Isolation of an endoglucanase gene from Bacteroides ruminicola subsp. brevis.

A gene coding for endo-1, 4-beta-glucanase activity has been isolated from Bacteroides ruminicola subsp. brevis by cloning in Escherichia coli. After restriction mapping of a 6.4 kb insert, a 2.2 kb DNA fragment was sub-cloned in pUC19 to produce the enzymically active clone pJW3. Recloning of the gene fragment in the reverse orientation in pUC18 (clone pJW4) indicated that a gene promoter was present in the cloned fragment and was able to function in E. coli. The clone pJW4 displayed increased activity which was attributed to expression from the lac promoter of pUC18. The enzyme encoded by pJW4 was optimally active at pH 5.5-6.0, and in the temperature range 37-42 degrees C. The preferred substrate was carboxymethylcellulose, but the enzyme displayed 50-60% of maximal activity on both acid-swollen cellulose and soluble xylan. No significant activity was detected on ball-milled filter paper or particulate xylan. Deletion experiments confirmed that both cellulase and xylanase activities were altered to a similar extent by deletion of DNA from the 3' end of the gene, suggesting that both are a function of the same polypeptide product.

Inducible bacteriophages from ruminal bacteria.

The incidence of temperate bacteriophage in a wide range of ruminal bacteria was investigated by means of induction with mitomycin C. Supernatant liquid from treated cultures was examined for phagelike particles by using transmission electron microscopy. Of 38 ruminal bacteria studied, nine organisms (23.7%) representing five genera (Eubacteria, Bacteroides, Butyrivibrio, Ruminococcus, and Streptococcus) produced phagelike particles. Filamentous particles from Butyrivibrio fibrisolvens are the first of this morphological type reported from ruminal bacteria. All of the other particles obtained possessed polyhedral heads and long, noncontractile tails (group B-type phage). The limited range of morphological types produced by mitomycin C induction cannot yet account for the much wider range of types found in ruminal contents by direct examination. The presence of viral genetic material in a significant percentage of the bacteria tested, as well as in a range of different genera, indicates that viral genetic material may be a normal constituent of the genome of appreciable numbers of ruminal bacteria.

An apparent phosphate selenium interaction in weaner sheep.

The severity of selenium dependent nutritional myopathy in weaner sheep was markedly increased by the ruminal infusion of soluble phosphate (6.0 g d-1). Liver selenium concentration was also significantly lower in the sheep infused with sodium phosphate than in sheep fed the same diet and infused with sodium chloride. There were no significant differences in the selenium concentrations of other tissues examined (kidney, heart, skeletal muscle, blood). Blood glutathione peroxidase (GSH.Px) activity in the sheep affected with nutritional myopathy was considerably higher than values generally accepted to indicate a selenium deficiency. The results indicated that there was an interaction in the animal between selenium and phosphate leading to lower liver selenium concentrations. Results obtained from sheep intraruminally dosed with 75[Se]selenate suggested that an interaction between selenium and phosphate could also occur in the gastrointestinal tract.

Selenite uptake and incorporation by Selenomonas ruminantium.

Selenite uptake and incorporation in Selenomonas ruminantium was constitutive with an inducible component. It was distinct from sulphate or selenate transport, since sulphate and selenate did not inhibit uptake, nor could sulphate or selenate uptake be demonstrated. Selenite uptake had an apparent Km of 1.28 mM and a Vmax of 148 ng Se min-1 mg-1 protein. Uptake was sensitive to inhibition by 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), azide, iodoacetic acid (IAA) and N-ethylmaleimide (NEM), but not chlorpromazine (CPZ), N,N'-dicyclohexyl-carbodiimide (DCCD), quinine, arsenate, or fluoride. Treatment of cells accumulating 75[Se]-Selenite with 2,4,DNP inhibited uptake, but did not cause efflux. Transport of selenite was inhibited by sulphite and nitrite, but not by nitrate, phosphate, sulphate of selenate. 75[Se]-Selenite was incorporated into selenocystine, selenoethionine, selenohomocysteine, and selenomethionine and was also reduced to red elemental selenium.