High-level expression and biochemical characterization of a novel cold-active lipase from <Emphasis Type="Italic">Rhizomucor endophyticus</Emphasis>

Objectives

To identify novel cold-active lipases from fungal sources and improve their production by heterologous expression in Pichia pastoris.

Results

A novel cold-active lipase gene (ReLipB) from Rhizomucor endophyticus was cloned. ReLipB was expressed at a high level in Pichia pastoris using high cell-density fermentation in a 5-l fermentor with the highest lipase activity of 1395 U/ml. The recombinant lipase (RelipB) was purified and biochemically characterized. ReLipB was most active at pH 7.5 and 25 °C. It was stable from pH 4.5–9.0. It exhibited broad substrate specificity towards p-nitrophenyl (pNP) esters (C₂–C₁₆) and triacylglycerols (C₂–C₁₂), showing the highest specific activities towards pNP laurate (231 U/mg) and tricaprylin (1840 U/mg), respectively. In addition, the enzyme displayed excellent stability with high concentrations of organic solvents including cyclohexane, n-hexane, n-heptane, isooctane and petroleum ester and surfactants.

Conclusions

A novel cold-active lipase from Rhizomucor endophyticus was identified, expressed at a high level and biochemically characterized. The high yield and unique enzymatic properties make this lipase of some potential for industrial applications.

     

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.Download video file.^{(85M, mov)}     

Probiotics in the intestinal tract of juvenile whiteleg shrimp Litopenaeus vannamei: modulation of the bacterial community

Molecular analysis of the 16S rDNA of the intestinal microbiota of whiteleg shrimp Litopenaeus vannamei was examined to investigate the effect of a Bacillus mix (Bacillus endophyticus YC3-b, Bacillus endophyticus C2-2, Bacillus tequilensisYC5-2) and the commercial probiotic (Alibio^®) on intestinal bacterial communities and resistance to Vibrio infection. PCR and single strain conformation polymorphism (SSCP) analyses were then performed on DNA extracted directly from guts. Injection of shrimp with V. parahaemolyticus at 2.5 × 10⁵ CFU g^?1 per shrimp followed 168 h after inoculation with Bacillus mix or the Alibio probiotic or the positive control. Diversity analyses showed that the bacterial community resulting from the Bacillus mix had the highest diversity and evenness and the bacterial community of the control had the lowest diversity. The bacterial community treated with probiotics mainly consisted of α- and γ-proteobacteria, fusobacteria, sphingobacteria, and flavobacteria, while the control mainly consisted of α-proteobacteria and flavobacteria. Differences were grouped using principal component analyses of PCR-SSCP of the microbiota, according to the time of inoculation. In Vibrio parahaemolyticus-infected shrimp, the Bacillus mix (~33 %) induced a significant increase in survival compared to Alibio (~21 %) and the control (~9 %). We conclude that administration of the Bacillus mix induced modulation of the intestinal microbiota of L. vannamei and increased its resistance to V. parahaemolyticus.     

Structural investigations on the lipopolysaccharide isolated from Vibrio cholera ogawa G-2102

A pure lipopolysaccharide was isolated from Vibrio cholera Ogawa G-2102. After removal of the lipid, the polysaccharide (PS) could be resolved on a column of Sephadex G-75 into antigenic PS and the core PS. Results of the structural investigations on these components are discussed.     

The enzymic synthesis of β-[32P]UDP-N-acetylglucosamine

Cells of Micrococcus sp. 2102 incorporate inorganic [³²P]phosphate from the medium into the sugar-phosphate polymer of the wall. Controlled acid hydrolysis of sodium dodecyl sulphate-extracted cells gives N-acetylglucosamine 6-[³²P]phosphate which can be purified by ion-exchange chromatography and incubated with UTP in the presence of crude preparations of phosphoacetylglucosamine mutase from Neurospora crassa and UTP: N-acetylglucosamine 1-phosphate phosphotransferase from Bacillus licheniformis which act in concert to synthesise β-[³²P]UDP-N-acetylglucosamine.     

Extraction and purification of lipoteichoic acids from gram-positive bacteria

Hot and cold, 80% aqueous phenol extraction procedures together with an aqueous extraction technique have been evaluated for the isolation of lipoteichoic acids from the cytoplasmic membrane of Gram-positive bacteria. Lipoteichoic acids of Staphylococcus aureusH, Micrococcus 2102, Bacillus subtilis 168, and Bacillus subtilis W-23 were examined as each of them emphasises a different problem of contamination. The purity of the lipoteichoic acids with respect to cell-wall material, nucleic acid, and protein is discussed together with the criteria of purity which enables critical structural analysis of lipoteichoic acids to be carried out.     

Construction of an expression system for the secretory production of recombinant α-agarase in yeast

α-Agarase hydrolyzes the α-1,3 linkage of agarose yielding agaro-oligosaccharides. It is less well characterized than β-agarase. AgaA gene (2.3 kb ORF), encoding the α-agarase from Thalassomonas JAMB A33, was subcloned into both a constitutive and an inducible expression vector. Both the constructed plasmids, pVT-AgaA (ADH1 promoter) and pYInu-AgaA (GAL10 promoter), were transformed into Saccharomyces cerevisiae SEY2102 and FY833 and pPIC9-AgaA harboring the AOX1 promoter was transformed into Pichia pastoris GS115. The recombinant α-agarases were over-expressed with activities from 0.3 to 1.6 unit/ml, the highest being in the SEY2102/pYInu-AgaA transformant. Most of the recombinant α-agarase was extracellular because each plasmid possesses a signal sequence for the secretory production of α-agarase. In contrast, the Pichia host-vector expression system was unsuitable for the production of recombinant α-agarase. This is the first report of recombinant production of α-agarase in yeast for industrial use.     

Control of MarRAB Operon in Escherichia coli via Autoactivation and?Autorepression

Choice of network topology for gene regulation has been a question of interest for a long time. How do simple and more complex topologies arise? In this work, we analyze the topology of the marRAB operon in Escherichia coli, which is associated with control of expression of genes associated with conferring resistance to low-level antibiotics to the bacterium. Among the 2102 promoters in E. coli, the marRAB promoter is the only one that encodes for an autoactivator and an autorepressor. What advantages does this topology confer to the bacterium? In this work, we demonstrate that, compared to control by a single regulator, the marRAB regulatory arrangement has the least control cost associated with modulating gene expression in response to environmental stimuli. In addition, the presence of dual regulators allows the regulon to exhibit a diverse range of dynamics, a feature that is not observed in genes controlled by a single regulator.     

Protection of Oilseed Rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) Toward Fungal Pathogens by Strains of Plant-associated <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis>

In this report, four Bacillus strains were tested for effects on plant fitness and disease protection of oilseed rape (Brassica napus). The strains belonged to newly discovered plant-associated Bacillus amyloliquefaciens and a recently proposed species, Bacillus endophyticus. The fungal pathogens tested represented different infection strategies and included Alternaria brassicae, Botrytis cinerea, Leptosphaeria maculans, and Verticillium longisporum. The B. amyloliquefaciens strains showed no or a weak plant growth promoting activity, whereas the B. endophyticus strain had negative effects on the plant as revealed by phenological analysis. On the other hand, two of the B. amyloliquefaciens strains conferred protection of oilseed rape toward all pathogens tested. In vitro experiments studying the effects of Bacillus exudates on fungal growth showed clear growth inhibition in several but not all cases. The protective effects of Bacillus can therefore, at least in part, be explained by production of antibiotic substances, but other mechanisms must also be involved probably as a result of intricate plant–bacteria interaction. The protective effects observed for certain Bacillus strains make them highly interesting for further studies as biocontrol agents in Brassica cultivation.     

Bacillus oceani sp. nov., a new slightly halophilic bacterium, isolated from a deep sea sediment environment

A strictly aerobic, Gram-stain positive, slightly halophilic strain, designated SCSIO 04524^T, was isolated from a deep sea sediment sample collected from the northern South China Sea at a depth of 3415 m. The isolate slightly embedded into the medium after 72 h incubation at 30 °C. Growth was found to occur on media with 0–10 % NaCl but extremely weak growth occurred without supplying NaCl. The predominant menaquinone was determined to be MK-7. The major cellular fatty acid identified was iso-C_15:0. The diagnostic polar lipids were determined to be diphosphatidylglycerol, phosphatidyl methylethanolamine, phosphatidylethanolamine and phosphatidylglycerol. The genomic DNA G+C content was determined to be 38 mol%. 16S rRNA gene sequences analysis showed that this strain had the highest similarities with Bacillus carboniphilus JCM 9731^T (94.7 %) and Bacillus endophyticus 2DT^T (94.3 %). Phylogenetic analysis revealed that strain SCSIO 04524^T formed a distinct lineage with Bacillus chungangensis CAU 348^T and B. carboniphilus JCM 9731^T. Physiological characteristics including utilization of sole nitrogen and carbon sources, and chemotaxonomic properties of cellular fatty acids and polar lipids could readily distinguish strain SCSIO 04524^T from its most closely related species. Based on this polyphasic taxonomic data, a new species, Bacillus oceani sp. nov., is proposed, with the type strain SCSIO 04524^T (=DSM 26213^T = KCTC 33077^T).     

2102Ep embryonal carcinoma cells have compromised respiration and shifted bioenergetic profile distinct from H9 human embryonic stem cells

Recent studies have shown that cellular bioenergetics may be involved in stem cell differentiation. Considering that during cancerogenesis cells acquire numerous properties of stem cells, it is possible to assume that the energy metabolism in tumorigenic cells might be differently regulated. The aim of this study was to compare the mitochondrial bioenergetic profile of normal pluripotent human embryonic stem cells (hESC) and relatively nullipotent embryonal carcinoma cells (2102Ep cell line).We examined three parameters related to cellular bioenergetics: phosphotransfer system, aerobic glycolysis, and oxygen consumption. Activities and expression levels of main enzymes that facilitate energy transfer were measured. The oxygen consumption rate studies were performed to investigate the respiratory capacity of cells.2102Ep cells showed a shift in energy distribution towards adenylate kinase network. The total AK activity was almost 3 times higher in 2102Ep cells compared to hESCs (179.85 ± 5.73 vs 64.39 ± 2.55 mU/mg of protein) and the expression of AK2 was significantly higher in these cells, while CK was downregulated. 2102Ep cells displayed reduced levels of oxygen consumption and increased levels of aerobic glycolysis compared to hESCs. The compromised respiration of 2102Ep cells is not the result of increased mitochondrial mass, increased proton leak, and reduced respiratory reserve capacity of the cells or impairment of respiratory chain complexes. Our data showed that the bioenergetic profile of 2102Ep cells clearly distinguishes them from normal hESCs. This should be considered when this cell line is used as a reference, and highlight the importance of further research concerning energy metabolism of stem cells.     

Production of agarase from a novel <Emphasis Type="Italic">Micrococcus</Emphasis> sp. GNUM-08124 strain isolated from the East Sea of Korea

The agar degrading bacterial strain GNUM-08124 was isolated from Enteromorpha compressa collected in the East Sea of Korea by using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. GNUM-08124 grows to produce a circular, smooth, yellow-colored, and raised colony. Its ability to hydrolyze agar was confirmed by staining the ASW agar plate with Lugol’s solution. In liquid culture, the cell density (A₆₀₀) increased exponentially and reached a maximum level on the third day of cultivation. The specific agarase activity also increased in proportion to the cell density and reached maximum agarolytic activity on the third day. The 16S rRNA sequence of GNUM-08124 showed a close relationship to Micrococcus luteus (99.65%) and Micrococcus endophyticus (99.15%), which led us to assign it to the genus Micrococcus. Physiological studies indicated that optimal growth conditions were between 30 and 40°C, pH 4 and 7, using media containing between 5 and 10% NaCl (w/v), respectively. The GNUM-08124 strain was a grampositive, urease-positive, and catalase-positive bacterium. It could not hydrolyze gelatin, cellulose, xylan, or starch, but fermented a broader range of substrates, including Dglucose, D-galactose, D-fructose, D-lactose, D-trehalose, D-mannitol, D-melibiose, D-raffinose, D-xylose, methyl-α-D-glucopyranoside, N-acetyl-glucosamine, and xylitol, than those fermented by M. luteus or M. endophyticus, suggesting GNUM-08124 is a novel agar hydrolyzing microorganism belonging to Genus Micrococcus. Micrococcus sp. GNUM-08124 showed the highest agarase activity when it was cultured in ASW-YP medium supplemented with 0.4% glucose, but demonstrated lower activity in rich media (LB or TSB), in spite of superior cell growth, implying that agarase production is tightly regulated in an agar-dependent manner and repressed in rich conditions.     

Gene cloning and characterization of novel antinociceptive peptide from the brain of the frog, Odorrana grahami

Amphibian opiate peptides including dermorphins and deltorpins have been recently found only in the skin of South American frogs belonging to the subfamily Phyllomedusinae (Phyllomedusa, Agalychnis and Pachymedusa species). No opiate peptides have ever been identified from other amphibians or organs except skin. Here we report the purification and characterization of a novel antinociceptive peptide named odorranaopin from the homogenates of the frog brains, Odorrana grahami, which is also the first antinociceptive peptide found in Ranidae amphibian. Odorranaopin comprises 17 amino acid residues with the sequence of DYTIRTRLHQESSRKVL (Mr 2102 Da). The cDNA encoding odorranaopin was cloned from the frog brain cDNA library, and it was confirmed to be a specific gene. The odorranaopin precursor deduced is composed of 61 amino acid residues including the predicted signal peptide, acidic spacer peptide and mature odorranaopin positioned at the C-terminus. Odorranaopin could inhibit nociceptive responses induced by formalin and acetic acid. It also inhibited the contractile responses of ileum smooth muscle induced by bradykinin, implying that the antinociceptive activity of odorranaopin possibly results from its blockade on bradykinin or bradykinin receptor functions. Odorranaopin is the first antinociceptive peptide found in Ranidae amphibian.     

Sex control by Zfy siRNA in the mouse

The objective of this work was to detect the influence of Y sperm forming of Mus musculus by silencing the Zfy gene during spermatogenesis. The recombination expression vectors pSilencer5.1/Zfy215 and pSilencer5.1/Zfy2102 were constructed. 64 male KunMing Mus were divided into four groups randomly and averagely. The two recombination expression vectors were injected into two groups, respectively, through testis. The other two groups were injected with the same volume of physiological saline and empty vector pSilencer5.1-H1 Retro, respectively. They were injected every ten days for a total of four injections. Seventeen days after the fourth injection, 8 male Mus of each group mated with 8 female Mus. The testis tissue of the other 8 male Mus of each group was collected, and the expression level of Zfy mRNA was determined by fluorescence quantitation real time PCR (qRT-PCR). The result showed that the expression of Zfy mRNA decreased significantly after injection of pSilencer5.1/Zfy2102 (P < 0.01), and that 72.3% of the offspring were female, a number significantly higher than in the control group (P < 0.01). In the pSilencer5.1/Zfy215 group, the expression of Zfy mRNA was significantly lower than in the control group (P < 0.05), but the female rate of offspring was not. It was concluded that the Zfy gene could play a role in the process of Y sperm formation.     

Recherches sur l'hérédité de la dilution et du blanchiment du pelage dans le genre cavia

Sans résuméTravail exécuté grâce à une subvention de la DonationGeorges etAntoine Claraz, instituta et curataJohannis Schinz professoris auspiciis Série zoologie n^o 66     

Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards industrial wheat straw hydrolysates

The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH.