Date of Completion

8-6-2013

Embargo Period

8-5-2017

Keywords

Microbiology

Major Advisor

David R. Benson

Associate Advisor

Daniel J. Gage

Associate Advisor

Spencer Nyholm

Field of Study

Microbiology

Degree

Doctor of Philosophy

Open Access

Campus Access

Abstract

Frankia strains are N2-fixing facultative symbionts of actinorhizal plants and potentially produce a large number of natural products. This research used “omic” analyses to examine frankial genomes, the transcriptome and proteome of strain CcI3, and the metabolomes of plant nodules and roots. Using a variety of bioinformatic tools and databases, natural product biosynthesis gene clusters were identified and compared in the genomes of strains CcI3, ACN, and EAN. Next-Gen transcriptome sequencing and liquid chromatography mass spectrometry (LC-MS) were used to determine the extent to which CcI3 synthesizes proteins involved in natural product production. Mass spectrometry was also used to perform metabolomic profiling on Casuarina and Alnus roots and nodules.

My analyses identified 19 gene clusters in the genome of strain CcI3, along with 23 in ACN and 23 in EAN, that are likely involved in natural product biosynthesis. The three strains have ten homologous clusters in common. Some clusters are unique to Frankia, while others are similar to clusters found in other actinobacteria or other bacterial phyla. Phylogenetic analyses suggest that at least 12 gene clusters have been horizontally transferred into the frankial genomes. Transcriptome analysis revealed that several genes clusters have relatively high expression levels in CcI3 cultures with N2-fixing cultures showing significantly higher expression than non-N2-fixing cultures. LC-MS proteomic data identified at least one protein per cluster for each of the 19 metabolites putatively produced by CcI3. Metabolomic profiling resulted in the detection of 331 metabolites in Casuarina and 443 in Alnus roots and nodules.

My data suggests that frankiae have a set of core secondary metabolite biosynthesis gene clusters, and clusters are not as numerous as previously suggested. Illumina transcriptome sequencing and proteomic results indicate that strain CcI3 expresses many genes encoding enzymes involved in natural product biosynthesis, both in culture and symbiosis. Metabolomic analyses indicate that frankial infections dramatically change the metabolic profile of roots. Amino acid concentrations were increased in roots, while sugars, acids, and sugar alcohols were decreased compared to nodules. Flavonoids such as riboflavin, catechin, and epicatechin were differentially expressed in nodules and likely play a role in the frankial-plant symbiosis.

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