Detection and subcellular localization of mature protein H, involved in excretion of cloacin DF13

Abstract To investigate whether the pCloDF13 encoded protein H is synthesized as a precursor with a signal sequence and to detect the mature protein H, radioactive labeling experiments with Escherichia coli minicells harboring different pCloDF13 derivatives were carried out. Analysis of the labeled polypeptides on special sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed that the polypeptide encoded by the pCloDF13 gene H is a precursor with an apparent M _r of about 5000. The mature polypeptide had an apparent M _r of about 2800. Localization studies revealed that the mature protein H is predominantly located in the membrane fraction, both in the cytoplasmic and in the outer membranes of the minicells.     

Mass transfer limitation as a tool to enhance the enantiomeric excess in the enzymatic synthesis of chiral cyanohydrins

The enantioselective synthesis of cyanohydrins catalyzed by R-hydroxynitrile lyase in an aqueous-organic liquid two-phase system using, mass transfer limitation to enhance enantiomeric excess at 5°C and pH 5.5 is described. Benzaldehyde, a good substrate, and cinnamaldehyde, a notoriously difficult substrate, were used as model substrates and compared in order to establish the mass transfer limitation concept in a two-liquid phase system, where the non-enzymatic-racemic reaction competes. Enzyme concentration and phase volume ratio between organic and buffer phase were geared to one another to enhance the enantiomeric excess for each substrate. In both cases, after optimization, excellent chemical conversion (>99% on a 60 mmol scale), high throughput and high enantiomeric excess (benzaldehyde >99% and cinnamaldehyde >96%) were achieved.     

Genetic mapping and QTL analysis of Botrytis resistance in <Emphasis Type="Italic">Gerbera hybrida</Emphasis>

Gerbera hybrida is an economically important cut flower. In the production and transportation of gerbera with unavoidable periods of high relative humidity, grey mould occurs and results in losses in quality and quantity of flowers. Considering the limitations of chemical use in greenhouses and the impossibility to use these chemicals in auction or after sale, breeding for resistant gerbera cultivars is considered as the best practical approach. In this study, we developed two segregating F1 populations (called S and F). Four parental linkage maps were constructed using common and parental specific SNP markers developed from expressed sequence tag sequencing. Parental genetic maps, containing 30, 29, 27 and 28 linkage groups and a consensus map covering 24 of the 25 expected chromosomes, could be constructed. After evaluation of Botrytis disease severity using three different tests, whole inflorescence, bottom (of disc florets) and ray floret, quantitative trait locus (QTL) mapping was performed using the four individual parental maps. A total of 20 QTLs (including one identical QTL for whole inflorescence and bottom tests) were identified in the parental maps of the two populations. The number of QTLs found and the explained variance of most QTLs detected reflect the complex mechanism of Botrytis disease response.     

Chaotropic heat treatment resolves native‐like aggregation of a heterologously produced hyperthermostable laminarinase

Production of hyperthermostable enzymes in mesophilic hosts frequently causes undesired aggregation of these proteins. During production of Pyrococcus furiosus endo‐β‐1,3 glucanase (LamA) in Escherichia coli, soluble and insoluble species form. Here, the authors address the composition of this mixture, including the nature of LamA conformers, and establish a method to increase the yield of native monomer. With gel electrophoresis, size‐exclusion chromatography, light scattering, circular dichroism and enzyme kinetics the authors show that approximately 50 % of heterologously produced LamA is soluble, and that 40 % of this fraction constitutes native‐like oligomers and non‐native monomers. Soluble oligomers display, like native LamA monomer, substrate inhibition, although with poor activity. Treatment of soluble oligomers with 3 M guanidinium hydrochloride at 80 °C yields up to 75 % properly active monomer. Non‐native monomer shows low specific activity without substrate inhibition. Incubating non‐native monomer with 3 M guanidinium hydrochloride at 80 °C causes formation of 25 % native LamA. Also, a large amount of insoluble LamA aggregates can be converted into soluble native monomer by application of this procedure. Thus, chaotropic heat treatment can improve the yield and quality of hyperthermostable proteins that form aberrant species during production in E. coli.     

Lipid production in wheypermeate by an unsaturated fatty acid mutant of the oleaginous yeastApiotrichum curvatum

Summary Growth rate and lipid production rate of an unsaturated fatty acid mutant (Ufa25), derived from the oleaginous yeastApiotrichum curvatum, in wheypermeate supplemented with rapeseed oil (as unsaturated fatty acid source), was comparable with wild type. The quality of the lipids produced by Ufa25 approached cocoa butter. Production of 1 kg lipid by Ufa25 will need about 6.3 kg lactose from whey and 0.5 kg rapeseed oil.     

Challenging Theophrastus: A common core list of plant traits for functional ecology

Abstract. Ecologists need a common language of plant traits in order to make comparisons across regions and scales, pool data, and maximize the utility of the data. To develop such a set of traits we began with the primary challenges faced by plants: dispersal, establishment, and persistence in order to identify fundamental traits. Most of these traits are hard to measure, but advances in comparative ecology have suggested a number of easy to measure analogs. Unfortunately, some of the fundamental traits have no simple analog. The common core list includes: seed mass, seed shape, dispersal mode, clonality, specific leaf area, leaf water content, height, above-ground biomass, life history, onset of flowering, stem density, and resprouting ability. Most of the traits can be measured quantitatively, but several traits on the list must still be measured qualitatively due to logistical problems or lack of an easy analog. Key problem areas include: dispersal ability, capacity for vegetative spread, germination, palatability, plasticity, and all the various below-ground traits. Comparative studies need to address these problem areas. The common core list is suggested as a common starting point for studies of functional ecology. The idiosyncrasies of regional floras and specific research agendas will dictate which traits can be ignored and those that need to be added.