Comparative membrane proteome analysis of three Borrelia species

The versatility of the surface of Borrelia, the causative agent of Lyme borreliosis, is very important in host-pathogen interactions allowing bacteria to survive in ticks and to persist in a mammalian environment. To identify the surface proteome of Borrelia, we have performed a large comparative proteomic analysis on the three most important pathogenic Borrelia species, namely B. burgdorferi (strain B31), B. afzelii (strain K78), and B. garinii (strain PBi). Isolation of membrane proteins was performed by using three different approaches: (i) a detergent-based fractionation of outer membrane proteins; (ii) a trypsin-based partial shedding of outer cell surface proteins; (iii) biotinylation of membrane proteins and preparation of the biotin-labelled fraction using streptavidin. Proteins derived from the detergent-based fractionation were further sub-fractionated by heparin affinity chromatography since heparin-like molecules play an important role for microbial entry into human cells. All isolated proteins were analysed using either a gel-based liquid chromatography (LC)-MS/MS technique or by two-dimensional (2D)-LC-MS/MS resulting in the identification of 286 unique proteins. Ninety seven of these were found in all three Borrelia species, representing potential targets for a broad coverage vaccine for the prevention of Lyme borreliosis caused by the different Borrelia species.     

Unusual signal peptide directs penicillin amidase from Escherichia coli to the Tat translocation machinery

The recently described Tat protein translocation system in Escherichia coli recognizes its protein substrates by the consensus twin arginine (SRRXFLK) motif in the signal peptide. The signal sequence of E. coli pre-pro-penicillin amidase bears two arginine residues separated by one aspargine and does not resemble the Tat-targeting motif but can nevertheless target the precursor to the Tat pathway. Mutational studies have shown that the hydrophobic core region acts in synergism with the positive charged N-terminal part of the signal peptide as a Tat recognition signal and contributes to the efficient Tat targeting of the pre-pro-penicillin amidase.     

Enzyme synthesis of oligosaccharides using cashew apple juice as substrate

The use of agriculture substrates in industrial biotechnological processes has been increasing because of their low cost. In this work, the use of clarified cashew apple juice was investigated as substrate for enzyme synthesis of prebiotic oligosaccharide. The results showed that cashew apple juice is a good source of reducing sugars and can be used as substrate for the production of dextransucrase by Leuconostoc citreum B-742 for the synthesis of oligosaccharides using the crude enzyme. Optimal oligosaccharide yield (approximately 80%) was obtained for sucrose concentrations lower than 60 g/L and reducing sugar concentrations higher than 100 g/L.     

The above-ground coarse wood productivity of 104 Neotropical forest plots

The net primary production of tropical forests and its partitioning between long‐lived carbon pools (wood) and shorter‐lived pools (leaves, fine roots) are of considerable importance in the global carbon cycle. However, these terms have only been studied at a handful of field sites, and with no consistent calculation methodology. Here we calculate above‐ground coarse wood carbon productivity for 104 forest plots in lowland New World humid tropical forests, using a consistent calculation methodology that incorporates corrections for spatial variations in tree‐size distributions and wood density, and for census interval length. Mean wood density is found to be lower in more productive forests. We estimate that above‐ground coarse wood productivity varies by more than a factor of three (between 1.5 and 5.5 Mg C ha^?1 a^?1) across the Neotropical plots, with a mean value of 3.1 Mg C ha^?1 a^?1. There appear to be no obvious relationships between wood productivity and rainfall, dry season length or sunshine, but there is some hint of increased productivity at lower temperatures. There is, however, also strong evidence for a positive relationship between wood productivity and soil fertility. Fertile soils tend to become more common towards the Andes and at slightly higher than average elevations, so the apparent temperature/productivity relationship is probably not a direct one. Coarse wood productivity accounts for only a fraction of overall tropical forest net primary productivity, but the available data indicate that it is approximately proportional to total above‐ground productivity. We speculate that the large variation in wood productivity is unlikely to directly imply an equivalent variation in gross primary production. Instead a shifting balance in carbon allocation between respiration, wood carbon and fine root production seems the more likely explanation.     

Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180?mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270?mg/L isobutanol and 40?mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14?g/L branched-chain alcohols over the duration of 50?days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.     

Analysis of the fibroblast growth factor system reveals alterations in a mouse model of spinal muscular atrophy

The monogenetic disease Spinal Muscular Atrophy (SMA) is characterized by a progressive loss of motoneurons leading to muscle weakness and atrophy due to severe reduction of the Survival of Motoneuron (SMN) protein. Several models of SMA show deficits in neurite outgrowth and maintenance of neuromuscular junction (NMJ) structure. Survival of motoneurons, axonal outgrowth and formation of NMJ is controlled by neurotrophic factors such as the Fibroblast Growth Factor (FGF) system. Besides their classical role as extracellular ligands, some FGFs exert also intracellular functions controlling neuronal differentiation. We have previously shown that intracellular FGF-2 binds to SMN and regulates the number of a subtype of nuclear bodies which are reduced in SMA patients. In the light of these findings, we systematically analyzed the FGF-system comprising five canonical receptors and 22 ligands in a severe mouse model of SMA. In this study, we demonstrate widespread alterations of the FGF-system in both muscle and spinal cord. Importantly, FGF-receptor 1 is upregulated in spinal cord at a pre-symptomatic stage as well as in a mouse motoneuron-like cell-line NSC34 based model of SMA. Consistent with that, phosphorylations of FGFR-downstream targets Akt and ERK are increased. Moreover, ERK hyper-phosphorylation is functionally linked to FGFR-1 as revealed by receptor inhibition experiments. Our study shows that the FGF system is dysregulated at an early stage in SMA and may contribute to the SMA pathogenesis.