Date of Award

Winter 2021

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Louis S Tisa

Second Advisor

Cheryl Andam

Third Advisor

Feixia Chu

Abstract

Introduction: Frankia is a genus of symbiotic, nitrogen fixing actinobacteria found in the soil and root tissue of actinorhizal plants throughout the world. Both Frankia and actinorhizal plants are resistant to a wide range of environmental and chemical stresses including heavy metals, high salinity, and drought. This makes Frankia and the actinorhizal symbiosis an interesting candidate for bioremediation applications. However, the microbial community found in the actinorhizal rhizosphere and surrounding soil has been implicated in some of the symbiotic and resistance traits attributed to Frankia. This necessitates thorough investigation of the genomic and genetic components of any bioremediation traits possessed by Frankia.

Aims: This work pursued three primary aims: 1). To expand the range of available whole genome sequences available for the genus Frankia and use them to assess the pan-genome characteristics of Frankia, 2). To determine the composition of the microbial communities associated with Frankia and actinorhizal plants, and 3). To assess the potential of Frankia to remediate dioxin-like compounds.

Results: Pan-genome analysis of 45 Frankia genomes revealed that the Frankia genus has small core genome made up of 44 genes with 9013 shell genes and over 62,000 cloud genes. Analysis also showed that Frankia has an open pan-genome structure with a Heap’s alpha value of 0.86. Analysis of the 5 clades that form the Frankia genus indicated that each clade has a closed pan-genome structure with core genome sizes ranging from 383 to 3414. Metagenomic analysis of the microbiomes associated with Coriaria myrtifolia, Casuarina glauca, and New Hampshire lithic environments identified highly diverse communities that included Frankia strains not previously known to be associated with these specific actinorhizal hosts. NMDS analysis showed that each tissue type assessed had a unique microbial community structure. This work also found that microbial diversity was positively correlated with proximity to an actinorhizal host plant. Bioinformatic analysis identified 5 Frankia strains with a putative bph operon. Two strains were assessed further and exhibited resistance to dioxin-like compounds (biphenyl, 4-chlorobiphenyl, and dibenzofuran). The same two strains, EuI1c and EUN1f, were able to grow with dioxin-like compounds as a sole source of carbon in minimal media. RNA sequencing and qPCR showed that the putative bph operon was up-regulated in Frankia in response to dioxin-like compound exposure. Lastly, GC-FID analysis showed that Frankia is able to degrade biphenyl in liquid culture. These results advance the basic understanding of Frankia biology, and show that Frankia has the ability to be used as a bioremediation agent.

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