Succinate is one of the primary carbon and energy sources within the nitrogen-fixing nodule. The rhizobia depend on energy from these plant-supplied substrates for nitrogen fixation. Succinate is oxidized by succinate dehydrogenase (Sdh) which participates directly in the electron transport chain. A critical role for Sdh in nitrogen fixation is supported by a R. meliloti strain lacking succinate dehydrogenase activity that forms ineffective nodules. Also, a correlation exists between succinate dehydrogenase activity and increased efficiency of nitrogen fixation (Emerich et al. 1978; Katznelson et al. 1957; Soberon et al. 1989; Tajima 1990). We propose that it would be possible to enhance nitrogen fixation by increasing the level of respiratory enzymes such as succinate dehydrogenase. Therefore, we are investigating regulatory factors influencing B. japonicum sdhCDAB expression. These regulatory factors would serve as targets for increasing sdhCDBA expression in B. japonicum.

Expression of sdhCDBA was measured using an operon fusion linking approximately 500 bases upstream of the sdh promoter and 100 bases downstream (including the entire untranslated leader region) to a promoterless lacZ gene. The sdh::lacZ fusion construct was introduced into B. japonicum strain USDA llOd. A low level of P-galactosidase activity was measured in xylose grown cells. However, the same strain grown in minimal medium containing malate and succinate, had 10-fold higher levels of P-galactosidase activity. Pyruvate and arabinose/gluconate resulted in intermediate levels of P-galactosidase activity.

B. japonicum containing the sdh::lacZ fusion construct was also grown on minimal medium containing each carbon source and incubated with either high aeration (aerobic) or in sealed test tubes (microaerobic) and assayed for sdh. lacZ expression. On all carbon sources, an increased level of p-galactosidase activity is detected under microaerobic conditions compared to aerobic conditions. Higher sdh expression under microaerobic conditions is unique to B. japonicum but would be consistent with the need for higher Sdh activity in the microaerobic nitrogen-fixing nodule.