Paul M. Zelisko

Endophytic insect-parasitic fungi translocate nitrogen directly from insects to plants.

Plants That Eat Animals Apart from some spectacular exceptions, such as pitcher plants and Venus fly traps, most plants are thought to acquire nitrogen passively from microbial decomposition and the activities of nitrogen-fixing bacteria. Metarhizium species are common endophytes—fungi that live within plant tissues without causing disease. This genus is also found ubiquitously in soil, where they parasitize insects. In a series of microcosm experiments, Behie et al. (p. 1576) investigated whether these fungi could couple their endophytic life-styles with their parasitic modes and be a conduit by which plants could obtain nitrogen from animals. Radio-labeled moth larvae were added to the microcosms in which bean and grass plants were grown, and when the larvae were inoculated with fungi, it was only a matter of days before the nitrogen label was detected in the plants. A fungal plant symbiont also consumes insects in surrounding soil and transfers animal nitrogen to the plant’s roots. Most plants obtain nitrogen through nitrogen-fixing bacteria and microbial decomposition of plant and animal material. Many vascular plants are able to form close symbiotic associations with endophytic fungi. Metarhizium is a common plant endophyte found in a large number of ecosystems. This abundant soil fungus is also a pathogen to a large number of insects, which are a source of nitrogen. It is possible that the endophytic capability and insect pathogenicity of Metarhizium are coupled to provide an active method of nitrogen transfer to plant hosts via fungal mycelia. We used soil microcosms to test the ability of M. robertsii to translocate insect-derived nitrogen to plants. Insects were injected with 15N-labeled nitrogen, and we tracked the incorporation of 15N into amino acids in two plant species, haricot bean (Phaseolus vulgaris) and switchgrass (Panicum virgatum), in the presence of M. robertsii. These findings are evidence that active nitrogen acquisition by plants in this tripartite interaction may play a larger role in soil nitrogen cycling than previously thought.

Biocatalytic synthesis of silicone polyesters.

The immobilized lipase B from Candida antarctica (CALB) was used to synthesize silicone polyesters. CALB routinely generated between 74-95% polytransesterification depending on the monomers that were used. Low molecular weight diols resulted in the highest rates of esterification. Rate constants were determined for the CALB catalyzed polytransesterifications at various reaction temperatures. The temperature dependence of the CALB-mediated polytransesterifications was examined. A lipase from C. rugosa was only successful in performing esterifications using carboxy-modified silicones that possessed alkyl chains greater than three methylene units between the carbonyl and the dimethylsiloxy groups. The proteases alpha-chymotrypsin and papain were not suitable enzymes for catalyzing any polytransesterification reactions.

Water-in-Silicone Oil Emulsion Stabilizing Surfactants Formed From Native Albumin and α,ω-Triethoxysilylpropyl-Polydimethylsiloxane

Contact with hydrophobic silicones frequently leads to protein denaturation. However, it is demonstrated that albumin in water-in-silicone oil emulsions retains its native structure in the presence of a functional, triethoxysilyl-terminated silicone polymer, TES-PDMS. Both HSA and TES-PDMS were essential for the formation of stable water-in-silicone oil emulsions: attempts to generate stable emulsions using independently either the protein or the functionalized silicone as a surfactant failed. Confocal microscopy indicated that the human serum albumin (HSA) preferentially adsorbed at the oil/water interface, even in the presence of another protein (glucose oxidase). A variety of experiments demonstrated that the hydrolysis of the Si-OEt groups on the functional silicone occurred only to a limited extent, consistent with the absence of a covalent linkage between the silicone and protein, or of cross-linked silicones at the interface. The fluorescence spectra of HSA extracted from the emulsions, front-faced fluorescence experiments on the HSA/silicone emulsion itself, and HSA/salicylate binding studies all demonstrated that the stability of the water/oil interface decreased as the protein began to unfold: unfolding of the protein in the emulsion was slower than in aqueous solution. The experimental evidence indicated that the interaction between HSA and TES-PDMS is not associated with either homomolecular (HSA/HSA; TES-PDMS/TES-PDMS) interactions or with covalent linkage between two the polymers. Rather, the data is consistent with the direct binding of unhydrolyzed Si(OEt) 3 groups to native HSA. The nature of these interactions is discussed.

Carbon translocation from a plant to an insect-pathogenic endophytic fungus

Metarhizium robertsii is a common soil fungus that occupies a specialized ecological niche as an endophyte and an insect pathogen. Previously, we showed that the endophytic capability and insect pathogenicity of Metarhizium are coupled to provide an active method of insect-derived nitrogen transfer to a host plant via fungal mycelia. We speculated that in exchange for this insect-derived nitrogen, the plant would provide photosynthate to the fungus. By using 13CO2, we show the incorporation of 13C into photosynthate and the subsequent translocation of 13C into fungal-specific carbohydrates (trehalose and chitin) in the root/endophyte complex. We determined the amount of 13C present in root-associated fungal biomass over a 21-day period by extracting fungal carbohydrates and analysing their composition using nuclear magnetic resonance (NMR) spectroscopy. These findings are evidence that the host plant is providing photosynthate to the fungus, likely in exchange for insect-derived nitrogen in a tripartite, and symbiotic, interaction.

A Comparison of Protease Active Sites and their Ability to Process Silicon-Based Substrates

Many enzymes have been identified that can participate in the hydrolysis of alkoxysilanes; each with a different degree of specificity. Our working hypothesis was that the nature of the active site of the enzyme (i.e., the compatibility of binding pockets with the substrate) could have a direct effect on the rate of catalysis. This communication reports our experiments on the relative rates of hydrolysis of a model alkoxysilane, phenyltrimethoxysilane (PTMS), by three proteases: trypsin, α-chymotrypsin, and pepsin. Trypsin which typically accepts amino acids bearing positively charged basic residues was not particularly proficient for the hydrolysis of PTMS. On the other hand, both α-chymotrypsin and pepsin, each of which contains a binding pocket, or two in the case of pepsin, suitable for accommodating aromatic residues, were more suitable for mediating hydrolysis. This report provides some preliminary data to support the hypothesis that the architecture of the enzyme active site is important in determining the proficiency with which an enzyme will process a given organosilicon substrate.

Chain length selectivity during the polycondensation of siloxane-containing esters and alcohols by immobilized Candida antarctica lipase B.

We have examined the chain length selectivity for a series of acyl donors by lipase B from Candida antarctica (CalB). CalB accepted aliphatic diesters of C4, C6 and C12 chain lengths equally. The introduction of a carbon-carbon double bond into the C4 esters dramatically lowered the rate constant associated with polymerization highlighting the role of geometry in catalysis; fumarate esters were polymerized at a reduced rate compared to the succinate esters, while the maleate esters were not polymerized above 5% over the course of 24h. A disiloxane-containing diester impeded catalysis by CalB. We examined a series of vinyl siloxane esters and alcohols, and learned that the Z arrangement around the double bond stalled esterification by CalB completely. The distance between the ester carbonyl and the dimethylsiloxy group was shown to be an important factor in mediating catalysis. The rate constants were similar when the methylene spacer was 3, 4, or 5 units in length; beyond 6 methylene units, the rate constants increased. This has been tentatively attributed to the local reduction on the steric bulk when the larger siloxane moiety lies outside of the active site of the enzyme.

Synthesis of a self-healing siloxane-based elastomer cross-linked via a furan-modified polyhedral oligomeric silsesquioxane: investigation of a thermally reversible silicon-based cross-link

We have successfully prepared an elastomeric material exhibiting excellent temperature-controlled self-healing characteristics. A synthetic procedure is provided to graft siloxane chains with maleimidocarboxyphenyl functional groups. A furan-modified polyhedral oligomeric silsesquioxane with flexible tethers was also synthesized to cross-link the siloxane chains via a Diels–Alder reaction. A model system was prepared to study the thermally reversible nature of the cross-linked entities using differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) experiments. Likewise, dynamic solid-state 1H NMR experiments confirmed the reversibility of the linkages in the polymeric materials. Elastomers that were completely severed could be reannealed to the point that signs of a defect were imperceptible even by scanning electron microscopy (SEM).

Cyclotetrasiloxane frameworks for the chemoenzymatic synthesis of oligoesters

Immobilized lipase B from Candida antarctica (Novozym® 435, N435) was utilized as part of a chemoenzymatic strategy for the synthesis of branched polyesters based on a cyclotetrasiloxane core in the absence of solvent. Nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were utilized to monitor the reactions between tetraester cyclotetrasiloxanes and aliphatic diols. The enzyme-mediated esterification reactions can achieve 65–80% consumption of starting materials in 24–48 h. Longer reaction times, 72–96 h, resulted in the formation of cross-linked gel-like networks. Gel permeation chromatography of the polymers indicated that the masses were Mw = 11400, 13100, and 19400 g mol−1 for the substrate pairs of C7D4 ester/octane-1,8-diol, C10D4 ester/pentane-1,5-diol and C10D4 ester/octane-1,8-diol respectively, after 48 h. Extending the polymerization for an additional 24 h with the C10D4 ester/octane-1,8-diol pair gave Mw = 86800 g mol−1. To the best of our knowledge this represents the first report using lipase catalysis to produce branched polymers that are built from a cyclotetrasiloxane core.

Macrocyclic Oligoesters Incorporating a Cyclotetrasiloxane Ring

Macrocyclic oligoester structures based on a cyclotetrasiloxane core consisting of tricyclic (60+ atoms) and pentacycylic (130+ atoms) species were identified as the major components of a lipase-mediated transesterification reaction. Moderately hydrophobic solvents with log P values in the range of 2-3 were more suitable than those at lower or higher log P values. Temperature had little effect on total conversion and yield of the oligoester macrocycles, except when a reaction temperature of 100 °C was employed. At this temperature, the amount of the smaller macrocycle was greatly increased, but at the expense of the larger oligoester. For immobilized lipase B from Candida antarctica (N435), longer chain length esters and diols were more conducive to the synthesis of the macrocycles. Langmuir isotherms indicated that monolayers subjected to multiple compression/expansion cycles exhibited a reversible collapse mechanism different from that expected for linear polysiloxanes.

Analysis of Trisiloxane Phosphocholine Bilayers

We have synthesized unique siloxane phosphocholines and characterized their aggregates in aqueous solution. The siloxane phosphocholines form nearly monodisperse vesicles in aqueous solution without the need for secondary extrusion processes. The area/lipid, lipid volume, and bilayer thickness were determined from small-angle X-ray scattering experiments. The impetus for the spontaneous formation of unilamellar vesicles by these compounds is discussed.