of yeast Cdc8 protein and produced brighter, higher definition images of fixed yeast cell actin structure than a fluorescein-Ab conjugate routinely produced in our laboratory. The QD-Ab conjugate was also significantly more resistant to photobleaching than the fluorescein-Ab conjugate. Results from other experiments involving EDC, the water-soluble carbodiimide 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulphonate (CMC), and EDC.HCl have suggested a new reaction mechanism for EDC coupling under basic aqueous conditions. In summary, a robust understanding of commercial QD-COOH surface chemistry and the variables involved in the materials' efficient conjugation with a bioligand using carbidiimide has been obtained along with an optimized approach for Ab-QD conjugate production. A novel assay has been developed for bioassay of QD-Ab conjugates and a new mechanism for EDC coupling under basic aqueous conditions is proposed.

Encapsulation of superparamagnetic nanoparticles into red blood cells as new carriers of MRI contrast agents.

AIMS: The half-life of superparamagnetic iron oxide nanoparticles in the bloodstream is very short since they are rapidly taken up by the reticuloendothelial system. In this article, we report the encapsulation of different magnetic nanoparticles into human erythrocytes to increase their blood circulation time. MATERIALS & METHODS: Newly synthesized and commercially available nanoparticles were evaluated for the encapsulation into red blood cells through the transient opening of membrane pores by controlled hypotonic dialysis and successive isotonic resealing and reannealing of cells. RESULTS: Commercial superparamagnetic iron oxide nanoparticles (SHU 555A, AMI 227 and PMP-50) dextran or carboxydextran coated can be successfully loaded into red blood cells; similarly, some of the new nanomaterials, such as Np-1 nanoparticles dispersed in the Disperbyk®-190 agent, can be efficiently encapsulated into red blood cells. CONCLUSION: A careful consideration of magnetic nanoparticles parameters, such as size, synthesis protocols, coating and/or dispersant agents, is required in order to obtain efficient loading through the cell membrane pores.

Design of water-based ferrofluids as contrast agents for magnetic resonance imaging.

We report the synthesis, characterization and relaxometric study of ferrofluids based on iron oxide, with potential for use as magnetic resonance imaging (MRI) contrast agents (CAs). The effect of different cost-effective, water-based surface modification approaches which can be easily scaled-up for the large scale synthesis of the ferrofluids has been investigated. Surface modification was achieved by silanization, and/or coating with non-toxic commercial dispersants (a lauric polysorbate and a block copolymer with pigment affinic groups, namely Tween 20 and Disperbyk 190) which were added after or during iron oxide nanoparticle synthesis. It was observed that all the materials synthesized functioned as negative contrast agents at physiological temperature and at frequencies covered by clinical imagers. The relaxometric properties of the magnetic nanoparticles were significantly improved after surface coating with stabilizers compared to the original iron oxide nanoparticles, with particular reference to the silica-coated magnetic nanoparticles. The results indicate that the optimization of the preparation of colloidal magnetic ferrofluids by surface modification is effective in the design of novel contrast agents for MRI by enabling better or more effective interaction between the coated iron oxide nanoparticles and protons present in their aqueous environment.

Inorganic materials for bone repair or replacement applications.

In recent years, excipient systems have been used increasingly in biomedicine in reconstructive and replacement surgery, as bone cements, drug-delivery vehicles and contrast agents. Particularly, interest has been growing in the development and application of controlled pore inorganic ceramic materials for use in bone-replacement and bone-repair roles and, in this context, attention has been focused on calcium-phosphate, bioactive glasses and SiO2- and TiO2-based materials. It has been shown that inorganic materials that most closely mimic bone structure and surface chemistry most closely function best in bone replacement/repair and, in particular, if a substance possesses a macroporous structure (pores and interconnections >100 microm diameter), then cell infiltration, bone growth and vascularization can all be promoted. The surface roughness and micro/mesoporosity of a material have also been observed to significantly influence its ability to promote apatite nucleation and cell attachment significantly. Pores (where present) can also be packed with pharmaceuticals and biomolecules (e.g., bone morphogenetic proteins [BMPs], which can stimulate bone formation). Finally, the most bio-efficient - in terms of collagen formation and apatite nucleation - materials are those that are able to provide soluble mineralizing species (Si, Ca, PO(4)) at their implant sites and/or are doped or have been surface-activated with specific functional groups. This article presents the context and latest advances in the field of bone-repair materials, especially with respect to the development of bioactive glasses and micro/mesoporous and macroporous inorganic scaffolds. It deals with the possible methods of preparing porous pure/doped or functionalized silicas or their composites, the studies that have been undertaken to evaluate their abilities to act as bone repair scaffolds and also presents future directions for work in that context.

Tn5530 from Burkholderia cepacia strain 2a encodes a chloride channel protein essential for the catabolism of 2,4-dichlorophenoxyacetic acid.

Chloride channel proteins (ClC) are found in living systems where they transport chloride ions across cell membranes. Recently, the structure/function of two prokaryotic ClC has been determined but little is known about the role of these proteins in the microbial metabolism of chlorinated compounds. Here we show that transposon Tn5530 from Burkholderia cepacia strain 2a encodes a ClC protein (BcClC) which is responsible for expelling Cl(-) ions generated during the catabolism of 2,4-dichlorophenoxyacetic acid (a chlorinated herbicide). We found that BcClC has the ability to transport Cl(-) ions across reconstituted proteoliposome membranes. We created two mutants in which the intrachannel glutamate residue of the protein, known to be responsible for opening and closing the channel (i.e. gating), was changed in order to create constitutively open and closed forms. We observed that cells carrying the closed-channel protein accumulated Cl(-) ions intracellularly leading to a decrease in intracellular pH, cell stasis and death. Further, we established that BcClC has the same gating mechanism as that reported for the ClC protein from Salmonella typhimurium. Our results show that the physiological role of ClC is to maintain cellular homeostasis which can be impaired by the catabolism of chlorinated compounds.

The use of magnetic nanoparticles in the development of new molecular detection systems.

Magnetic nanoparticles have been widely used in biomolecular separation and discrimination which coincidentally also represents the basis for most current day molecular diagnostic procedures. The specificity, affinity, and binding capacity of magnetic nanoparticles depends on their size, form, dispersion, and surface chemistry. In this review, we will briefly analyze how these factors affect biomolecular separations and focus on the use of magnetic nanoparticles in monitoring the microbial biodiversity in the environment. We found that magnetic nanoparticles are especially effective for biomolecular separations in environmental samples collected and preserved with fixatives. This feature, together with the high sample throughput capability and the generic low cost, makes magnetic nanoparticles particularly suitable for environmental microbial monitoring. Furthermore, key features that permit the optimization of magnetic nanoparticles-based separations and that can be useful in the development of new analytical procedures are also discussed.

Surface modification of magnetic nanoparticles with alkoxysilanes and their application in magnetic bioseparations.

A versatile and inexpensive method for the introduction of amine groups onto the surface of silica-coated magnetite composite nanoparticles has been established based on the condensation of (aminopropyl)triethoxysilane (APTS). The process was observed to be sensitive to a range of variables, and a range of silane surface-modified nanoparticles was synthesized under various reaction conditions, that is, solvent systems [water, tetrahydrofuran (THF), ethanol, or 1:1 mixtures of them], reaction times (from 1 to 24 h), and temperatures (18, 50, and 70 degrees C), with water as the catalyst and silane at either 0.2% or 2% (w/v) in an attempt to optimize the process. The products of the various reactions were characterized in terms of their possession of surface -NH2 groups, morphologies, and properties with respect to DNA binding and elution before being modified with a single-stranded oligonucleotide capture sequence. It was observed that careful manipulation of temperature, time, and solvent conditions was important for optimal silanization of the nanoparticles, and in our experiments best results were obtained when silanization of the particles in suspension involved use of water as the solvent and APTS at 0.2% (w/v) and when the reaction was conducted at room temperature for 5 h and was preceded by ultrasonication of the particle suspension. The materials produced were used in experiments to selectively capture complementary nucleic acid sequences by hybridization after grafting with an oligonucleotide. The efficiency of the oligonucleotide-modified particles in the capture experiments was observed to be directly related to the original density of amine groups present at the surface of the support. The results indicate that surface engineering of the nanoparticles was possible by silanization under defined, optimized conditions. This approach could be extended to the activation of such surfaces and other materials with other functional groups.