Complete killing of Caenorhabditis elegans by Burkholderia pseudomallei is dependent on prolonged direct association with the viable pathogen

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. Much remains to be known about the contributions of genotypic variations within the bacteria and the host, and environmental factors that lead to the manifestation of the clinical symptoms of melioidosis.

Methodology/Principal Findings

In this study, we showed that different isolates of B. pseudomallei have divergent ability to kill the soil nematode Caenorhabditis elegans. The rate of nematode killing was also dependent on growth media: B. pseudomallei grown on peptone-glucose media killed C. elegans more rapidly than bacteria grown on the nematode growth media. Filter and bacteria cell-free culture filtrate assays demonstrated that the extent of killing observed is significantly less than that observed in the direct killing assay. Additionally, we showed that B. pseudomallei does not persistently accumulate within the C. elegans gut as brief exposure to B. pseudomallei is not sufficient for C. elegans infection.

Conclusions/Significance

A combination of genetic and environmental factors affects virulence. In addition, we have also demonstrated that a Burkholderia-specific mechanism mediating the pathogenic effect in C. elegans requires proliferating B. pseudomallei to continuously produce toxins to mediate complete killing.     

Assignment of the dipeptidylpeptidase IV (DPP4) gene to pig Chromosome 15q21

A porcine 2-kb partial dipeptidylpeptidase IV (DPP4, EC 3.4.14.5) cDNA clone and a porcine 16-kb genomic fragment containing parts of the DPP4 gene were isolated, characterized, and used as probes to map the DPP4 gene to pig Chr (Chr) 15q21 by fluorescence in situ hybridization. A two-allele RFLP was revealed for the DPP4 gene. This polymorphism was utilized in a linkage test against the erythrocyte antigen G (EAG), previously assigned to Chr 15, and the microsatellite S0088, which is linked to EAG. The linkage analyses revealed significant evidence for linkage confirming the assignment of DPP4 to Chr 15.     

Molecular phylogeny and diversity of penaeid shrimps (Crustacea: Decapoda) from South-East Asian waters

The decapod family Penaeidae comprises most of the economically important marine shrimp species. Its members are widespread throughout the world, with its highest species diversity centred in the Indo-West Pacific region. Despite this importance, their taxonomy, classification and phylogeny are not yet settled due in part to incongruence among hypotheses proposed from molecular versus morphological studies. In this study, using a thorough taxonomic sampling of especially the South-East Asian species, we aim to (a) utilize a reconstructed phylogeny to test the monophyly of the Penaeidae and its currently recognized genera and (b) explore its species diversity in South-East Asian waters. To infer the phylogeny, a combined gene data set (including 109 ingroup and six outgroup taxa) of mitochondrial genes, COI and 16S rRNA, and two nuclear genes, NaK and PEPCK, was utilized. To explore its diversity, another data set that included 371 COI gene sequences (231 newly generated and 140 retrieved from public sources) was compiled and subsequently analysed with two different tools (ABGD and bPTP) for species delimitation. Other than supporting the non-monophyly of the Penaeidae with the Sicyoniidae nested within the penaeid tribe Trachypenaeini, the genera Penaeus, Mierspenaeopsis and Parapenaeopsis were also revealed to be polyphyletic. Our species delimitation analysis inferred that 94 putative species actually existed within the 71 morphospecies reviewed, indicating an underestimated biodiversity in this family and the potential presence of new species within the following morphospecies: Kishinouyepenaeopsis cornuta, K. incisa, Mierspenaeopsis sculptilis, M hardwicki, Parapenaeopsis coromandelica and Penaeus monodon.     

Size and shape of two intestinal dipeptidases

Physicochemical parameters were determined on glycyl-L-leucine hydrolase (glycy-leucine dipeptidase, EC 3.4.13.2) and aminoacyl-L-proline hydrolase (proline dipeptidase, EC 3.4.13.9), purified from pig small intestine. The native molecular weights were found to be 115,000 and 113,000, respectively, as determined by a sedimentation equilibrium technique. Under denaturing conditions the molecular weights were found to be 51,000 and 63,200, respectively, using the same technique. It is concluded that each dipeptidase is composed of two subunits of equal molecular weight. The two dipeptidases have the same Stokes radius, 4.2 nm, analysed by gel chromatography. The sedimentation coefficients were found to be 5.8. S and 6.5 S and the intrinsic viscosities 5.4 ml/g and 5.8 ml/g, respectively. For both dipeptidases the measured physicochemical parameters are in accordance with the model of a prolate ellipsoid of revolution, having an axial ratio of about 5.     

Production and partial characterization of biosurfactant produced by Streptomyces sp. R1

The present study focused on developing a wild-type actinomycete isolate as a model for a non-pathogenic filamentous producer of biosurfactants. A total of 33 actinomycetes isolates were screened and their extracellular biosurfactants production was evaluated using olive oil as the main substrate. Out of 33 isolates, 32 showed positive results in the oil spreading technique (OST). All isolates showed good emulsification activity (E₂₄) ranging from 84.1 to 95.8%. Based on OST and E₂₄ values, isolate R1 was selected for further investigation in biosurfactant production in an agitated submerged fermentation. Phenotypic and genotypic analyses tentatively identified isolate R1 as a member of the Streptomyces genus. A submerged cultivation of Streptomyces sp. R1 was carried out in a 3-L stirred-tank bioreactor. The influence of impeller tip speed on volumetric oxygen transfer coefficient (k _L a), growth, cell morphology and biosurfactant production was observed. It was found that the maximum biosurfactant production, indicated by the lowest surface tension measurement (40.5 ± 0.05 dynes/cm) was obtained at highest k _L a value (50.94 h⁻¹) regardless of agitation speed. The partially purified biosurfactant was obtained at a concentration of 7.19 g L⁻¹, characterized as a lipopeptide biosurfactant and was found to be stable over a wide range of temperature (20–121 °C), pH (2–12) and salinity [5–20% (w/v) of NaCl].

     

Esterification of Levulinic Acid Using ZrO<Subscript>2</Subscript>-Supported Phosphotungstic Acid Catalyst for Ethyl Levulinate Production

Ethyl levulinate (EL) is a versatile bio-based chemical with various applications such as fragrance and flavoring agents and also fuel blending component. EL can be produced through catalytic esterification of levulinic acid (LA) with ethanol. Herein, a series of zirconia (ZrO₂)-supported phosphotungstic acid (HPW) (HPW/Zr) catalyst, 15-HPW/Zr, 20-HPW/Zr, and 25-HPW/Zr, were prepared, characterized, and tested for EL production. The physicochemical properties of catalysts were characterized using Fourier-transformed infrared (FTIR) spectroscopy, x-ray diffraction (XRD), field emission scanning electron microscope (FESEM), N₂ physisorption, and ammonia temperature-programmed desorption (NH₃-TPD). The effect of reaction parameters: reaction time, temperature, catalyst loading, and molar ratio of LA to ethanol was inspected on LA conversion and EL yield. The catalyst with high surface area and high acidity seemed suitable for EL production. Among the catalysts tested, 20-HPW/Zr exhibited the highest EL yield of 97.3% at the following conditions: 150 °C, 3 h, 1.0 g of 20-HPW/Zr and 1:17 M ratio of LA to ethanol. The 20-HPW/Zr could be reused for at least four times with insignificant decrease in the EL yield. This study demonstrates the potential of ZrO₂-supported HPW for bio-based alkyl levulinate production at mild process conditions.     

Optimization of Biomass Conversion to Levulinic Acid in Acidic Ionic Liquid and Upgrading of Levulinic Acid to Ethyl Levulinate

Levulinic acid (LA) is a versatile platform chemical that can be derived from biomass as an alternative to fossil fuel resources. Herein, the optimization of LA production from glucose and oil palm fronds (OPF) catalyzed by an acidic ionic liquid; 1-sulfonic acid-3-methyl imidazolium tetrachloroferrate ([SMIM][FeCl₄]) have been investigated. Response surface methodology based on Box-Behnken design was employed to optimize the LA yield and to examine the effect and interaction of reaction parameters on the LA production. The reaction parameters include reaction temperature, reaction time, feedstock loading, and catalyst loading. From the optimization study, the predicted mathematical models for LA production from glucose and OPF covered more than 90 % of the variability in the experimental data. At optimum conditions, 69.2 % of LA yield was obtained from glucose, while 24.8 % of LA yield was attained from OPF and registered 77.3 % of process efficiency. The recycled [SMIM][FeCl₄] gave sufficient performance for five successive cycles. Furthermore, the optimum LA produced from glucose and OPF can be directly converted to ethyl levulinate through esterification over the [SMIM][FeCl₄] catalyst. This study highlights the potential of [SMIM][FeCl₄] for biorefinery processing of renewable feedstocks at mild process conditions.