O-027. Shotgun Proteomics Sheds Light on Frankia Metabolism in Culture

Y. Huang, J. E. Mastronunzio, D. R. Benson;
Univ. of Connecticut, Storrs, CT.

Actinobacteria of the genus Frankia fix nitrogen in root nodule symbioses with actinorhizal plants. Shotgun proteomics, when partnered with available genomic data, is a potent tool to identify the proteins expressed by bacteria under different conditions. This study focuses on Frankia vesicles, the specialized structures in which nitrogen fixation occurs, and extracellular proteins, which may include proteins that interact directly with host plants. We compare these two subproteomes to whole cell extracts of Frankia strains CcI3 and CpI1 (isolated from different hosts), grown in liquid culture. Protein extracts were digested with trypsin and peptides were identified by liquid chromatography coupled to tandem mass spectrometry (LC MS/MS). The resulting proteomic profiles highlight metabolic pathways active in cultures grown in minimal media with pyruvate (strain CcI3) or succinate (strain CpI1). Major pathways of glycolysis, citric acid cycle, oxidative phosphorylation, and amino acid metabolism are well-represented, as well as minor pathways such as for hopanoid biosynthesis. A total of 68 proteins in whole-cell extracts overlapped from both strains, while 43 vesicle proteins and 30 extracellular proteins were shared. Sixty-nine vesicle-specific proteins were identified in CcI3 and 100 in CpI1. As expected, enzymes involved in nitrogen fixation (nitrogenases) and ammonia assimilation (glutamine synthetase II) were detected in vesicles in both strains. There were 154 proteins in CcI3 and 61 in CpI1 found only in the extracellular fraction, but less than 10% of all extracellular proteins detected had predicted signal peptides, which suggests that few proteins are constitutively secreted by Frankia cells. This research has revealed unique frankial proteins localized to distinct cellular fractions, and establishes a baseline reference for the Frankia proteome in culture. These proteomic data on free-living cells will serve as a control for comparison to symbiosis-specific proteins expressed in root nodules.