Identification of Ferredoxin II As a Major Calcium Binding Protein in the Nitrogen-fixing Symbiotic Bacterium Mesorhizobium Loti

Background: Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca(2+) concentration, which is a fundamental prerequisite for any Ca(2+)-based signalling system, is accomplished by complex mechanisms including Ca(2+) binding proteins acting as Ca(2+) buffers. In this work we investigated the occurrence of Ca(2+) binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus.

Results: A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca(2+)-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca(2+)-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca(2+) binding ability of the M. loti protein was demonstrated by (45)Ca(2+)-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca(2+) adducts.

Conclusions: The present data indicate that ferredoxin II is a major Ca(2+) binding protein in M. loti that may participate in Ca(2+) homeostasis and suggest an evolutionarily ancient origin for protein-based Ca(2+) regulatory systems.