A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)⁹ addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native β-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF6^8,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced^10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFDα and β, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFDα and β improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.     

Pathogenicity of Fungi to Eggs of Heterodera glycines

Twenty-one isolates of 18 fungal species were tested on water agar for their pathogenicity to eggs of Heterodera glycines. An egg-parasitic index (EPI) for each of these fungi was recorded on a scale from 0 to 10, and hatch of nematode eggs was determined after exposure to the fungi on water agar for 3 weeks at 24 C. The EPI for Verticillium chlamydosporium was 7.6, and the fungus reduced hatch 74%. Pyrenochaeta terrestris and two sterile fungi also showed a high EPI and reduced hatch 42-73%. Arthrobotrys dactyloides, Fusarium oxysporum, Paecilomyces lilacinus, Stagonospora heteroderae, Neocosmospora vasinfecta, Fusarium solani, and Exophiala pisciphila were moderately pathogenic to eggs (EPI was 2.0-4.5, and hatch was reduced 21-56%). Beauveria bassiana, Hirsutella rhossiliensis, Hirsutella thompsonii, Dictyochaeta heteroderae, Dictyochaeta coffeae, Gliocladium catenulatum, and Cladosporium sp. showed little parasitism of nematode eggs but reduced hatch. A negative correlation was observed between hatch and fungal parasitism of eggs. Fusarium oxysporum, H. rhossiliensis, P. lilacinus, S. heteroderae, V. chlamydosporium, and sterile fungus 1 also were tested in soil in a greenhouse test. After 3 months, the nematode densities were lower in soil treated with H. rhossiliensis and V. chlamydosporium than in untreated soil. The nematode population densities were correlated negatively with the EPI, but not with the percentage of cysts colonized by the fungi. Plant weights and heights generally increased in the soil treated with the fungi.     

Review of the Palaearctic Acomopterella Zaitzev (Diptera,Sciaroidea, Mycetophilidae)

The distribution of Acomopterella species in the Palaearctic region has been re-examined in this study, using recently collected material. The European species was found to be distributed in the eastern Palaearctic as well. A second Palaearctic species from Honshu (Japan) is herein described. The morphology of adult specimens was studied by light microscopy and scanning electron microscopy. The shape of functional specialized setae on mid tibiae in Acomopterella and seven further fungus gnat genera is described and the suitability of this character for systematic studies is discussed. Details of a “hind tibial organ” are described.The position of Acomopterella in the tribe Gnoristini is briefly discussed. Acomopterella is found to be more closely related to Speolepta Edwards, 1925, than to any other recent genus.     

Interrelationship of Heterodera schachtii and Meloidogyne hapla on Tomato

Invasion of tomato (Lycopersicon esculentum L.) roots by combined and sequential inoculations of Meloidogyne hapla and a tomato population of Heterodera schachtii was affected more by soil temperature than by nematode competition. Maximum invasion of tomato roots, by M. hapla and H. schachtii occurred at 30 and 26 C, respectively. Female development and nematode reproduction (eggs per plant) of M. hapla was adversely affected by H. schachtii in combined inoculations of the two nematode species. Inhibition of M. hapla development and reproduction on tomato roots from combined nematode inoculations was more pronounced as soil temperature was increased over a range of 18-30 C and with prior inoculation of tomato with H. schachtii. M. hapla minimally affected H. schachtii female development, but there was significant reduction in the buildup of H. schachtii when M. hapla inoculation preceded that of H. schachtii by 20 days.     

Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species

A PCR-based diagnostic assay was developed for early detection and identification of Aphelenchoides fragariae directly in host plant tissues using the species-specific primers AFragFl and AFragRl that amplify a 169-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA. These species-specific primers did not amplify DNA from Aphelenchoides besseyi or Aphelenchoides ritzemabosi. The PCR assay was sensitive, detecting a single nematode in a background of plant tissue extract. The assay accurately detected A. fragariae in more than 100 naturally infected, ornamental plant samples collected in North Carolina nurseries, garden centers and landscapes, including 50 plant species not previously reported as hosts of Aphelenchoides spp. The detection sensitivity of the PCR-based assay was higher for infected yet asymptomatic plants when compared to the traditional, water extraction method for Aphelenchoides spp. detection. The utility of using NaOH extraction for rapid preparation of total DNA from plant samples infected with A. fragariae was demonstrated.     

Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3

Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. GlcNAc, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T, and HepG2 cell lines including WT cells, SLC35A2 knockouts, SLC35A3 knockouts, and double-knockout cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knockout CHO cells, only limited changes were observed; GlcNAc was still incorporated into N-glycans, but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knockout HEK293T cells, UDP-GlcNAc transport was significantly decreased but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells, the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knockout HEK293T and HepG2 cells, GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between WT and SLC35A3 knockout cells nor between SLC35A2 knockout and double-knockout cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.     

Evaluation of Antimicrobial Activity of Bauhinia purpurea Leaves Under In Vitro Conditions

This study was undertaken with an objective of testing the antibacterial and antifungal activities of Bauhinia purpurea leaves and identifying the bioactive compounds. The antimicrobial activity of leaf extract was determined in aqueous and organic extracts and the minimum inhibitory concentration (MIC) against six species of pathogenic and non-pathogenic microorganisms: Bacillus subtilis, Staphylococcus aureus, Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa and Candida albicans using the disk diffusion method. The chemical constituents of organic plant extract were separated by thin layer chromatography and purified by column chromatography and further identified by gas chromatography–mass spectrometry (GC–MS) analysis. Significant inhibitory activity was observed with methanol extracts of plant against the test microorganisms while less antibacterial activity was observed in hexane, acetone and aqueous extracts. MIC of B. purpurea extract was ≤1,500 μg/ml against S. aureus and B. subtilis while this extract showed no inhibition against Gram-negative S. typhi, E. coli and P. aeruginosa or against fungus C. albicans. Eleven compounds were identified in B. purpurea leaf extract by GC–MS analysis. The composition of B. purpurea revealed the presence of lupeol, stigmasterol, lanosterol, ergosterol, beta-tocopherol, phytol, hexadeconic acids, hexadeconic acids methyl esters, octadecadienoic acids and octadecatrienoic acid. Stigmasterol and lupeol were the most abundant (34.48 and 15.63 %). Other phytosterols like lanosterol (4.15 %) and ergosterol (2.82 %) were also found to be present in this extract.     

Changes in Heterodera glycines Egg Population Density in Continuous Glycine max over Four Years

Soybean cyst nematode, Heterodera glycines, is found throughout soybean production areas of the United States, but the nematode''s distribution is not uniform within states, counties, and individual fields. The goal of this research was to determine the spatial pattern of H. glycines population density in a field in southeastern Missouri and whether it changed over time in the absence of management practices. Geostatistical methods were used to describe and map the distribution of H. glycines over 4 years in a soybean (Glycine max) field in southeastern Missouri. Semivariograms and kriging, an interpolation method, were used to prepare isoarithmic contour maps and associated error maps. In the field studied, fall H. glycines population density (Pf) was poorly related to density the following spring (Pi). The distribution of peak H. glycines population density within the field changed from year to year, although high densities were often detected in the same general region of the field. The patchiness of H. glycines distribution within a field was verified. Yield was not related to H. glycines egg population density at planting, indicating that unmeasured variables were also reducing yield.     

Molecular Diagnosis of an Ocular Toxocariasis Patient in Vietnam

An ocular Toxocara canis infection is reported for the first time in Vietnam. A 34-year-old man residing in a village of Son La Province, North Vietnam, visited the National Eye Hospital (NEH) in August 2011. He felt a bulge-sticking pain in his left eye and loss of vision occurred over 3 months before visiting the hospital. The eye examination in the hospital showed damage of the left eye, red eye, retinal fibrosis, retinal detachment, inflammation of the eye tissues, retinal granulomas, and a parasitic cyst inside. A larva of Toxocara was collected with the cyst by a medical doctor by surgery. Comparison of 264 nucleotides of internal transcribed spacer 2 (ITS2) of ribosomal DNA was done between our Vietnamese Toxocara canis and other Toxocara geographical isolates, including Chinese T. canis, Japanese T. canis, Sri Lankan T. canis, and Iranian T. canis. The nucleotide homology was 97-99%, when our T. canis was compared with geographical isolates. Identification of a T. canis infection in the eye by a molecular method was performed for the first time in Vietnam.     

Metabolomic profiling and antioxidant activity of some Acacia species

Metabolomic profiling of different parts (leaves, flowers and pods) of Acacia species (Acacia nilotica, Acacia seyal and Acacia laeta) was evaluated. The multivariate data analyses such as principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) were used to differentiate the distribution of plant metabolites among different species or different organs of the same species. A.nilotica was characterized with a high content of saponins and A.seyal was characterized with high contents of proteins, phenolics, flavonoids and anthocyanins. A.laeta had a higher content of carbohydrates than A. nilotica and A. seyal. On the basis of these results, total antioxidant capacity, DPPH free radical scavenging activity and reducing power of the methanolic extracts of studied parts were evaluated. A.nilotica and A.seyal extracts showed less inhibitory concentration 50 (IC₅₀) compared to A.laeta extracts which means that these two species have the strongest radical scavenging activity whereas A. laeta extracts have the lowest radical scavenging activity. A positive correlation between saponins and flavonoids with total antioxidant capacity and DPPH radical scavenging activity was observed. Based on these results, the potentiality of these plants as antioxidants was discussed.     

Essential Role for Vacuolar Acidification in Candida albicans Virulence

Fungal infections are on the rise, with mortality above 30% in patients with septic Candida infections. Mutants lacking V-ATPase activity are avirulent and fail to acidify endomembrane compartments, exhibiting pleiotropic defects in secretory, endosomal, and vacuolar pathways. However, the individual contribution of organellar acidification to virulence and its associated traits is not known. To dissect their separate roles in Candida albicans pathogenicity we generated knock-out strains for the V₀ subunit a genes VPH1 and STV1, which target the vacuole and secretory pathway, respectively. While the two subunits were redundant in many vma phenotypes, such as alkaline pH sensitivity, calcium homeostasis, respiratory defects, and cell wall integrity, we observed a unique contribution of VPH1. Specifically, vph1Δ was defective in acidification of the vacuole and its dependent functions, such as metal ion sequestration as evidenced by hypersensitivity to Zn²⁺ toxicity, whereas stv1Δ resembled wild type. In growth conditions that elicit morphogenic switching, vph1Δ was defective in forming hyphae whereas stv1Δ was normal or only modestly impaired. Host cell interactions were evaluated in vitro using the Caco-2 model of intestinal epithelial cells, and murine macrophages. Like wild type, stv1Δ was able to inflict cellular damage in Caco-2 and macrophage cells, as assayed by LDH release, and escape by filamentation. In contrast, vph1Δ resembled a vma7Δ mutant, with significant attenuation in host cell damage. Finally, we show that VPH1 is required for fungal virulence in a murine model of systemic infection. Our results suggest that vacuolar acidification has an essential function in the ability of C. albicans to form hyphae and establish infection.     

Mortality of Pratylenchus penetrans by Volatile Fatty Acids from Liquid Hog Manure

As part of our research program assessing the use of liquid hog manure (LHM) to control root-lesion nematodes, Pratylenchus penetrans, a series of acute toxicity tests was conducted to: (i) examine if non-ionized forms of volatile fatty acids (VFA) are responsible for the mortality of P. penetrans exposed to LHM under acidic conditions, (ii) determine if Caenorhabditis elegans can be a surrogate for P. penetrans in screening tests by comparing their sensitivities to VFA, (iii) characterize the nematicidal effect of individual VFA in LHM to P. penetrans, and (iv) determine whether individual VFA in LHM interact in their toxicity to P. penetrans. LHM was significantly (P ≤ 0.05) more toxic to P. penetrans than a mixture of its main VFA components at concentrations of 5% and 10% (vol. VFA or LHM /vol. in buffer). Pratylenchus penetrans was more sensitive to acetic acid than C. elegans, whereas the sensitivity of both nematode species to n-caproic acid was similar. Individual VFA vary in their lethality to P. penetrans. n-valeric acid was the most toxic (LC₉₅= 6.8 mM), while isobutyric acid was the least toxic (LC₉₅ = 45.7 mM). Individual VFA did not interact in their toxicity to P. penetrans, and their effects were considered additive. VFA account for the majority of the lethal effect of LHM to P. penetrans under acidic conditions. Caenorhabditis elegans cannot be used as a surrogate to P. penetrans in toxicity studies using VFA. The efficacy of LHM to control P. penetrans can be evaluated by assessing its VFA content prior to application, and this evaluation is facilitated by the fact that the interaction of individual VFA appears to be simply additive.     

Redescription of arenicolous dipluran Parajapyx pauliani (Diplura,Parajapygidae) and DNA barcoding analyses of Parajapyx from China

Littoral dipluran Parajapyx pauliani Pagés, 1959 was redescribed based on the specimens collected in Hainan Island, South China. The littoral habitat was confirmed for the species, as the first report of arenicolous dipluran in China. DNA barcoding fragment was sequenced for five Parajapyx species (18 individuals) from China, and this is the first report on DNA barcodes used for dipluran identification. The mean intra- and interspecific divergencesare 1.9% and 19.1% respectively. Synonymy of Parajapyx paucidentis and Parajapyx isabellae was confirmed.     

Genetic and palaeo-climatic evidence for widespread persistence of the coastal tree species Eucalyptus gomphocephala (Myrtaceae) during the Last Glacial Maximum

Background and Aims

Few phylogeographic studies have been undertaken of species confined to narrow, linear coastal systems where past sea level and geomorphological changes may have had a profound effect on species population sizes and distributions. In this study, a phylogeographic analysis was conducted of Eucalyptus gomphocephala (tuart), a tree species restricted to a 400 × 10 km band of coastal sand-plain in south west Australia. Here, there is little known about the response of coastal vegetation to glacial/interglacial climate change, and a test was made as to whether this species was likely to have persisted widely through the Last Glacial Maximum (LGM), or conforms to a post-LGM dispersal model of recovery from few refugia.

Methods

The genetic structure over the entire range of tuart was assessed using seven nuclear (21 populations; n = 595) and four chloroplast (24 populations; n = 238) microsatellite markers designed for eucalypt species. Correlative palaeodistribution modelling was also conducted based on five climatic variables, within two LGM models.

Key Results

The chloroplast markers generated six haplotypes, which were strongly geographically structured (G_ST = 0·86 and R_ST = 0·75). Nuclear microsatellite diversity was high (overall mean H_E 0·75) and uniformly distributed (F_ST = 0·05), with a strong pattern of isolation by distance (r² = 0·362, P = 0·001). Distribution models of E. gomphocephala during the LGM showed a wide distribution that extended at least 30 km westward from the current distribution to the palaeo-coastline.

Conclusions

The chloroplast and nuclear data suggest wide persistence of E. gomphocephala during the LGM. Palaeodistribution modelling supports the conclusions drawn from genetic data and indicates a widespread westward shift of E. gomphocephala onto the exposed continental shelf during the LGM. This study highlights the importance of the inclusion of complementary, non-genetic data (information on geomorphology and palaeoclimate) to interpret phylogeographic patterns.     

Effect of Ditylenchus dipsaci and Pratylenchus penetrans on Verticillium Wilt of Alfalfa

Verticillium albo-atrum wilt symptoms appeared faster and were significantly more severe in the presence of Ditylenchus dipsaci in Vernal, a wilt-susceptible cultivar, than in Marls Kabul, a wilt-resistant cultivar. Winter kill in the field was not affected by the nematode during the first winter, but 50% of plants were killed in the second winter. Forage yield from nematode-infected plants was significantly reduced the second year. Interaction with V. albo-atrum did not significantly reduce forage yields below that of D. dipsaci alone. Pratylenchus penetrans did not increase the severity of wilt symptoms in the presence of V. albo-atrum, nor did it affect forage yield in the greenhouse. It did, however, reduce alfalfa yields in presence of V. albo-atrum under field conditions. D. dipsaci and P. penetrans reproduced faster in Vernal than in Maris Kabul when the fungus was present.     

Substrate Specificity and Subcellular Localization of the Aldehyde-Alcohol Redox-coupling Reaction in Carp Cones

Our previous study suggested the presence of a novel cone-specific redox reaction that generates 11-cis-retinal from 11-cis-retinol in the carp retina. This reaction is unique in that 1) both 11-cis-retinol and all-trans-retinal were required to produce 11-cis-retinal; 2) together with 11-cis-retinal, all-trans-retinol was produced at a 1:1 ratio; and 3) the addition of enzyme cofactors such as NADP(H) was not necessary. This reaction is probably part of the reactions in a cone-specific retinoid cycle required for cone visual pigment regeneration with the use of 11-cis-retinol supplied from Müller cells. In this study, using purified carp cone membrane preparations, we first confirmed that the reaction is a redox-coupling reaction between retinals and retinols. We further examined the substrate specificity, reaction mechanism, and subcellular localization of this reaction. Oxidation was specific for 11-cis-retinol and 9-cis-retinol. In contrast, reduction showed low specificity: many aldehydes, including all-trans-, 9-cis-, 11-cis-, and 13-cis-retinals and even benzaldehyde, supported the reaction. On the basis of kinetic studies of this reaction (aldehyde-alcohol redox-coupling reaction), we found that formation of a ternary complex of a retinol, an aldehyde, and a postulated enzyme seemed to be necessary, which suggested the presence of both the retinol- and aldehyde-binding sites in this enzyme. A subcellular fractionation study showed that the activity is present almost exclusively in the cone inner segment. These results suggest the presence of an effective production mechanism of 11-cis-retinal in the cone inner segment to regenerate visual pigment.