Production of xylitol by metabolically engineered strains of Bacillus subtilis

Xylitol-phosphate dehydrogenase (XPDH) genes from several Gram-positive bacteria were isolated and expressed in Bacillus subtilis. The substrate specificities of the recombinant XPDH enzymes were compared and it was found that the XPDH enzymes of Lactobacillus rhamnosus and Clostridium difficile had the highest selectivity towards D-xylulose 5-phosphate. Expression of these two XPDH enzymes in D-ribulose and D-xylulose producing B. subtilis strain resulted in strains of B. subtilis capable of converting D-glucose into xylitol at around 23% yield.     

A critical three-way junction is conserved in budding yeast and vertebrate telomerase RNAs

The telomerase ribonucleoprotein copies a short template within its integral RNA moiety onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Non-template regions of telomerase RNA (TER) are also crucial for telomerase function, yet they are highly divergent in sequence among species and their roles are largely unclear. Using both phylogenetic and mutational analyses, we predicted secondary structures for TERs from Kluyveromyces budding yeast species. A comparison of these secondary structure models with the published model for the Saccharomyces cerevisiae TER reveals a common arrangement into three long arms, a templating domain in the center and several conserved elements in the same positions within the structure. One of them, a three-way junction element, is highly conserved in budding yeast TERs. This element also shows sequence and structure similarity to the critical CR4-CR5 activating domain of vertebrate TERs. Mutational analysis in Kluyveromyces lactis confirmed that this element, and in particular the residues conserved across yeast and vertebrates, is critical for telomerase action both in vivo and in vitro. These findings demonstrate that despite the extreme divergence of TER sequences from different organisms, they do share conserved elements, which presumably carry out common roles in telomerase function.     

The nucleolar channel system of human endometrium is related to endoplasmic reticulum and R-rings

The nucleolar channel system (NCS) is a well-established ultrastructural hallmark of the postovulation endometrium. Its transient presence has been associated with human fertility. Nevertheless, the biogenesis, composition, and function of these intranuclear membrane cisternae are unknown. Membrane systems with a striking ultrastructural resemblance to the NCS, termed R-rings, are induced in nuclei of tissue culture cells by overexpression of the central repeat domain of the nucleolar protein Nopp140. Here we provide a first molecular characterization of the NCS and compare the biogenesis of these two enigmatic organelles. Like the R-rings, the NCS consists of endoplasmic reticulum harboring the marker glucose-6-phosphatase. R-ring formation initiates at the nuclear envelope, apparently by a calcium-mediated Nopp140-membrane interaction, as supported by the calcium-binding ability of Nopp140, the inhibition of R-ring formation by calcium chelators, and the concentration of Nopp140 and complexed calcium in R-rings. Although biogenesis of the NCS may initiate similarly, the reduced presence of complexed calcium and Nopp140 suggests the involvement of additional factors.     

Chromosomal toxin-antitoxin loci can diminish large-scale genome reductions in the absence of selection

Superintegrons (SIs) are chromosomal genetic elements containing assemblies of genes, each flanked by a recombination sequence (attC site) targeted by the integron integrase. SIs may contain hundreds of attC sites and intrinsic instability is anticipated; yet SIs are remarkably stable. This implies that either selective pressure maintains the genes or mechanisms exist which favour their persistence in the absence of selection. Toxin/antitoxin (TA) systems encode a stable toxin and a specific, unstable antitoxin. Once activated, the continued synthesis of the unstable antitoxin is necessary for cell survival. A bioinformatic search of accessible microbial genomes for SIs and TA systems revealed that large SIs harboured TA gene cassettes while smaller SIs did not. We demonstrated the function of TA loci in different genomic contexts where large-scale deletions can occur; in SIs and in a 165 kb dispensable region of the Escherichia coli genome. When devoid of TA loci, large-scale genome loss was evident in both environments. The inclusion of two TA loci, relBE1 and parDE1, which we identified in the Vibrio vulnificus SI rendered these environments refractory to gene loss. Thus, chromosomal TA loci can stabilize massive SI arrays and limit the extensive gene loss that is a hallmark of reductive evolution.     

Eurasian otter (<Emphasis Type="Italic">Lutra lutra</Emphasis>) density estimate based on radio tracking and other data sources

Estimating animal population size is a critical task in both wildlife management and conservation biology. Precise and unbiased estimates are nonetheless mostly difficult to obtain, as estimates based on abundance over unit area are frequently inflated due to the “edge effect” bias. This may lead to the implementation of inappropriate management and conservation decisions. In an attempt to obtain an as accurate and conservative as possible picture of Eurasian otter (Lutra lutra) numbers, we combined radio tracking data from a subset of tracked individuals from an extensive project on otter ecology performed in Southern Portugal with information stemming from other data sources, including trapping, carcasses, direct observation of tagged and untagged individuals, relatedness estimates among genotyped individuals, and a minor contribution from non-invasive genetic sampling. In 158 km of water network, which covers a sampling area of 161 km² and corresponds to the minimum convex polygon constructed around the locations of five radio-tracked females, 21 animals were estimated to exist. They included the five radio-tracked, reproducing females and six adult males. Density estimates varied from one otter per 3.71–7.80 km of river length (one adult otter per 7.09–14.36 km) to one otter per 7.67–7.93 km² of range, depending on the method and scale of analysis. Possible biases and implications of methods used for estimating density of otters and other organisms living in linear habitats are highlighted, providing recommendations on the issue.     

Internalization of staphylococcal leukotoxins that bind and divert the C5a receptor is required for intracellular Ca2+ mobilization by human neutrophils

A growing number of receptors, often associated with the innate immune response, are being identified as targets for bacterial toxins of the beta‐stranded pore‐forming family. These findings raise the new question of whether the receptors are activated or merely used as docking points facilitating the formation of a pore. To elucidate whether the Staphylococcus aureus Panton‐Valentine leukocidin and the leukotoxin HlgC/HlgB act through the C5a receptor (C5aR) as agonists, antagonists or differ from the C5a complement‐derived peptide, their activity is explored on C5aR‐expressing cells. Both leukotoxins equally bound C5aR in neutrophils and in stable transfected U937 cells and initiated mobilization of intracellular Ca²⁺. HlgC/HlgB requires the presence of robust intracellular acidic Ca²⁺ stores in order to evoke a rise in free [Ca²⁺]_i, while the LukS‐PV/LukF‐PV directly altered reticular Ca²⁺ stores. Intracellular target specificity is conferred by the F‐subunit associated to the S‐subunit binding the receptor. Furthermore, internalization of the two leukotoxin components (S‐ and F‐subunits) associated to C5aR is required for the initiation of [Ca²⁺]_i mobilization. Electrophysiological recordings on living cells demonstrated that LukS‐PV/LukF‐PV does not alter the membrane resistance of C5aR‐expressing cells. The present observations suggest that part of the pore‐forming process occurs in distinct intracellular compartments rather than at the plasma membrane.     

Living on the verge: are roads a more suitable refuge for small mammals than streams in Mediterranean pastureland?

The retention of natural habitat corridors is a useful and practical conservation tool that can attenuate the effects of habitat loss and fragmentation on wildlife. Linear structures may contribute to the conservation of biodiversity by providing additional habitats for small fauna living in highly modified environments. We assessed the importance of road verges as refuge areas for small mammals, in highly intensified grazed pastures, within a Mediterranean landscape and compared the role of road verges as refuges with that of riparian galleries, which have been described as important shelter locations for small fauna. For this purpose, a small mammal trapping study was undertaken on two road verges and beside two small streams in southern Portugal. We captured 457 individuals of five different species, with Mus spretus the most common species captured, followed by Crocidura russula. Captures were 4.6-fold higher immediately beside both roads and streams than 12 m away in the surrounding matrix. Individuals captured in the matrix presented a smaller body size and lower body condition, suggesting that this suboptimal habitat is occupied mainly by subadults. M. spretus was 46% more abundant by roads than by streams, while C. russula was present in similar numbers in both habitats. M. spretus individuals were larger near streams but exhibited no difference in body condition between habitats. C. russula had a better body condition and slightly higher body lengths at roadsides. Our results show that roadside verges in intensively grazed Mediterranean landscapes act as important refuges and constitute equally vital habitats for small mammals as do riparian vegetation strips in landscapes where other suitable habitats are scarce.     

Solid-phase treatment with the fungus Trametes versicolor substantially reduces pharmaceutical concentrations and toxicity from sewage sludge

For safe biosolid-land-applying, sludge should be contaminant-free. However, it may contain important amounts of micropollutants, not removed in the wastewater-treatment-processes. An alternative treatment with the fungus Trametes versicolor was applied in sterile solid-phase systems consisting of sludge and a lignocellulosic substrate. Fungal colonization and activity were demonstrated during the process, according to monitoring of ergosterol, laccase activity and the naproxen-degradation test (ND24). Fourteen out of 43 analyzed pharmaceuticals were found in the raw sludge. After treatment, phenazone, bezafibrate, fenofibrate, cimetidine, clarithromycin, sulfamethazine and atenolol were completely removed, while removals between 42% and 80% were obtained for the remaining pharmaceuticals. Toxicological analyses (Daphnia magna, Vibrio fischeri and seed germination) showed an important reduction in sludge toxicity after treatment. Results suggest that a solid-phase treatment with T. versicolor may reduce the ecotoxicological impact of micropollutants present in sewage sludge. This is the first report of a fungal-approach for elimination of emerging pollutants from biosolids.     

Evaluation of the acetaldehyde production and degradation potential of 26 enological <Emphasis Type="Italic">Saccharomyces</Emphasis> and non-<Emphasis Type="Italic">Saccharomyces</Emphasis> yeast strains in a resting cell model system

Acetaldehyde is relevant for wine aroma, wine color, and microbiological stability. Yeast are known to play a crucial role in production and utilization of acetaldehyde during fermentations but comparative quantitative data are scarce. This research evaluated the acetaldehyde metabolism of 26 yeast strains, including commercial Saccharomyces and non-Saccharomyces, in a reproducible resting cell model system. Acetaldehyde kinetics and peak values were highly genus, species, and strain dependent. Peak acetaldehyde values varied from 2.2 to 189.4 mg l⁻¹ and correlated well (r ² = 0.92) with the acetaldehyde production yield coefficients that ranged from 0.4 to 42 mg acetaldehyde per g of glucose in absence of SO₂. S. pombe showed the highest acetaldehyde production yield coefficients and peak values. All other non-Saccharomyces species produced significantly less acetaldehyde than the S. cerevisiae strains and were less affected by SO₂ additions. All yeast strains could degrade acetaldehyde as sole substrate, but the acetaldehyde degradation rates did not correlate with acetaldehyde peak values or acetaldehyde production yield coefficients in incubations with glucose as sole substrate.