Succinate metabolism by azorhizobium caulinodans ORS 571.
thesisposted on 19.11.2015, 09:08 by Michael William. Evans
Azorhizobium caulinodans ORS 571 was grown in continuous culture (30°C pH 6.8) over a range of dilution rates with ammonia as the sole nitrogen source and either succinate, oxygen or sulphate as the growth-limiting nutrient. Molar growth yields of succinate-limited cultures indicated a P/O quotient of 2 (Ymax succ and Ymax02 ; 42.4 and 27.7 g cell dry wt mol-1 respectively). Molar growth yields of oxygen-limited and sulphate-limited cultures were lower and varied inversely with the steady-state concentration of unmetabolised succinate and K+.The deleterious effect of succinic acid probably reflects its ability to diffuse into the cell and act as an uncoupling agent. A. caulinodans synthesised a membrane-bound respiratory chain which contained b- and c-type cytochromes plus cytochrome oxidases aa3 and o-type (probably co). 0-type cytochrome oxidases were partially or completely repressed by excess oxygen, whereas cytochrome oxidase aa3 was induced by excess oxygen. Nitrogen-fixing cultures (DOT ?1%) showed decreased levels of cytochrome c and increased levels of cytochrome b. Molar growth yields were lower than those of cells assimilating ammonia, and decreased with increasing DOT. The calculated ATP/N2 molar ratio was 17 (assuming a P/O quotient of 2). Washed cells from succinate-limited cultures incorporated [2,3- 14C]succinate at a fast initial rate followed by a slower rate of metabolism (57 nmol min-1 [mg cell dry wt]-1) which remained constant for at least 30 min, and reflected succinate metabolism rather than transport. [2,3-14C]maleate was metabolised only very slowly and was therefore a better substrate for measuring dicar-boxylate transport, which occurred at a minimum rate of 144 nmol min-1 [mg cell dry wt]-1). [14C]succinate incorporation was inhibited by unlabelled fuma-rate, malate and maleate, abolished by the uncoupling agent FCCP and exhibited an accumulation ratio of approximately 20. Dicarboxylate transport/ incorporation was accompanied by net alkalisation of the culture medium, and partially repressed by excess succinate or a product of its metabolism and induced by succinate, fumarate and malate. These results indicate that A. caulinodans actively transported dicarboxylates using a common proton symport system.