Title

Genomic and proteomic analyses of extracellular and symbiosis-related proteins in Frankia

Date of Completion

January 2009

Keywords

Biology, Microbiology

Degree

Ph.D.

Abstract

A variety of trees and woody shrubs, called actinorhizal plants, form N2-fixing root nodules when in symbiosis with actinobacteria of the genus Frankia. The availability of genome sequences from three Frankia strains has faciliated bioinformatics and proteomics approaches to investigate the molecular interactions governing this symbiosis. This research focuses first on extracellular proteins, which likely play a role in the intimate interaction between the plant and bacterium, then on proteins detected in symbiotic root nodules. Using signal peptide detection algorithms to predict secreted proteins in each strain, we found that the predicted secretomes of Frankia sp. are relatively small (4-5% of the proteome) compared to other soil bacteria, and contain few hydrolytic enzymes. No plant polysaccharide-degrading enzymes were present in all three genomes, but esterolytic, lipolytic and proteolytic enzymes, as well as solute-binding proteins, were found in all three strains and may be required for symbiosis. The extracellular proteome of strain CcI3 was then investigated by analyzing proteins in the culture supernatant using two-dimensional gel electrophoresis and direct liquid chromatography coupled to tandem mass spectrometry (LC MS/MS). Most of the proteins in the CcI3 culture supernatant were cytoplasmic, which is likely due to lysis of older hyphae in this filamentous organism.^ Direct LC MS/MS proteomic techniques were then applied to CcI3 inside Casuarina root nodules, leading to the identification of over 1000 proteins expressed in symbiosis. Nitrogen fixation proteins were the most abundant, and nodule-specific secreted proteins, transporters, cell surface proteins, and signalling proteins were also identified, providing clues to the lifestyle of the bacterium in symbiosis. The proteomes of symbionts from field-collected actinorhizal root nodules of Alnus incana, Ceanothus americanus, and Elaeagnus angustifolia were also analyzed by LC MS/MS, demonstrating that heterologous genome sequences can be used to study Frankia in the environment. This work has addressed long-standing questions and generated many hypotheses about the Frankia-actinorhizal plant symbiosis.^