Growth and pectate-lyase activity of the ruminal bacterium Lachnospira multiparus in the presence of short-chain organic acids.

AIMS: Acetic, propionic, butyric and lactic acids are end products of feed fermentation by rumen microbes. The effects of these short chain acids on growth and pectate-lyase (PL) activity of Lachnospira multiparus were studied. METHODS AND RESULTS: The bacterial strain used was L. multiparus D32. Acids were tested between 50 and 300 mmol l(-1). Growth and PL activity were measured by the increase in total protein content and by the increase in absorbance at 235 nm in the reaction medium respectively. With the exception of lactic acid, all acids decreased bacterial growth rates; generally, these effects were more pronounced at higher concentrations and with acids of longer chains. PL activity was inhibited by all the acids except by butyric acid at 50 and 100 mmol l(-1). Enzyme inhibition increased with the concentrations of the acids and lactic acid was the most inhibitory. CONCLUSIONS: High concentrations of short chain acids can differentially inhibit the growth rate and the PL activity of L. multiparus. SIGNIFICANCE AND IMPACT OF THE STUDY: Products of fermentation generated by the ruminal microbiota could modify the degradation of pectic substances by this bacterium.

Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rumen bacterium Lachnospira multiparus.

AIMS: Lachnospira multiparus belongs to the main rumen pectinolytic bacteria. Its carbohydrate metabolism was studied in growth experiments on laboratory fermenters, and using assays of activities of enzymes involved in pectin fermentation. METHODS AND RESULTS: The type strain of this species and two substrates were used. Lachnospira multiparus ATCC 19207 grew on pectin and glucose at a similar rate and had no preference for one or the other substrate. Pectin-grown cultures, however, produced significantly more acetate and less formate, lactate, ethanol, hydrogen, cell dry matter and protein than corresponding cultures grown on glucose. Extracellular exopectate lyase (EC 4.2.2.9) was the principal enzyme degrading the pectin macromolecule. Cell extracts possessed 2-keto-3- deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) and fructosediphosphate aldolase (EC 4.1.2.13) activity. The former enzyme catalyses the final reaction in the Entner-Doudoroff pathway; the latter is the key enzyme of glycolysis and the pentose phosphate pathway. CONCLUSION: These results are consistent with the assumption that acidic products of pectin degradation are catabolized via a modified Entner-Doudoroff pathway. Phosphogluconate was not metabolized by cell extracts of the strain studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This suggests that the conventional Entner-Doudoroff pathway of glucose utilization does not operate in this bacterium, presumably because of the lack of 6-phosphogluconate dehydrase (EC 4.2.1.12) activity.

Structural basis for the extreme thermostability of D-glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: analysis based on homology modelling.

D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a hyperthermophilic eubacterium, Thermotoga maritima, is remarkably heat stable (Tm = 109 degrees C). In this work, we have applied homology modelling to predict the 3-D structure of Th.maritima GAPDH to reveal the structural basis of thermostability. Three known GAPDH structures were used as reference proteins. First, the rough model of one subunit was constructed using the identified structurally conserved and variable regions of the reference proteins. The holoenzyme was assembled from four subunits and the NAD molecules. The structure was refined by energy minimization and molecular dynamics simulated annealing. No errors were detected in the refined model using the 3-D profile method. The model was compared with the structure of Bacillus stearothermophilus GAPDH to identify structural details underlying the increased thermostability. In all, 12 extra ion pairs per subunit were found at the protein surface. This seems to be the most important factor responsible for thermostability. Differences in the non-specific interactions, including hydration effects, were also found. Minor changes were detected in the secondary structure. The model predicts that a slight increase in alpha-helical propensities and helix-dipole interactions also contribute to increased stability, but to a lesser degree.

Fermentative degradation of glutarate via decarboxylation by newly isolated strictly anaerobic bacteria.

Two strains of new strictly anaerobic, gram-negative bacteria were enriched and isolated from a freshwater (strain WoG13) and a saltwater (strain CuG11) anoxic sediment with glutarate as sole energy source. Strain WoG13 formed spores whereas strain CuG11 did not. Both strains were rod-shaped, motile bacteria growing in carbonate-buffered, sulfide-reduced mineral medium supplemented with 2% of rumen fluid. Both strains fermented glutarate to butyrate, isobutyrate, CO2, and small amounts of acetate. With methylsuccinate, the same products were formed, and succinate was fermented to propionate and CO2. No sugars, amino acids or other organic acids were used as substrates. Molar growth yields (Ys) were very small (0.5-0.9 g cell dry mass/mol dicarboxylate). Cells of strain WoG13 contained no cytochromes, and the DNA base ratio was 49.0 +/- 1.4 mol% guanine-plus-cytosine. Enzyme activities involved in glutarate degradation could be demonstrated in cell-free extracts of strain WoG13. A pathway of glutarate fermentation via decarboxylation of glutaconyl-CoA to crotonyl-CoA is suggested which forms butyrate and partly isobutyrate by subsequent isomerization.

The pectinolytic enzyme of Selenomonas ruminantium.

A cell-bound pectinolytic enzyme was isolated from cells of Selenomonas ruminantium and purified about 360-fold. The optimum pH and temperature for enzyme activity was 7.0 and 40 degrees C. The enzyme degraded polymeric substrates by hydrolysis of digalacturonic acid units from the non-reducing end; the best substrate was nonagalacturonic acid. Unsaturated trigalacturonate was also degraded, but 30% slower than the saturated analogue. The enzyme was classified as a poly (1,4-alpha-D-galactosiduronate) digalacturono-hydrolase; EC 3.2.1.82. Another enzyme, hydrolysing digalacturonic acid to monomers, was also produced in a very small amount by this organism.

An exopectate lyase of Butyrivibrio fibrisolvens from the bovine rumen.

An extracellular pectinolytic enzyme produced by Butyrivibrio fibrisolvens isolated from the bovine rumen was studied. The enzyme had a pH optimum of 8.0 to 8.5 and was stimulated by Ca2+ and inhibited by EDTA. The products of pectinolysis had an absorption peak at 235 nm and reacted with thiobarbituric acid, indicating a lyase type of action. The enzyme cleaved the substrates terminally from the reducing end; action on poly- and oligogalacturonates resulted in the formation of an unsaturated trigalacturonate. The enzyme was classified as an exopectate lyase (EC 4.2.2.9). A pectinesterase was also produced by B. fibrisolvens but polygalacturonase was not detected.

A polygalacturonate lyase produced by Lachnospira multiparus isolated from the bovine rumen.

A poly(1,4-alpha-D-galacturonide) lyase (EC 4.2.2.2) from the culture fluid of Lachnospira multiparus was purified about 20-fold. The optimum pH and temperature for enzyme activity were 8.0 and 40 degrees C. The enzyme required Ca2+ and was inhibited by EDTA; it preferred polygalacturonate as substrate, cleaving 1,4-alpha-glycosidic linkages randomly to form unsaturated galacturonates, mainly the unsaturated digalacturonate. Some properties of the crude and purified enzyme preparations are described. An exopolygalacturonase is also produced by this organism.