Effects of nitrogen source and concentration upon glutamine synthetase and protease activity in the rumen bacterium Prevotella ruminicola strain B1 4

Abstract

A series of experiments was conducted to determine the effects of varying nitrogen sources and concentrations upon glutamine synthetase and protease activities in Prevotella ruminicola strain B,4. P. ruminicola was grown on a defined basal medium (mineral salts, essential volatile fatty acids, vitamin cofactors, 12 mM glucose, cysteine-HCL as a reductant and sodium carbonate) with variable amounts of ammonium chloride, pepticase or casein as the nitrogen source. The results indicated that when ammonium chloride or pepticase are used as the sole source of nitrogen P. ruminicola becomes nitrogen limited when nitrogen concentration is at 0.5 mM. However, when casein is the sole source of nitrogen the culture becomes nitrogen limited at 2.5 mM. These three nitrogen concentrations were then used as the nitrogen limited groups for the glutamine synthetase and protease assays. Nitrogen concentrations of 15.0 mM, 15.0 mM, and 12.5 mM respectively, were used for the non-limited group. Glutamine synthetase activity was measured from mid-log phase cells grown in either nitrogenlimited or non-limited conditions. No activity was observed in the non-limited treatments. However, in the limited treatments, pepticase had the highest activity (20.755 units) followed by ammonium chloride (18.715 units) and casei n (1 4.42 u n its), and all treatments were significantly different (p < 0. 05). Experiments with MSX, a potent inhibitor of glutamine synthetase, indicated the relative importance of glutamine synthetase during nitrogen-limited conditions. The results of growth studies showed that when MSX was used in concentrations of 1.0, 1 0.0, and 50.0 m no growth was observed under nitrogen-limiting conditions. However, in the non-limited conditions MSX had no significant effect upon growth. The protease activity assays indicated that nitrogen-limited cultures had higher proteolytic activity than non-limited cultures. Moreover, these activities appeared to be positively correlated with the previously observed glutamine synthetase activities. The results of this study indicate that protease activity in the rumen may be influenced by nitrogen concentration such that activity increases when nitrogen availability decreases. This may provide some insight into the regulation of rumen bacterial proteases that could facilitate the development of practical and effective bacterial protease inhibitors.