Date of Award

Winter 2007

Project Type

Dissertation

Program or Major

Microbiology

Degree Name

Doctor of Philosophy

First Advisor

Louisa S Tisa

Abstract

Frankia, a nitrogen-fixing actinomycete, forms a symbiotic association with a variety of woody dicotyledonous plants. The lack of standard genetic tools for this important bacterium has hindered studies on the molecular biology of this symbiosis. We are interested in developing tools for the genetic analysis of Frankia physiology and its interactions with its host plants. Our approach has focused on the development of a physical and genetic map of the Frankia chromosome by macrorestriction analysis. Agarose-embedded chromosomal DNA plugs were prepared from three Frankia isolates: Eul1c, EAN1pec and Ccl3. The genome sizes of the three strains were determined by pulse-field gel electrophoresis (PFGE) and estimated to be 5.5Mb for Ccl3, 9.1 Mb for EAN1pec and 8.5Mb for Eul1c. Several members of the actinomycetales have linear chromosomes; however, our experiments on topology show that the Frankia chromosome is a single circular chromosome. The physical map was achieved with the enzymes AseI, SspI and SwaI for Ccl3 and EAN1pec and the enzymes AseI and PmeI for Eul1c isolate. Also a partial genetic map was constructed for isolate, Eul1c, by locating the known Frankia genes including: gln A, glnII, a truncated hemoglobin, and the 16srRNA. The development of these physical maps of Frankia has provided a stepping-stone to facilitate the sequencing of the entire genome of isolates, EAN1pec and Ccl3.

Another important step in further understanding the genetics of this important symbiosis is establishing a protocol for genetic transfer and a reliable system for standardize mutagenesis. This was achieved through the use of a conjugative transposon, Tn916, which was introduced into Frankia isolate Eul1c by mating with Enterococcus faecalis GC110. The generation of stable tetracycline and novobiocin resistant transconjugants suggests that the transposon inserted into the genome. Results from PCR and Southern Blot Hybridization experiments confirmed the insertion of the tetM gene. The transposon insertion sites have been mapped to the physical map of the Frankia chromosome that we developed by macrorestriction analysis. The development of the physical map along with the first successful transfer and expression of foreign DNA into Frankia is a major stepping-stone in the advancement of knowledge of this important prokaryote.

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