Retama species growing in different ecological–climatic areas of northeastern Algeria have a narrow range of rhizobia that form a novel phylogenetic clade within the Bradyrhizobium genus

Sixty-seven isolates were isolated from nodules collected on roots of Mediterranean shrubby legumes Retama raetam and Retama sphaerocarpa growing in seven ecological–climatic areas of northeastern Algeria. Genetic diversity of the Retama isolates was analyzed based on genotyping by restriction fragment length polymorphism of PCR-amplified fragments of the 16S rRNA gene, the intergenic spacer (IGS) region between the 16S and 23S rRNA genes (IGS), and the symbiotic genes nifH and nodC. Eleven haplotypes assigned to the Bradyrhizobium genus were identified. Significant biogeographical differentiation of the rhizobial populations was found, but one haplotype was predominant and conserved across the sites. All isolates were able to cross-nodulate the two Retama species. Accordingly, no significant genetic differentiation of the rhizobial populations was found in relation to the host species of origin. Sequence analysis of the 16S rRNA gene grouped the isolates with Bradyrhizobium elkanii, but sequence analyses of IGS, the housekeeping genes (dnaK, glnII, recA), nifH, and nodC yielded convergent results showing that the Retama nodule isolates from the northeast of Algeria formed a single evolutionary lineage, which was well differentiated from the currently named species or well-delineated unnamed genospecies of bradyrhizobia. Therefore, this study showed that the Retama species native to northeastern Algeria were associated with a specific clade of bradyrhizobia. The Retama isolates formed three sub-groups based on IGS and housekeeping gene phylogenies, which might form three sister species within a novel bradyrhizobial clade.     

Ethanol production from olive cake biomass substrate

The inexpensive production of sugars from lignocellulose is an essential step for the use of biomass to produce fuel ethanol. Olive cake is an abundant by-product of the olive oil industry and represents a potentially significant lignocellulosic source for bioethanol production in the Mediterranean basin. Furthermore, converting olive cake to ethanol could add further value to olive production. In the present study, olive cake was evaluated as a feedstock for ethanol production. To this end, the lignocellulosic component of the olive cake was dilute-acid pretreated at a 13.5% olive-cake loading with 1.75% (w/v) sulfuric acid and heating at 160°C for 10 min. This was followed by chemical elimination of fermentation inhibitors. Soluble sugars resulting from the pretreatment process were fermented using E. coli FBR5, a strain engineered to selectively produce ethanol. 8.1 g of ethanol/L was obtained from hydrolysates containing 18.1 g of soluble sugars. Increasing the pretreatment temperature to 180°C resulted in failed fermentations, presumably due to inhibitory by-products released during pretreatment.     

Biooxidation of n-hexanol by alcohol oxidase and catalase in biphasic and micellar systems without solvent

Alcohol oxidase from Pichia pastoris together with catalase from bovine liver was used to oxidize n-hexanol to hexanal. For this purpose, an aqueous buffer solution was mixed with large amounts of hexanol by simple agitation, yielding a biphasic system, or by adding the nonionic surfactant Brij 35. Initial velocities and reaction yields after 24 h were measured as a function of various parameters such as the amounts of enzymes, hexanol, or surfactant. High enzymatic activity was determined for hexanol concentrations of between 20 mass% and 80 mass% without using any additional organic solvent. The homogenization of the biphasic systems with the help of Brij 35 did not yield a significant improvement of the bioconversion, which would justify the use of surfactants.     

Characterisation of Pythium paroecandrum and its antagonism towards Botrytis cinerea, the causative agent of grey mould disease of grape

Pythium paroecandrum (B-30), an oomycete, was isolated from soil samples taken from a wheat field in Genlis in the Burgundy region of France and was found to check the growth and development of Botrytis cinerea, a serious grapevine pathogen. The oomycete is a fast-growing organism, living on vegetable debris, and can be recognised by its catenulate hyphal swellings, catenulate oogonia, and monoclinous antheridia. When grown together with B. cinerea, the causal agent of the grey mould disease of the grapevine, P. paroecandrum shows a pronounced antagonism and suppresses its growth and its aptitude to provoke the grey mould symptoms. Morphological features of this oomycete, its antagonism to B. cinerea, the sequences of the internal transcribed spacer region of its nuclear ribosomal DNA, and its comparison with related species are discussed in this article.     

A phosphate-stimulated NAD(P)+-dependent glyceraldehyde-3-phosphate dehydrogenase in Bacillus cereus

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of central carbon metabolism, was studied in a Bacillus cereus strain isolated from the phosphate layer from Morocco. Enzymatic assays with cell extracts demonstrated that when grown on Luria-Bertani (LB) medium, B. cereus contains a major NAD+-dependent GAPDH activity and only traces of NADP+-dependent activity, but in cells grown on Pi-supplemented LB medium a strong increase of the NADP+-dependent activity, that became predominant, occurs concurrently with a GAPDH protein increase. Our results show that B. cereus possesses two GAPDH activities, namely NAD+- and NADP+-dependent, catalyzed by two enzymes with distinct coenzyme specificity and different phosphate regulation patterns. The finding of a phosphate-stimulated NADP+-dependent GAPDH in B. cereus indicates that this bacterium can modulate its primary carbon metabolism according to phosphate availability.     

Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene

The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) has been purified to homogeneity from skeletal muscle of the newt Pleurodeles waltl (Amphibia, Urodela). The purification procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulted in a 24-fold increase in specific activity and a final yield of approximately 46%. The native protein exhibited an apparent molecular weight of approximately 146 kDa with absolute specificity for NAD(+). Only one GAPDH isoform (pI 7.57) was obtained by chromatofocusing. The enzyme is an homotetrameric protein composed of identical subunits with an apparent molecular weight of approximately 37 kDa. Monospecific polyclonal antibodies raised in rabbits against the purified newt GAPDH immunostained a single 37-kDa GAPDH band in extracts from different tissues blotted onto nitrocellulose. A 510-bp cDNA fragment that corresponds to an internal region of a GapC gene was obtained by RT-PCR amplification using degenerate primers. The deduced amino acid sequence has been used to establish the phylogenetic relationships of the Pleurodeles enzyme--the first GAPDH from an amphibian of the Caudata group studied so far--with other GAPDHs of major vertebrate phyla.     

Novel major bacterial candidate division within a municipal anaerobic sludge digester

In a previous study, we analyzed the molecular diversity of Planctomycetales by PCR amplification and sequencing of 16S rRNA clone libraries generated from a municipal wastewater plant, using planctomycete-specific and universal primer sets (R. Chouari, D. Le Paslier, P. Daegelen, P. Ginestet, J. Weissenbach, and A. Sghir, Appl. Environ. Microbiol. 69:7354-7363, 2003). Only a small fraction (4%) of the 16S rRNA gene sequences of the digester clone library corresponded to the Planctomycetales division. Importantly, 85.9% of the digester clone sequences are grouped into two different clusters named WWE1 (81.4% of the sequences) and WWE2 (4.5%) and are distantly affiliated with unidentified bacterial sequences retrieved from a methanogenic reactor community and from a termite gut, respectively. In phylogenetic analysis using 16S rRNA gene sequence representatives of the main phylogenetic bacterial divisions, the two clusters are monophyletic, branch apart from each other, and are distantly related to Planctomycetales and other bacterial divisions. A novel candidate division is proposed for WWE1, while the WWE2 cluster strongly affiliates with the recently proposed Lentisphearae phylum. We designed and validated a 16S rRNA probe targeting WWE1 16S rRNA sequences by both fluorescent in situ hybridization (FISH) and dot blot hybridization (DBH). Results of FISH analysis show that WWE1 representative microorganisms are rods or filamentous shaped, while DBH shows that WWE1 accounts for 12% of the total bacterial rRNA within the anaerobic digester. The remaining 16S rRNA gene sequences are affiliated with Verrucomicrobia or recently described candidate divisions with no known pure culture representatives, such as OD1, BRC1, or NBL-UPA2, making up less than 3.5% of the clone library, respectively. This inventory expands the known diversity of the latter bacterial division-level lineages.     

Hemodynamic Changes following Aortic Valve Bypass: A Mathematical Approach

Aortic valve bypass (AVB) has been shown to be a viable solution for patients with severe aortic stenosis (AS). Under this circumstance, the left ventricle (LV) has a double outlet. The objective was to develop a mathematical model capable of evaluating the hemodynamic performance following the AVB surgery. A mathematical model that captures the interaction between LV, AS, arterial system, and AVB was developed. This model uses a limited number of parameters that all can be non-invasively measured using patient data. The model was validated using in vivo data from the literature. The model was used to determine the effect of different AVB and AS configurations on flow proportion and pressure of the aortic valve and the AVB. Results showed that the AVB leads to a significant reduction in transvalvular pressure gradient. The percentage of flow through the AVB can range from 55.47% to 69.43% following AVB with a severe AS. LV stroke work was also significantly reduced following the AVB surgery and reached a value of around 1.2 J for several AS severities. Findings of this study suggest: 1) the AVB leads to a significant reduction in transvalvular pressure gradients; 2) flow distribution between the AS and the AVB is significantly affected by the conduit valve size; 3) the AVB leads to a significant reduction in LV stroke work; and 4) hemodynamic performance variations can be estimated using the model.     

Molecular Mechanical Differences between Isoforms of Contractile Actin in the Presence of Isoforms of Smooth Muscle Tropomyosin

The proteins involved in smooth muscle''s molecular contractile mechanism – the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin – are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for -actin (A), -actin-tropomyosin- (A-Tm), -actin-tropomyosin- (A-Tm), -actin (A), -actin-tropomyosin- (A-Tm), and -actin-tropomoysin- (A-Tm). Actin sliding analysis with our specifically developed video analysis software followed by statistical assessment (Bootstrapped Principal Component Analysis) indicated that the in vitro motility of A, A, and A-Tm is not distinguishable. Compared to these three ‘baseline conditions’, statistically significant differences () were: A-Tm – actin sliding velocity increased 1.12-fold, A-Tm – motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, A-Tm – run time elevated 1.7-fold. We constructed a mathematical model, simulated actin sliding data, and adjusted the kinetic parameters so as to mimic the experimentally observed differences: A-Tm – myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, A-Tm – mechanical coupling between myosins is stronger, A-Tm – the secondary power stroke is decelerated and mechanical coupling between myosins is weaker. In summary, our results explain the different regulatory effects that specific combinations of actin and smooth muscle tropomyosin have on smooth muscle actin-myosin interaction kinetics.     

Actinopolyspora righensis sp. nov., a novel halophilic actinomycete isolated from Saharan soil in Algeria

A novel halophilic actinomycete strain, H23^T, was isolated from a Saharan soil sample collected in Djamâa (Oued Righ region), El-Oued province, South Algeria. Strain H23^T was identified as a member of the genus Actinopolyspora by a polyphasic approach. Phylogenetic analysis showed that strain H23^T had 16S rRNA gene sequence similarities ranging from 97.8 % (Actinopolyspora xinjiangensis TRM 40136^T) to 94.8 % (Actinopolyspora mortivallis DSM 44261^T). The strain grew optimally at pH 6.0–7.0, 28–32 °C and in the presence of 15–25 % (w/v) NaCl. The substrate mycelium was well developed and fragmented with age. The aerial mycelium produced long, straight or flexuous spore chains with non-motile, smooth-surfaced and rod-shaped spores. Strain H23^T had MK-10 (H₄) and MK-9 (H₄) as the predominant menaquinones. The whole micro-organism hydrolysates mainly consisted of meso-diaminopimelic acid, galactose and arabinose. The diagnostic phospholipid detected was phosphatidylcholine. The major cellular fatty acids were anteiso-C_17:0 (37.4 %), iso-C_17:0 (14.8 %), iso-C_15:0 (14.2 %), and iso-C_16:0 (13.9 %). The genotypic and phenotypic data show that the strain represents a novel species of the genus Actinopolyspora, for which the name Actinopolyspora righensis sp. nov. is proposed, with the type strain H23^T (=DSM 45501^T = CCUG 63368^T = MTCC 11562^T).