Isolation and characterization of a new poly(3-hydroxybutyrate)-degrading, denitrifying bacterium from activated sludge

One hundred and forty isolates of thermophilic bacteria from the genus Thermus were screened for the presence of restriction endonuclease activity. Thermostable isoschizomers of restriction endonucleases, such as AceIII, BbvI, BglI, BsePI, FnuDII, HgiAI, MaeII, MboI, MseI, PvuII, StuI, TaqI, Tsp4CI, TspEI, XhoI and XmaIII, were isolated. Two restriction enzymes, TatI and TauI, recognizing novel degenerate sequences 5'-W (downward arrow)GTACW-3' and 5'-GCSG (downward arrow)C-3' respectively were partially purified and the recognition and cleavage sites were determined.     

A genetic overhaul of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 424A(LNH-ST) to improve xylose fermentation

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD⁺-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.     

Exclusive transmission of the embryonic stem cell‐derived genome through the mouse germline

Gene targeting in embryonic stem (ES) cells remains best practice for introducing complex mutations into the mouse germline. One aspect in this multistep process that has not been streamlined with regard to the logistics and ethics of mouse breeding is the efficiency of germline transmission: the transmission of the ES cell‐derived genome through the germline of chimeras to their offspring. A method whereby male chimeras transmit exclusively the genome of the injected ES cells to their offspring has been developed. The new technology, referred to as goGermline, entails injecting ES cells into blastocysts produced by superovulated homozygous Tsc22d3 floxed females mated with homozygous ROSA26‐Cre males. This cross produces males that are sterile due to a complete cell‐autonomous defect in spermatogenesis. The resulting male chimeras can be sterile but when fertile, they transmit the ES cell‐derived genome to 100% of their offspring. The method was validated extensively and in two laboratories for gene‐targeted ES clones that were derived from the commonly used parental ES cell lines Bruce4, E14, and JM8A3. The complete elimination of the collateral birth of undesired, non‐ES cell‐derived offspring in goGermline technology fulfills the reduction imperative of the 3R principle of humane experimental technique with animals. genesis 54:326–333, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.     

Assessment of the potential of Napier grass (Pennisetum purpureum) varieties as trap plants for management of Chilo partellus

Ten Napier grass [Pennisetum purpureum Schumach (Poaceae)] varieties, used in various parts of Kenya as animal fodder, were tested for their potential role as a trap crop in the management of the gramineous spotted stemborer, Chilo partellus (Swinhoe) (Lepidoptera: Crambidae), through habitat manipulation. Oviposition preference and larval survival and development were determined for each of these varieties under laboratory and screen‐house conditions. Two‐choice tests revealed that seven of the varieties tested were preferentially chosen by gravid female moths for oviposition over a susceptible maize cv. Inbred A. Larval survival was significantly lower on the majority of the Napier grass varieties (about 10%) than on maize cv. Inbred A (about 60%). Similarly, larval development was about 2 weeks longer on the majority of the Napier grass varieties. It was concluded that four of the varieties tested (Bana, Ex‐Machakos, Gold Coast, and Ex‐Nyanza‐2) had potential for use as trap crops in C. partellus management because they were more preferred by the borer moths for oviposition and allowed minimal survival of the larvae. This minimal larval survival is desirable in the conservation of C. partellus natural enemies when the cultivated host plant is out of season.     

QTL mapping of fire blight resistance in apple

Fire blight caused by the bacterium Erwinia amylovora is a severe threat to apple and pear orchards worldwide. Apple varieties exhibit a wide range of relative susceptibility/tolerance to fire blight. Although, no monogenic resistance against fire blight has been identified yet, recent evidence indicates the existence of quantitative resistance. Potential sources of fire blight resistance include several wild Malus species and some apple cultivars. F1 progenies of ‘Fiesta’בDiscovery’ were inoculated with the Swiss strain Ea 610 and studied under controlled conditions to identify quantitative trait loci (QTLs) for fire blight resistance. Disease was evaluated at four time points after inoculation. Shoot lesion length and the area under disease progress curve (AUDPC) values were used for QTL analysis. One significant (LOD score of 7.5–8.1, p<0.001) QTL was identified on the linkage group 7 of ‘Fiesta’ (F7). The F7 QTL explained about 37.5–38.6% of the phenotypic variation.     

Synergistic Interactions of Eugenol-tosylate and Its Congeners with Fluconazole against Candida albicans

We previously reported the antifungal properties of a monoterpene phenol “Eugenol” against different Candida strains and have observed that the addition of methyl group to eugenol drastically increased its antimicrobial potency. Based on the results and the importance of medicinal synthetic chemistry, we synthesized eugenol-tosylate and its congeners (E1-E6) and tested their antifungal activity against different clinical fluconazole (FLC)- susceptible and FLC- resistant C. albicans isolates alone and in combination with FLC by determining fractional inhibitory concentration indices (FICIs) and isobolograms calculated from microdilution assays. Minimum inhibitory concentration (MIC) results confirmed that all the tested C. albicans strains were variably susceptible to the semi-synthetic derivatives E1-E6, with MIC values ranging from 1–62 μg/ml. The test compounds in combination with FLC exhibited either synergy (36%), additive (41%) or indifferent (23%) interactions, however, no antagonistic interactions were observed. The MICs of FLC decreased 2–9 fold when used in combination with the test compounds. Like their precursor eugenol, all the derivatives showed significant impairment of ergosterol biosynthesis in all C. albicans strains coupled with down regulation of the important ergosterol biosynthesis pathway gene-ERG11. The results were further validated by docking studies, which revealed that the inhibitors snugly fitting the active site of the target enzyme, mimicking fluconazole, may well explain their excellent inhibitory activity. Our results suggest that these compounds have a great potential as antifungals, which can be used as chemosensitizing agents with the known antifungal drugs.     

Detection and identification of phytoplasma associated with witches’ broom and little leaf disease in Arundo donax: first report from India

During the survey of two successive years 2012–2013, in nearby places of Gorakhpur districts, Uttar Pradesh, India, Arundo donax plants were found to be exhibiting witches’ broom, excessive branching accompanied with little leaf symptoms with considerable disease incidence. Nested PCR carried out with universal primers pair R16F2n/R16R2 employing the PCR (P1/P7) product as a template DNA (1:20) resulted in expected size positive amplification ~1.2 kb in all symptom-bearing plants suggested the association of phytoplasma with witches’ broom disease of Narkat plants. BLASTn analysis of the 16S rRNA gene sequence showed the highest (99%) sequence identity with Candidatus phytoplasma asteris (16SrI group). In phylogenetic analysis, the sequence data showed close relationships with the members of 16SrI phytoplasma and clustered within a single clade of 16SrI group and closed to B subgroup representatives. This is a first report of 16Sr I-B group phytoplasma associated with witches’ broom accompanied with little leaf disease of Narkat in India.