The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding to Metabolic Intermediates

Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low μm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates.     

Monosaccharide precursors for boosting chondroitin-like capsular polysaccharide production

Chondroitin sulfate is a well-known bioactive molecule, widely used as an anti-osteoarthritis drug, that is nowadays mainly produced by animal tissue sources with unsafe extraction procedures. Recent studies have explored an integrated biotechnological–chemical strategy to obtain a chondroitin sulfate precursor from Escherichia coli K4 capsular polysaccharide, demonstrating the influence of environmental and growth conditions on capsule synthesis. In this research work, the flexibility of the strain biosynthetic machinery was investigated to enhance the K4 capsular polysaccharide production by supplementing the growth medium with the monosaccharides (glucuronic acid, galactosamine and fructose) that constitute the chain. Shake flask experiments were performed by adding the sugars singularly or together, by testing monosaccharide different concentrations and times of addition and by observing the bacterial sugar consumption. A K4 capsular polysaccharide production enhancement, compared to the control, was observed in all cases of supplementation and, in particular, significant 68 and 57 % increases were observed when adding 0.385 mM glucuronic acid plus galactosamine or 0.385 mM fructose, respectively. Increased expression levels of the gene kfoC, coding for a K4 polymerase, evaluated in different growth conditions, confirmed the results at the molecular level. Furthermore, batch fermentations, performed in lab-scale reactors (2 L), allowed to double the K4 capsular polysaccharide production values obtained in shake flask conditions, by means of a strict control of the growth parameters.     

Purification of clinical-grade disulfide stabilized antibody fragment variable—Pseudomonas exotoxin conjugate (dsFv-PE38) expressed in Escherichia coli

Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion—toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the V_H or V_L. Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, V_L and V_H PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins—MR1-1 and HA22.     

The multiple forms of bovine seminal ribonuclease: Structure and stability of a C-terminal swapped dimer

Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111–116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed.     

Activation of Bacillus spores at moderately elevated temperatures (30–33 °C)

The time/temperature profiles experienced by spores on the track from their natural sporulation environment to consumable food products may be highly diverse. Temperature has been documented as an important factor that may activate spores, i.e. potentiates spores to germinate. There is, however, limited knowledge about the relationship between the expected temperature history and the subsequent germination characteristics of bacterial spores. We show here that the germination rate of five different Bacillus spore populations, represented by strains of Bacillus cereus, Bacillus weihenstephanensis, Bacillus pumilus, Bacillus licheniformis and Bacillus subtilis could be increased following 1 week storage at moderately elevated temperatures, 30–33 °C, compared to spores stored at 3–8 °C. The results imply that spores contamination routes to foods, specifically the temperature history, could be highly relevant data in predictive modeling of food spoilage and safety. Activation at these moderately elevated temperatures may be a native form of spore activation in their natural habitats, knowledge that also could be useful in development of decontamination strategies for mildly heated foods.     

Microbial biofilm structure and organic matter use in mediterranean streams

River and stream biofilms in mediterranean fluvial ecosystems face both extreme seasonality as well as arrhythmic fluctuations. The hydrological extremes (droughts and floods) impose direct changes in water availability but also in the quantity and quality of organic matter and nutrients that sustain the microbial growth. This review analyzes how these ecological pulses might determine unique properties of biofilms developing in mediterranean streams. The paper brings together data from heterotrophic and autotrophic community structure, and extracellular enzyme activities in biofilms in mediterranean streams. Mediterranean stream biofilms show higher use of peptides during the favorable period for epilithic algae development (spring), and preferential use of cellulose and hemicellulose in autumn as a response to allochthonous input. The drying process causes the reduction in bacterial production and chlorophyll biomass, but the rapid recovery of both autotrophs and heterotrophs with rewetting indicates their adaptability to fluctuations. Bacteria surviving the drought are mainly associated with sediment and leaf litter which serve as “humid refuges”. Some algae and cyanobacteria show resistant strategies to cope with the drought stress. The resistance to these fluctuations is strongly linked to the streambed characteristics (e.g., sediment grain size, organic matter accumulation, nutrient content).