Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.)

Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The full-length sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of near full-length cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and N-deglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-β-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-β-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-β-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-β-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-β-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and other members of the GH16 family suggested that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.Barbora Stratilová, Zuzana Firáková, Jaroslav Klaudiny; Sergej Šesták, Stanislav Kozmon, Dana Strouhalová, Soňa Garajová, Fairouz Ait‑Mohand, Ágnes Horváthová, Vladimír Farkaš, Eva Stratilová, Maria Hrmov

Biological activities of the fermentation extract of the endophytic fungus Alternaria alternata isolated from Coffea arabica L.

A total of 22 endophytic fungi isolated from coffee (Coffea arabica L.) were cultivated in vitro and their crude extracts tested. The screening was carried out using the agar diffusion method against Staphylococcus aureus, Escherichia coli and Candida albicans. The most effective isolate was Alternaria alternata, and subsequently, its extract was assayed. The total phenolic content was 3.44 &#956;g GAE/mg of the crude extract. For the antibacterial and antifungal activity assays, minimum inhibitory concentrations (MIC) and minimum bactericidal and fungicidal concentrations (MBC and MFC) were determined. The ranges of MIC values were 50-100 &#956;g/mL for S. aureus and 400-800 &#956;g/mL for E. coli. The extract did not show activity in the tested concentrations for C. albicans. The fungal crude extract was assayed for antioxidant activities. Its ability to scavenge DPPH radicals and antioxidant activity by &#946;-carotene/linoleic acid system oxidation was not significant. In addition, antitumor activity was studied using the MTT assay. At a dilution of 400 &#956;g/mL, the extract displayed a cytotoxic activity of approximately 50% towards HeLa cells in vitro. The results indicate that endophytic fungi could be a promising source of bioactive compounds and warrant further study.<br>Total de 22 fungos endofíticos isolados de café (Coffea arabica L.) foi cultivado in vitro e seus extratos testados. A triagem foi conduzida pelo método de difusão em agar contra bactérias Gram-positiva, Gram-negativa e uma levedura. O isolado mais efetivo foi Alternaria alternata e, subsequentemente, seu extrato foi analisado. O conteúdo de fenólicos totais do extrato bruto foi de 3,44 &#956;g EAG/mg de extrato. Para os testes de atividade antimicrobiana, a concentração inibitória mínima (CIM) e concentração bactericida e fungicida mínima (CBM e CFM) contra Staphylococcus aureus, Escherichia coli e Candida albicans foram determinadas. Resultados da CIM variaram entre 50-100 &#956;g/mL para S. aureus e 400-800 &#956;g/mL para E. coli. O extrato bruto não apresentou atividade nas concentrações testadas para C. albicans. Foram analisadas as atividades antioxidantes do extrato bruto. Sua habilidade para seqüestrar radicais DPPH e a atividade antioxidante pela oxidação do sistema &#946;-caroteno/ácido linoléico não foram significativas. Além disso, a atividade antitumoral foi estudada pelo teste do MTT. À diluição de 400 &#956;g/mL, o extrato apresentou atividade de aproximadamente 50% sobre as células HeLa in vitro. Os resultados indicam que fungos endófitos poderiam ser uma fonte promissora de compostos bioativos necessitando de estudos futuros

Defining the core Arabidopsis thaliana root microbiome

Land plants associate with a root microbiota distinct from the complex microbial community present in surrounding soil. The microbiota colonizing the rhizosphere (immediately surroundingthe root) and the endophytic compartment (within the root) contribute to plant growth, productivity, carbon sequestration and phytoremediation(1-3). Colonization of the root occurs despite a sophisticated plant immune system(4,5), suggesting finely tuned discrimination of mutualists and commensals from pathogens. Genetic principles governing the derivation of host-specific endophyte communities from soil communities are poorly understood. Here we report the pyrosequencing of the bacterial 16S ribosomal RNA gene of more than 600 Arabidopsis thaliana plants to test the hypotheses that the root rhizosphere and endophytic compartmentmicrobiota of plants grown under controlled conditions in natural soils are sufficiently dependent on the host to remain consistent across different soil types and developmental stages, and sufficiently dependent on host genotype to vary between inbred Arabidopsis accessions. We describe different bacterial communities in two geochemically distinct bulk soils and in rhizosphere and endophytic compartments prepared from roots grown in these soils. The communities in each compartment are strongly influenced by soil type. Endophytic compartments fromboth soils feature overlapping, low-complexity communities that are markedly enriched in Actinobacteria and specific families from other phyla, notably Proteobacteria. Some bacteria vary quantitatively between plants of different developmental stage and genotype. Our rigorous definition of an endophytic compartment microbiome should facilitate controlled dissection of plantmicrobe interactions derived from complex soil communities