Title

Growth Kinetics and Characteristics of Frankia sp. CcI3 and Proteomic Analyses of Nitrogen-fixing Vesicles

Date of Completion

January 2012

Keywords

Biology, Microbiology|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Filamentous actinobacteria from the genus Frankia form N2-fixing root nodules on diverse actinorhizal plants. They grow by hyphal tip extension and branching leading to a complex mycelium formation. To understand hyphal development, especially at an early growth stage, we compared the growth of strain CcI3 in liquid cultures with and without proteose peptone #3 (PP3) using time-lapse photomicrography and image analysis. The growth characteristics of Frankia resemble features in other filamentous actinomycetes, like Streptomyces, where individual hyphae show a pseudolinear increase in length at early stages of development and an exponential growth at the mycelial level. Adding PP3 to Frankia CcI3 minimal cultures promotes more frequent branching and denser mycelium, but does not alter the overall specific growth rate in culture. ^ Taking the advantage of Frankia genome information, we performed comparative proteomic analyses (2D-LC MS/MS) of CcI3 in culture conditions and in Casuarina root nodules. Over 1000 proteins were identified in each sample indicating at least 25% of genome was activated under each growth condition. Proteins involved in house-keeping functions were expressed in all fractions and they were often presented abundantly. The overall observation suggests that symbiotic frankiae do not perceive N-starvation while nitrogen fixation takes place in nodules. Unlike the situation in free-living cultures, where the vesicle protects nitrogenase from the oxygen damage, the infected plant cell creates microaerobic conditions for Frankia in symbiosis. Study in culture also demonstrated that CcI3 possesses the ability to fix nitrogen under low oxygen conditions and vesicle development is repressed in such environments. Moreover, short hyphal branches with swollen tips observed in low oxygen cultures may indicate the incompletely developed vesicles which could represent the specialized intracellular hyphae observed in Casuarina root nodules. Overall, this dissertation provides evidence for Frankia CcI3 growth characteristics in liquid cultures and also their life in symbiosis. ^