Pathogenesis of Potato Gangrene Caused by Phoma exigua var. foveata: II. Activities of some Hydrolases and Dehydrogenases

The location of enzyme activity in gangrene‐diseased tubers was determined using the nitrocellulose blotting method. The activity of aminopeptidase and esterase was located in tissues adjacent to dry rot caused by Phoma exigua var. foveata and in other apparently healthy tissues. The activity of glucuronidase, succinic and glucose‐6‐phosphate dehydrogenases (G‐6‐PDH), however, was confined to tissues adjacent to the rotted tissue. The pathogen produces very active α‐ and β‐glycosidases, so their highest activity occurred in rotten tissue that was filled with fungal mycelium. Results suggest that all these enzymes are involved in alteration of cell metabolism and the destruction of diseased tuber tissue.     

Glycosyl hydrolases of cell wall are induced by sugar starvation in Arabidopsis

Three Arabidopsis genes encoding a putative beta-galactosidase (At5g56870), beta-xylosidase (At5g49360) and beta-glucosidase (At3g60140) are induced by sugar starvation. The deduced proteins belong to the glycosyl hydrolase families 35, 3 and 1, respectively. They are predicted to be secretory proteins that play roles in modification of cell wall polysaccharides based on amino acid similarity. The beta-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved conditions with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose, as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These findings suggest that the cell wall may function as a storage reserve of carbon in addition to providing physical support for the plant body.     

Atypical regioselective biohydrolysis on steroidal oxiranes by Aspergillus niger whole cells: some stereochemical features

5,6-Epoxycholestan-3beta-ol derivatives were hydrolyzed in a diastereoconvergent manner by growing and resting cells of several strains of Aspergillus niger, particularly A. niger ATCC 11394. These strains displayed opposite regioselectivity toward each isomer in an alpha and beta epoxide mixture, thus, the nucleophilic attack took place at the less substituted and the most substituted carbon atom on each diasteromer, respectively. These biocatalysts opened trisubstituted oxiranes but were unable to hydrolyze the disubstituted oxiranes in the tested sterol derivatives. These findings suggest that A. niger strains possess another hydrolytic ability different from the commercial A. niger epoxide hydrolase (EH) that did not accept this kind of steroidal oxiranes as substrates.     

Identification of the glycosyl transferase required for synthesis of the principal glycolipid characteristic of heterocysts of Anabaena sp. strain PCC 7120

Nitrogenase is oxygen-labile. Cyanobacterial heterocysts can fix N(2) in an oxic milieu because their interior is micro-oxic, for which the glycolipid layer of the heterocyst envelope is required. ORF all5341 of the Anabaena sp. genome predicts a glycosyl transferase. An insertional mutant of all5341 synthesized only a nonglycosylated form of heterocyst envelope glycolipid, and lacked a glycolipid layer. All5341 appears to be the transferase required to glycosylate the glycolipid aglycone.     

The <Emphasis Type="Italic">pssA</Emphasis> gene encodes UDP-glucose: Polyprenyl phosphate-glucosyl phosphotransferase initiating biosynthesis of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> exopolysaccharide

Symbiotic nitrogen-fixing bacteria Rhizobium leguminosarum bv. viciae VF39 secrete an acidic heteropolysaccharide, the biosynthesis of which involves the stage of polyprenyl diphosphate octasaccharide formation with its carbohydrate fragment corresponding to the repeating polymer unit. The amino acid analysis of the product of the pssA gene, we have earlier identified, showed its homology to bacterial polyisoprenyl phosphate hexose 1-phosphate transferases catalyzing the formation of phosphodiester bonds between polyprenyl phosphates and hexose 1-phosphates, whose donors are nucleotide sugars. The immunoblotting demonstrated that Rhizobium cells synthesize a protein with a molecular mass of 25 kDa, which implies the translation of the open reading frame occurring from the second initiating codon followed by the protein processing. It was shown that PssA is an integral membrane-bound protein involved in glucose 1-phosphate transfer from UDP-glucose to polyprenyl phosphate to form polyprenyl diphosphate glucose. These results suggest that the pssA gene encodes UDP-glucose:polyprenyl phosphate-glucosyl phosphotransferase.     

An efficient method for transient gene expression in monocots applied to modify the Brachypodium distachyon cell wall

Background

Agrobacterium-mediated transformation is widely used to produce insertions into plant genomes. There are a number of well-developed Agrobacterium-mediated transformation methods for dicotyledonous plants, but there are few for monocotyledonous plants.

Methods

Three hydrolase genes were transiently expressed in Brachypodium distachyon plants using specially designed vectors that express the gene product of interest and target it to the plant cell wall. Expression of functional hydrolases in genotyped plants was confirmed using western blotting, activity assays, cell wall compositional analysis and digestibility tests.

Key Results

An efficient, new, Agrobacterium-mediated approach was developed for transient gene expression in the grass B. distachyon, using co-cultivation of mature seeds with bacterial cells. This method allows transformed tissues to be obtained rapidly, within 3–4 weeks after co-cultivation. Also, the plants carried transgenic tissue and maintained transgenic protein expression throughout plant maturation. The efficiency of transformation was estimated at around 5 % of initially co-cultivated seeds. Application of this approach to express three Aspergillus nidulans hydrolases in the Brachypodium cell wall successfully confirmed its utility and resulted in the expected expression of active microbial proteins and alterations of cell wall composition. Cell wall modifications caused by expression of A. nidulans α-arabinofuranosidase and α-galactosidase increased the biodegradability of plant biomass.

Conclusions

This newly developed approach is a quick and efficient technique for expressing genes of interest in Brachypodium plants, which express the gene product throughout development. In the future, this could be used for broad functional genomics studies of monocots and for biotechnological applications, such as plant biomass modification for biofuel production.     

Physiological roles of plant glycoside hydrolases

The functions of plant glycoside hydrolases and transglycosidases have been studied using different biochemical and molecular genetic approaches. These enzymes are involved in the metabolism of various carbohydrates containing compounds present in the plant tissues. The structural and functional diversity of the carbohydrates implies a vast spectrum of enzymes involved in their metabolism. Complete genome sequence of Arabidopsis and rice has allowed the classification of glycoside hydrolases in different families based on amino acid sequence data. The genomes of these plants contain 29 families of glycoside hydrolases. This review summarizes the current research on plant glycoside hydrolases concerning their principal functional roles, which were attributed to different families. The majority of these plant glycoside hydrolases are involved in cell wall polysaccharide metabolism. Other functions include their participation in the biosynthesis and remodulation of glycans, mobilization of energy, defence, symbiosis, signalling, secondary plant metabolism and metabolism of glycolipids.     

Effect of an exponential feeding regime on the production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica

Aims: To evaluate the effect of and exponential feeding regime on the production of epoxide hydrolase (EH) enzyme in recombinant Yarrowia lipolytica in comparison to a constant feed strategy. Methods and Results: An exponential feed model was developed and fermentations were fed at six different exponential rates. A twofold increase in EH productivity and a 15% increase in volumetric EH activity was obtained by applying exponential glucose feed rates in fed‐batch cultivation. These responses were modelled to obtain a theoretical optimum feed rate that was validated in duplicate fermentations. The model optimum of 0·06 h^?1 resulted in a volumetric EH activity of c. 5500 U l^?1 h^?1 and a maximum activity of 206 000 U l^?1. This correlated well with model predictions, with a variance of <10%. Conclusions: The use of an exponential feed strategy at a rate of 0·06 h ^{? 1} yielded best results for all key responses which show a clear improvement over a constant feed strategy. Significance and Impact of the Study: The study was the first evaluation of an exponential feed strategy on recombinant Y. lipolytica for the production of EH enzyme. The results suggest a strategy for the commercial production of a valuable pharmaceutical enzyme.     

Functional Characterization of the slr1944 Gene of Cyanobacterium Synechocystis sp. PCC 6803

The properties of Slr1944 protein encoded by the slr1944 gene and participating in the metabolism of lipophilic compounds in a cyanobacterium Synechocystis were under study. Located in the periplasm, this protein comprises a conserved pentapeptide G-X-S-X-G characteristic of lipases, acetylcholinesterases, and thioesterases. An attempt to delete the gene from the cyanobacterial genome failed; this fact presumes an essential function of Slr1944 protein under the optimum growth conditions. Expression of the slr1944 gene in Escherichia coli cells demonstrated a high affinity of the product for lipophilic compounds. An enhanced slr1944 expression deprived Synechocystis cells of the ability to restore the activity of the photosynthetic electron-transport chain following photoinactivation. The authors believe that Slr1944 participates in the biogenesis of the lipophilic components of photosynthetic complexes.