Efficient screening of potential cellulases and hemicellulases produced by <Emphasis Type="Italic">Bosea</Emphasis> sp. FBZP-16 using the combination of enzyme assays and genome analysis

Identification of bacteria that produce carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. Twenty lignocellulose-degrading bacterial isolates from Algerian compost and different soils were screened for their potential to produce different enzymes involved in biomass deconstruction. Based on 16S rRNA gene sequencing, the isolates belonged to Proteobacteria and Actinobacteria. Differences among species were reflected both as the presence/absence of enzymes or at the level of enzyme activity. Among the most active species, Bosea sp. FBZP-16 demonstrated cellulolytic activity on both amorphous cellulose (CMC) and complex lignocellulose (wheat straw) and was selected for whole-genomic sequencing. The genome sequencing revealed the presence of a complex enzymatic machinery required for organic matter decomposition. Analysis of the enzyme-encoding genes indicated that multiple genes for endoglucanase, xylanase, β-glucosidase and β-mannosidase are present in the genome with enzyme activities displayed by the bacterium, while other enzymes, such as certain cellobiohydrolases, were not detected at the genomic level. This indicates that a combination of functional screening of bacterial cultures with the use of genome-derived information is important for the prediction of potential enzyme production. These results provide insight into their possible exploitation for the production of fuels and chemicals derived from plant biomass.     

Morphological characterization and biological control of Alitropus typus (Isopoda: Aegidae)

The Alitropus typus infestation has a serious influence on fish farming in Pakistan. The present study focuses on the external morphology and control of highly infested ectoparasitic isopod Alitropus typus. Sixteen morphological parameters of the whole body were measured and statistically analyzed for mean, standard error and coefficient of variation by using the student t test. Non‐significant variations were observed in the size of pereopods which depict the significance of swimming and attached to its host. For biological control, different concentrations (1000, 5000 and 10 000 ppm) of a leaf extract of four plants (i.e. Euphorbia helioscopia, Ajuga bracteosa, Cannabis sativa and Callistemon citrinus) were tested against A. typus, and the mortality rates were recorded after 12, 24 and 36 h. One way Anova and Tukey tests were used to analyze the results. The recorded percentage mortality was in the order of E. helioscopia > A. bracteosa > C. sativa, whereas C. citrinus did not showed any toxicity. Concentrations of biochemical components like carbohydrates (mg/g), proteins (mg/g), and lipids (mg/dL) of treated isopods were estimated by the phenol‐sulfuric acid process, Lowry's method and biochemistry analyzer, respectively. The protein contents of the insects tested had decreased markedly as compared to control rates, and this might be due to insecticidal stress caused by the extracts. In another biocontrol experiment, crabs were introduced along with isopods into an aquarium. It was then noted that one crab consumes an average of seven isopods/week and can act as a scavenger for dead organisms.     

Chemical Composition and Allelopathic Potential of Essential Oils from Citharexylum spinosum L. Grown in Tunisia

Citharexylum spinosum L. (Verbenaceae) also known as Citharexylum quadrangulare Jacq . or Citharexylum fruticosum L. is an exotic tree introduced many years ago in Tunisia, specially used as a street and park ornamental tree. Essential oils (EOs) were obtained by hydrodistillation of the different parts (roots, stems, leaves, flowers and fruits; drupes) collected from trees grown in the area of Monastir (Tunisia). In total, 84 compounds, representing 90.1 – 98.4% of the whole oil composition, were identified by GC‐FID and GC/MS analyses. The root EO was distinguished by its high content in monoterpene hydrocarbons (α‐phellandrene; 30.8%) whereas that obtained from stems was dominated by sesquiterpene hydrocarbons (cuparene; 16.4%). The leaf oil was rich in an apocarotenoid derivative (hexahydrofarnesylacetone; 26%) and an aliphatic hydrocarbon (nonadecane; 14.5%). Flowers oil was rich in esters (2‐phenylethyl benzoate; 33.5%). Finally, drupes oil was rich in oxygenated sesquiterpenes (β‐eudesmol; 33.1%). Flowers oil showed a significant phytotoxic effect against lettuce seeds germination, it induces a total inhibition when tested at 1 mg/ml. Root and shoot elongation seemed to be more affected than germination. The inhibition of the shoot length varied from 3.6% to 100% and that of the root from 16.1% to 100%. The highest inhibition of 100% was detected for flower oil tested at 1 mg/ml. Our in vitro studies suggest a possible and new alternative use of C. spinosum EOs in herbicidal formulations, further experiments involving field conditions are necessary to confirm its herbicidal potential.     

Nitrite effect on nitrous oxide emission from denitrifying activated sludge

Laboratory-scale experiments were conducted to examine the N₂O emission during the denitrification process. For each of the 6 runs carried out, synthetic effluent was fed in a 10 l batch mixed liquor to investigate the effect of nitrite on N₂O emission and Helium was continuously bubbled through the reactor at constant rate (0.12 l/min) to favour N₂O transfer and detection. An increasing COD/NO₃⁻-N influent ratio from 3 to 7 was firstly applied (runs 1–3). Secondly, NO₂⁻ pulse additions were performed during run 4 and 5 (10 and 20 mg N/l, respectively). Finally, the reactor was fed with influent containing both NO₂⁻ and NO₃⁻. We showed that N₂O emission was detected shortly after NO₂⁻ accumulation, few minutes after the substrate feeding. The highest emission occurred at the lower COD/NO₃⁻-N ratio (=3) and at the higher NO₂⁻ addition (20 mg N/l). In addition, the higher nitrogen conversion to N₂O gas (14.4%) was obtained with an influent containing initially both NO₂⁻ and NO₃⁻. Our results suggest a direct effect of the NO₂⁻ concentration on the N₂O emission. We have also confirmed the inhibitory effect of NO₂⁻ concentration on N₂O reduction.     

Removal and recovery of Cu(II) and Zn(II) using immobilized Mentha arvensis distillation waste biomass

The potential use of the immobilized Mentha arvensis distillation waste (IMADW) biomass for removal and recovery of Cu(II) and Zn(II) from aqueous was evaluated in the present study. Biosorption capacity of Cu(II) and Zn(II) on IMADW increased with increase in pH reaching a maximum at 5 for Cu(II) and 6 for Zn(II). The equilibrium sorption data agreed well with Langmuir isotherm model and pseudo-second-order kinetic model in batch mode. Cu(II) and Zn(II) uptake by IMADW was best described by pseudo-first-order kinetic model in continuous mode. Maximum Cu(II) and Zn(II) uptake by IMADW was 104.48 and 107.75 mg/g, respectively. Fourier Transform Infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) were also carried out to investigate functional groups and surface changes of biomass. The results showed that IMADW biomass is a potential biomaterial to remove Cu(II) and Zn(II) ions with a high biosorption capacity from aqueous solutions.     

Extracellular Secretion of Phytase from Transgenic Wheat Roots Allows Utilization of Phytate for Enhanced Phosphorus Uptake

A significant portion of organic phosphorus comprises of phytates which are not available to wheat for uptake. Hence for enabling wheat to utilize organic phosphorus in form of phytate, transgenic wheat expressing phytase from Aspergillus japonicus under barley root-specific promoter was developed. Transgenic events were initially screened via selection media containing BASTA, followed by PCR and BASTA leaf paint assay after hardening. Out of 138 successfully regenerated T_o events, only 12 had complete constructs and thus further analyzed. Positive T1 transgenic plants, grown in sand, exhibited 0.08–1.77, 0.02–0.67 and 0.44–2.14 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, after 4 weeks of phosphorus stress. Based on these results, T2 generation of four best transgenic events was further analyzed which showed up to 1.32, 56.89, and 15.40 fold increase in phytase activity in root extracts, intact roots and external root solution, respectively, while in case of real-time PCR, maximum fold increase of 19.8 in gene expression was observed. Transgenic lines showed 0.01–1.18 fold increase in phosphorus efficiency along with higher phosphorus content when supplied phytate or inorganic phosphorus than control plants. Thus, this transgenic wheat may aid in reducing fertilizer utilization and enhancing wheat yield.     

Recessive mutations in DOCK6, encoding the guanidine nucleotide exchange factor DOCK6, lead to abnormal actin cytoskeleton organization and Adams-Oliver syndrome

Adams-Oliver syndrome (AOS) is defined by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). It is usually inherited as an autosomal-dominant trait, but autosomal-recessive inheritance has also been documented. In an individual with autosomal-recessive AOS, we combined autozygome analysis with exome sequencing to identify a homozygous truncating mutation in dedicator of cytokinesis 6 gene (DOCK6) which encodes an atypical guanidine exchange factor (GEF) known to activate two members of the Rho GTPase family: Cdc42 and Rac1. Another homozygous truncating mutation was identified upon targeted sequencing of DOCK6 in an unrelated individual with AOS. Consistent with the established role of Cdc42 and Rac1 in the organization of the actin cytoskeleton, we demonstrate a cellular phenotype typical of a defective actin cytoskeleton in patient cells. These findings, combined with a Dock6 expression profile that is consistent with an AOS phenotype as well as the very recent demonstration of dominant mutations of ARHGAP31 in AOS, establish Cdc42 and Rac1 as key molecules in the pathogenesis of AOS and suggest that other regulators of these Rho GTPase proteins might be good candidates in the quest to define the genetic spectrum of this genetically heterogeneous condition.     

Study of rust resistance genes in wheat germplasm with DNA markers

Wheat is a vital dietary component for human health and widely consumed in the world. Wheat rusts are dangerous pathogens and contribute serious threat to its production. In present study, PCR-Based DNA Markers were employed to check the rust resistance genes among 20 wheat genotypes and 22 markers were amplified. NTSYS-pc 2.2 was used to calculate genetic diversity and Nei and Li''s coefficients ranged from 0.55 to 0.95. Cluster analysis was obtained using UPGMA (Unweighted Pair Group Method of Arithmetic Average) algorithm. Maximum no. of genes (23) was amplified from TW-760010 genotype whereas minimum no of genes (14) were amplified from TW-76005 genotype. The data gained from present study open up new ways to produce new varieties by breeding rust resistant germplasm to avoid the economic and food loss and varieties with improved characteristics.     

The ESAT-6 Protein of Mycobacterium tuberculosis Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage

ESAT-6, an abundantly secreted protein of Mycobacterium tuberculosis (M. tuberculosis) is an important virulence factor, inactivation of which leads to reduced virulence of M. tuberculosis. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (β2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90–95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with β2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters β2M to inhibit cell surface expression of MHC-I-β2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:β2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which M. tuberculosis may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis.