OSMOREGULATION IN RHIZOBIUM-MELILOTI - CHARACTERIZATION OF ENZYMES INVOLVED IN GLUTAMATE SYNTHESIS

Rhizobium meliloti, like many bacteria, accumulates elevated levels of glutamate when osmotically stressed. The biochemical basis for this increase in glutamate production was investigated. Enzymes involved in glutamate synthesis, including glutamine synthetase, glutamate synthase, and glutamic dehydrogenase, were characterized in dialyzed crude cell-free extracts. A transaminase activity, which uses branched chain amino acids for the amination of 2-ketoglutaric acid, was also characterized. With the exception of glutamic dehydrogenase, the specific activity of the enzymes did not vary more than 4-fold in response to the available source of nitrogen or supplemental glutamate. Glutamic dehydrogenase activity was 13-fold greater when cells grew with 10 mM NH4+ than when cells grew with 0.5 mM NH4+. Glutamate synthase was repressed 2-fold when cells grew with supplemental glutamate. Conversely, this enzyme was derepressed 2x when cells grew with 0.5 mM NH4+ or nitrate. Growing cells in minimal defined medium with 400 mM NaCl to cause osmotic stress had little effect on the specific activity of any of the enzymes. The addition of K+ to the reactions stimulated heat-stable glutamine synthetase activity, but inhibited the other enzymes. Glutamate synthase was inhibited to a limited extent by several intermediates in the Krebs' cycle and very severely by glyoxylate. The addition of 10 mM glutamate to the reaction inhibited glutamate synthase 20%, but had no effect on the other enzymes.