Expression patterns of cell wall-modifying enzymes during grape berry development.

During ripening of grape (Vitis vinifera L.) berries, softening occurs concomitantly with the second growth phase of the fruit and involves significant changes in the properties of cell wall polysaccharides. Here, the activities of enzymes that might participate in cell wall modification have been monitored throughout berry development. Alpha-galactosidase (EC 3.2.1.22), beta-galactosidase (EC 3.2.1.23) and pectin methylesterase (EC 3.1.1.11) activities were present, but no polygalacturonase (EC 3.2.1.15), cellulase (EC 3.2.1.4), xyloglucanase (xyloglucan-specific cellulase EC 3.2.1.4) or galactanase (EC 3.2.1.89) could be detected. The accumulation of mRNAs encoding wall-modifying enzymes was examined by northern hybridization analysis. Transcripts for beta-galactosidase, pectin methylesterase, polygalacturonase, pectate lyase (EC 4.2.2.2) and xyloglucan endotransglycosylase (EC 2.4.1.207) were present during ripening, although polygalacturonase activity had not been detected in berry extracts. Cellulases could not be detected in ripening berries, either at the enzyme or mRNA levels. The increase in beta-galactosidase activity and mRNA is consistent with the observed decrease in type-I arabinogalactan content of the walls during ripening, and the detection of polygalacturonase and pectate lyase mRNAs might explain the increased solubility of galacturonan in walls of ripening grapes. Thus, the modification of cell wall polysaccharides during softening of grape berries is a complex process involving subtle changes to different components of the wall, and in many cases only small amounts of enzyme activity are required to effect these changes.

Definition of a minimal munc18c domain that interacts with syntaxin 4.

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein-protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c(1-592), munc18c(1-139) and munc18c(1-225), but not munc18c(226-592), munc18c(1-100), munc18c(43-139) or munc18c(66-139), interacted with the cytoplasmic portion of syntaxin 4, Stx4(2-273), as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by beta-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx4(29-157), failed to interact with full-length munc18c(1-592), indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein-protein interaction studies between Stx4(2-273) and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c(1-592), munc18c(1-139) and munc18c(1-225) interacted with Stx4(2-273) whereas munc18c(1-100) did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

Enzymatic synthesis and purification of uridine diphospho-beta-l-arabinopyranose, a substrate for the biosynthesis of plant polysaccharides.

Many plant cell wall components such as the polysaccharides xylans and pectins or the glycoproteins arabinogalactan proteins and extensins contain arabinosyl residues. The arabinosyl substituents are thought to be incorporated into these wall polymers by the action of arabinosyltransferases using UDP-l-arabinose as the precursor. UDP-l-arabinose is not commercially available and therefore a procedure for generating UDP-l-arabinose was developed for use in studies on the biosynthesis of the arabinose-containing polymers. In this procedure UDP-d-xylose is incubated with an enzyme preparation from wheat germ and the nucleotide sugars in the reaction mixture are extracted. High-performance anion-exchange chromatography of the extract resolves two major UV-absorbing components: one corresponding to UDP-xylose and a second that elutes earlier. TLC analysis of collected and hydrolyzed fractions demonstrated the presence of l-arabinose in the early eluting fraction. Further analysis by NMR identified the compound as UDP-beta-l-arabinopyranose. The procedure reported here provides an efficient method for preparing either radioactive UDP-l-[(14)C]arabinose or nonradioactive UDP-l-arabinose and can also be used as an assay for UDP-xylose-4-epimerase activity.

Chrysiogenes arsenatis gen. nov., sp. nov., a new arsenate-respiring bacterium isolated from gold mine wastewater.

A new strictly anaerobic bacterium (strain BAL-1T) has been isolated from a reed bed at Ballarat Goldfields in Australia. The organism grew by reducing arsenate [As(V)] to arsenite [As(III)], using acetate as the electron donor and carbon source; acetate alone did not support growth. When BAL-1T was grown with arsenate as the terminal electron acceptor, acetate could be replaced by pyruvate, L- and D-lactate, succinate, malate, and fumarate but not by H2, formate, citrate, glutamate, other amino acids, sugars, or benzoate. When acetate was the electron donor, arsenate could be replaced by nitrate or nitrite but not by sulfate, thiosulfate, or iron oxide. Nitrate was reduced to ammonia via nitrite. The doubling time for growth on acetate (5 mM) plus arsenate (5 mM) or nitrate (5 mM) was 4 h. The G+C content of the DNA is 49 mol%. The 16S rRNA sequence data for the organism support the hypothesis that this organism is phylogenetically unique and at present is the first representative of a new deeply branching lineage of the Bacteria. This organism is described as Chrysiogenes arsenatis gen. nov., sp. nov.

Pilot-Scale Selenium Bioremediation of San Joaquin Drainage Water with Thauera selenatis.

This report describes a simple method for the bioremediation of selenium from agricultural drainage water. A medium-packed pilot-scale biological reactor system, inoculated with the selenate-respiring bacterium Thauera selenatis, was constructed at the Panoche Water District, San Joaquin Valley, Calif. The reactor was used to treat drainage water (7.6 liters/min) containing both selenium and nitrate. Acetate (5 mM) was the carbon source-electron donor reactor feed. Selenium oxyanion concentrations (selenate plus selenite) in the drainage water were reduced by 98%, to an average of 12 (plusmn) 9 (mu)g/liter. Frequently (47% of the sampling days), reactor effluent concentrations of less than 5 (mu)g/liter were achieved. Denitrification was also observed in this system; nitrate and nitrite concentrations in the drainage water were reduced to 0.1 and 0.01 mM, respectively (98% reduction). Analysis of the reactor effluent showed that 91 to 96% of the total selenium recovered was elemental selenium; 97.9% of this elemental selenium could be removed with Nalmet 8072, a new, commercially available precipitant-coagulant. Widespread use of this system (in the Grasslands Water District) could reduce the amount of selenium deposited in the San Joaquin River from 7,000 to 140 lb (ca. 3,000 to 60 kg)/year.