Barco (Netherlands)

We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOS(FA,Ac)) from corn relative to a nonesterified fraction of corn oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1, and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and Axe1-6A), were heterologously expressed in Escherichia coli, purified, and biochemically characterized. All four enzymes showed the highest activity at physiologically relevant pH (6 to 7) and temperature (30 to 40°C) ranges. The P. ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme) released ferulic acid from methylferulate, wheat bran, corn fiber, and XOS(FA,Ac), a corn fiber-derived substrate enriched in O-acetyl and ferulic acid esters, but exhibited negligible activity on sugar acetates. As expected, the P. ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase family domains (CE1 and CE6), released ferulic acid from the same substrates as Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that had activity toward a broad range of mostly nonpolymeric acetylated substrates along with AcXOS. All enzymes were inhibited by the proximal location of other side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The unique diversity of carbohydrate esterases in P. ruminicola 23 likely gives it the ability to hydrolyze substituents on the xylan backbone and enhances its capacity to efficiently degrade hemicellulose.

A set of 15 Italian (Lolium multiflorum), three hybrid (L. boucheanum) and 33 perennial ryegrass (L. perenne) cultivars were evaluated for their rust susceptibility in the field. The cultivars were grown in 2001, 2004 and 2007 at 29, 32 and 27 European sites, respectively. Rust incidence was scored during different growth cycles using a scale from one (no rust) to nine (susceptible). Crown rust (Puccinia coronata f. sp. lolii) was the most frequently observed rust species on Italian, hybrid and perennial ryegrass during all three experimental years. There was a highly significant difference in mean crown rust scores among Italian and perennial ryegrass cultivars, respectively, in each of the 3 years. The ranking of the cultivars was very consistent across the different sites within each year (with some exceptions), despite a significant interaction of cultivar response with sites. Moreover, the rank order correlations of mean cultivar rust scores were highly significant (r > 0.9) between the years. Stem rust (P. graminis f. sp. graminicola) occurred almost exclusively on perennial ryegrass. The cultivars showed significant variation in stem rust susceptibility. Rank order correlations of mean cultivar stem rust scores were mostly significant between sites within each year and highly significant between the years. The ranking of perennial ryegrass cultivars was different depending on whether crown or stem rust was scored. However, the rank order correlation between the mean disease scores of the cultivars for the two pathogens was low but still significant. At a particular site the assessment of crown or stem rust was in most cases very consistent over the period of experimentation. Therefore there was no evidence that rust resistance of an individual cultivar was overcome by the rust pathogen at a particular site over the 7 years of experimentation.