Submitted to: North American Conference on Symbiotic Nitrogen Fixation
Publication Type: Abstract Only
Publication Acceptance Date: 6/3/2002
Publication Date: N/A
Technical Abstract: Citrate synthase, a key metabolic enzyme that condenses acetyl-CoA and oxaloacetate to citrate, plays an important role in nodulation and nitrogen fixation. We have isolated a citrate synthase gene by screening a Sinorhizobium fredii USDA257 cosmid library with a heterologous probe from S. meliloti. The S. fredii USDA257 citrate synthase gene was located in a 3.5 kb PstI fragment. Sequence analysis revealed an open-reading frame that is highly homologous to other citrate synthase genes from other bacteria. We have overexpressed citrate synthase in Escherichia coli and have raised antibodies against the purified recombinant citrate synthase. To test if overexpression of citrate synthase in S. fredii USDA improves its nodulation and nitrogen fixation ability, we have mobilized additional copies of citrate synthase into S. fredii USDA257. Western blot analysis confirmed that S. fredii USDA257 carrying extra copies of citrate synthase contained about three-fold increase in citrate synthase content. The symbiotic performance of S. fredii USDA257 carrying extra copies of citrate synthase is currently being examined.