Document Type

Dissertation - Open Access

Award Date

2020

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

First Advisor

Ruanbao Zhou

Keywords

biofuels, cyanobacteria, genetic engineering, heterocysts, hydrocarbon production, nitrogen fixation

Abstract

Anabaena sp. PCC 7120 is a filamentous, nitrogen-fixing cyanobacterium that uses spatial separation to perform photosynthesis and nitrogen fixation simultaneously. Under fixed nitrogen limiting conditions, Anabaena 7120 forms specialized cells, called heterocysts, to fix nitrogen. In this research, I sought to add to the knowledge surrounding the process of heterocystous nitrogen fixation in Anabaena 7120. Cyanobacteria universally produce alkanes, which have been suggested to play a role in helping the organism adapt to abiotic stress, such as diazotrophic conditions. In my first study, I sought to identify the genes required for production of the hydrocarbon heptadecane (C17H36). Through using a double crossover approach and determining loss of function, I identified two genes (alr5283 and alr5284) responsible for heptadecane production in Anabaena 7120. In my second study, I employed the alkane knockout mutant to study the impact of alkanes in nitrogen-fixing conditions. My results showed that heptadecane production spikes immediately following nitrogen starvation. Correspondingly, the mutant, which is unable to produce heptadecane, exhibited a fragmented phenotype with a lower nitrogenase activity in the days immediately following nitrogen starvation. These results suggested that heptadecane may play an important role in maintaining membrane integrity for cell-to-cell connections, especially during the initial response to nitrogen starvation. In my last study, I knocked out 16 genes whose encoded proteins had been found only in heterocysts (Qiu 2018). Two of these gene mutants (for all3132 and alr0731) were unable to grow under aerobic nitrogen-fixing conditions. I was able to complement the alr0731 mutant, and it regained its ability to grow in diazotrophic conditions. Therefore, I categorized alr0731 as a Fox gene (unable to fix nitrogen in the presence of oxygen). This gene encodes an anaerobic ribocucleoside triphosphate reductase activating protein that is needed for conversion of ribonucleoside triphosphates (NTPs) to deoxyribonucleoside triphosphates (dNTPs). Because cell division (requiring DNA replication) and gene rearrangements have been linked to heterocyst differentiation, Alr0731 may be primarily important in the process of heterocyst differentiation. Future work will continue to add to the understanding of key regulators and contributors to the process of heterocyst differentiation and nitrogen fixation in cyanobacteria.

Library of Congress Subject Headings

Anabaena.
Cyanobacteria.
Nitrogen -- Fixation.
Alkanes.
Hydrocarbons.
Genetic engineering.

Format

application/pdf

Number of Pages

167

Publisher

South Dakota State University

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Rights Statement

In Copyright