The Rhizobium etli FixL protein differs in structure from other known FixL proteins.

The central heme-binding domain in the FixL proteins of Sinorhizobium meliloti, Bradyrhizobium japonicum, Rhizobium leguminosarum biovar viciae and Azorhizobium caulinodans, is highly conserved. The similarity with the corresponding domain in the Rhizobium etli FixL protein is considerably less. This observation prompted us to analyze the heme-binding capacities of the R. etli FixL protein. The R. etli fixL gene was overexpressed in Escherichia coli. In the presence of S. meliloti FixJ, the overexpressed R. etli FixL protein was able to enhance FixJ-mediated activation of an S. meliloti pnifA-lacZ fusion, indicating that the R. etli FixL protein possesses an active conformation in E. coli. Subsequently, using a non-denaturing gel assay for heme, we analyzed the heme-binding capacity of the R. etli FixL protein expressed in E. coli, taking the S. meliloti FixL protein as a positive control. The R. etli FixL protein expressed in E. coli does not contain a heme group, in contrast to the S. meliloti FixL protein. Therefore we conclude that the R. etli FixL is a non-heme protein in the nif regulatory cascade.