Xylose reductase activity in <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis> UFV-170 cultivated in semi-synthetic medium and cotton husk hemicellulose hydrolyzate

To develop a new enzymatic xylose-to-xylitol conversion, deeper knowledge on the regulation of xylose reductase (XR) is needed. To this purpose, a new strain of Debaryomyces hansenii (UFV-170), which proved a promising xylitol producer, was cultivated in semi-synthetic media containing different carbon sources, specifically three aldo-hexoses (d-glucose, d-galactose and d-mannose), a keto-hexose (d-fructose), a keto-pentose (d-xylose), three aldo-pentoses (d-arabinose, l-arabinose and d-ribose), three disaccharides (maltose, lactose and sucrose) and a pentitol (xylitol). The best substrate was lactose on which cell concentration reached about 20 g l⁻¹ dry weight (DW), while the highest specific growth rates (0.58–0.61 h⁻¹) were detected on lactose, d-mannose, d-glucose and d-galactose. The highest specific activity of XR (0.24 U mg⁻¹) was obtained in raw extracts of cells grown on d-xylose and harvested in the stationary growth phase. When grown on cotton husk hemicellulose hydrolyzates, cells exhibited XR activities five to seven times higher than on semi-synthetic media.     

Probing the conformation of the resting state of a bacterial multidrug ABC transporter,BmrA, by a site‐directed spin labeling approach

Previously published 3‐D structures of a prototypic ATP‐binding cassette (ABC) transporter, MsbA, have been recently corrected revealing large rigid‐body motions possibly linked to its catalytic cycle. Here, a closely related multidrug bacterial ABC transporter, BmrA, was studied using site‐directed spin labeling by focusing on a region connecting the transmembrane domain and the nucleotide‐binding domain (NBD). Electron paramagnetic resonance (EPR) spectra of single spin‐labeled cysteine mutants suggests that, in the resting state, this sub‐domain essentially adopts a partially extended conformation, which is consistent with the crystal structures of MsbA and Sav1866. Interestingly, one of the single point mutants (Q333C) yielded an immobilized EPR spectrum that could arise from a direct interaction with a vicinal tyrosine residue. Inspection of different BmrA models pointed to Y408, within the NBD, as the putative interacting partner, and its mutation to a Phe residue indeed dramatically modified the EPR spectra of the spin labeled Q333C. Moreover, unlike the Y408F mutation, the Y408A mutation abolished both ATPase activity and drug transport of BmrA, suggesting that a nonpolar bulky residue is required at this position. The spatial proximity of Q333 and Y408 was also confirmed by formation of a disulfide bond when both Q333 and T407 (or S409) were replaced jointly by a cysteine residue. Overall, these results indicate that the two regions surrounding Q333 and Y408 are close together in the 3‐D structure of BmrA and that residues within these two sub‐domains are essential for proper functioning of this transporter.     

Time-resolved fluorescence resonance energy transfer shows that the bacterial multidrug ABC half-transporter BmrA functions as a homodimer

Members of the ATP-binding cassette (ABC) transporters share the same basic architecture, with a four-core domain made of two transmembrane plus two nucleotide-binding domains. However, a supramolecular organization has been detected in some ABC transporters, which might be relevant to physiological regulation of substrate transport. Here, the oligomerization status of a bacterial half-ABC multidrug transporter, BmrA, was investigated. Each BmrA monomer containing a single cysteine residue introduced close to either the Walker A or the ABC signature motifs was labeled using two probes, 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid (fluorescence donor) or 4-dimethylaminophenylazophenyl-4'-maleimide (fluorescence acceptor). Reconstitution into proteoliposomes of BmrA monomers labeled separately with either the fluorescence donor or the fluorescence acceptor allowed measurement of time-resolved fluorescence resonance energy transfer between the two probes, showing that efficient reassociation of the singly labeled BmrA monomers occurred upon reconstitution. The efficiency of energy transfer studied as a function of increasing concentration of BmrA-labeled with the fluorescence acceptor argues for a dimeric association of BmrA instead of a tetrameric one. Furthermore, the efficiency of energy transfer allowed estimation of the distances between the two bound probes. Results suggest that, in the resting state, BmrA in a lipid bilayer environment preferentially adopts a closed conformation similar to that found in the BtuCD crystal structure and that the presence of different effectors does not substantially modify its global conformation.     

Ribosomal Interaction of Bacillus stearothermophilus Translation Initiation Factor IF2: Characterization of the Active Sites

InfB-encoded translation initiation factor IF2 contains a non-conserved N-terminal domain and two conserved domains (G and C) constituted by three (G1, G2 and G3) and two (C1 and C2) sub-domains. Here, we show that: (i) Bacillus stearothermophilus IF2 complements in vivo an Escherichia coli infB null mutation and (ii) the N-domain of B. stearothermophilus IF2, like that of E. coli IF2, provides a strong yet dispensable interaction with 30 S and 50 S subunits in spite of the lack of any size, sequence or structural homology between the N-domains of the two factors. Furthermore, the nature of the B. stearothermophilus IF2 sites involved in establishing the functional interactions with the ribosome was investigated by generating deletion, random and site-directed mutations within sub-domains G2 or G3 of a molecule carrying an H301Y substitution in switch II of the G2 module, which impairs the ribosome-dependent GTPase activity of IF2. By selecting suppressors of the dominant-lethal phenotype caused by the H301Y substitution, three independent mutants impaired in ribosome binding were identified; namely, S387P (in G2) and G420E and E424K (in G3). The functional properties of these mutants and those of the deletion mutants are compatible with the premise that IF2 interacts with 30 S and 50 S subunits via G3 and G2 modules, respectively. However, beyond this generalization, because the mutation in G2 resulted in a functional alteration of G3 and vice versa, our results indicate the existence of extensive “cross-talking” between these two modules, highlighting a harmonic conformational cooperation between G2 and G3 required for a functional interaction between IF2 and the two ribosomal subunits. It is noteworthy that the E424K mutant, which completely lacks GTPase activity, displays IF2 wild-type capacity in supporting initiation of dipeptide formation.     

Effects of light intensity and dilution rate on the semicontinuous cultivation of Arthrospira (Spirulina) platensis. A kinetic Monod-type approach

Semicontinuous cultures were carried out at different dilution rates (D) and light intensities (I) to determine the maximum productivity of Arthrospira platensis cultivated in helicoidal photobioreactor up to the achievement of pseudo-steady-state conditions. At I=108 μmol photons m(-2) s(-1), the semicontinuous regime ensured the highest values of maximum cell concentration (X(m)=5772±113 mg L(-1)) and productivity (P(XS)=1319±25 mg L(-1) d(-1)) at the lowest (D=0.1 day(-1)) and the highest (D=0.3 day(-1)) dilution rates, respectively. A kinetic model derived from that of Monod was proposed to determine the relationship between the product of light intensity to dilution rate (ID) and the cell productivity, which were shown to exert a combined influence on this parameter. This result put into evidence that pseudo-steady-state conditions could be modified according to circumstances, conveniently varying one or other of the two independent variables.     

Retracted: Advances in the physiological and pathological implications of cholesterol

Cholesterol has evolved to fulfill sophisticated biophysical, cell signalling, and endocrine functions in animal systems. At the cellular level, cholesterol is found in membranes where it increases both bilayer stiffness and impermeability to water and ions. Furthermore, cholesterol is integrated into specialized lipid‐protein membrane microdomains with critical topographical and signalling functions. At the organismal level, cholesterol is the precursor of all steroid hormones, including gluco‐ and mineralo‐corticoids, sex hormones, and vitamin D, which regulate carbohydrate, sodium, reproductive, and bone homeostasis, respectively. This sterol is also the immediate precursor of bile acids, which are important for intestinal absorption of dietary lipids as well as energy homeostasis and glucose regulation. Complex mechanisms maintain cholesterol within physiological ranges and the dysregulation of these mechanisms results in embryonic or adult diseases, caused by either excessive or reduced tissue cholesterol levels. The causative role of cholesterol in these conditions has been demonstrated by genetic and pharmacological manipulations in animal models of human disease that are discussed herein. Importantly, the understanding of basic aspects of cholesterol biology has led to the development of high‐impact pharmaceutical therapies during the past century. The continuing effort to offer successful treatments for prevalent cholesterol‐related diseases, such as atherosclerosis and neurodegenerative disorders, warrants further interdisciplinary research in the coming decades.     

Genetic structure of Italian population of the grapevine downy mildew agent,Plasmopara viticola

Downy mildew, caused by the Oomycete Plasmopara viticola, is one of the most important diseases affecting the Eurasian grapevine, Vitis vinifera. The pathogen originated in Northern America and its presence was signalled for the first time into Europe in 1878. In this study, the genetic variability and structure of Italian P. viticola population was investigated by genotyping 106 P. viticola strains belonging to 12 different regions, at 31 microsatellite markers. As a result of the high percentage of missing data, 96 strains and 19 loci were retained for the data analysis. The overall Italian population presents absence of clones, evidence of sexual and asexual reproduction and a low genetic diversity, as expected for an introduced pathogen, but a slightly higher genetic diversity than that found in other European populations, based on allelic diversity at the investigated microsatellite loci. Out of 19 loci, half shows deviation from Hardy–Weinberg equilibrium and, indeed, structure analysis indicates the presence of two separate genetic clusters, with little but significantly different distribution according to geography (west–east gradient) and climatic conditions. Overall, the analysis of the P. viticola population, 140 years after its appearance in Italy, provides indication on the pathogen adaptability. This should be taken into consideration in the future for preserving the durability of disease resistant varieties in open field. In this view, all the disease control methods available should be integrated in order to reduce the selection of pathogen strains able to overcome plant resistance.     

Carbon material and bioenergetic balances of xylitol production from corncobs by Debaryomyces hansenii

The effect of oxygenation on xylitol production by the yeast Debaryomyces hansenii has been investigated in this work using the liquors from corncob hydrolysis as the fermentation medium. The concentrations of consumed substrates (glucose, xylose, arabinose, acetate and oxygen) and formed products (xylitol, arabitol, ethanol, biomass and carbon dioxide) have been used, together with those previously obtained varying the hydrolysis technique, the level of adaptation of the microorganism, the sterilization procedure and the initial substrate and biomass concentrations, in carbon material balances to evaluate the percentages of xylose consumed by the yeast for the reduction to xylitol, alcohol fermentation, respiration and cell growth. The highest xylitol concentration (71 g/L) and volumetric productivity (1.5 g/L.h) were obtained semiaerobically using detoxified hydrolyzate produced by autohydrolysis-posthydrolysis, at starting levels of xylose (S(0)) and biomass (X(0)) of about 100 g/L and 12 g(DM)/L, respectively. No less than 80% xylose was addressed to xylitol production under these conditions. The experimental data collected in this work at variable oxygen levels allowed estimating a P/O ratio of 1.16 mol(ATP)/mol(O). The overall ATP requirements for biomass production and maintenance demonstrated to remarkably increase with X(0) and for S(0) >or= 130 g/L and to reach minimum values (1.9-2.1 mol(ATP)/C-mol(DM)) just under semiaerobic conditions favoring xylitol accumulation.     

Urea increases fed‐batch growth and γ‐linolenic acid production of nutritionally valuable Arthrospira (Spirulina) platensis cyanobacterium

In this article we investigate the simultaneous influence of feeding time and amount of urea added as a nitrogen source on the fed‐batch growth and composition of Arthrospira (Spirulina) platensis. Cultivations were performed in 5‐L minitanks at constant temperature (25°C) and light intensity (42 μmol photons/m²s), using exponentially increasing rate of urea addition, and varying the above independent variables in the ranges 9–15 days and 4.6–12.1 mM, respectively. Special emphasis was placed on the content of added high value fatty acids (e.g., γ‐linolenic acid) of concern for the food industry. To this purpose, a 2²‐plus star central composite design was employed, and maximum cell concentration, cell productivity, yield of biomass on nitrogen added, protein content and fatty acids profile were evaluated by multiple regression analysis. The highest cell concentration (1759 mg/L) was obtained at feeding time of 14 days and amount of urea per unit reactor volume of 5.8 mM, while the highest contents of γ‐linolenic acid (27.5% of the lipid fraction) and proteins (77.2%) were obtained at 10 and 14 days and 5.8 and 10.8 mM, respectively. The results confirm the possibility of using urea as cheap nitrogen source to culture this nutritionally valuable cyanobacterium.