The symbiotic relationship between wood-eating termites and hindgut protists is crucial for termite digestion, with protists aiding in lignocellulose degradation. This relationship, dating back to the late Jurassic, resembles the ancestral association between termites and wood roaches, Cryptocercus, established over 150 million years ago. Paraneotermes simplicicornis and Kalotermes flavicollis, members of the Kalotermitidae family, harbor diverse symbiotic communities pivotal for wood digestion and nitrogen fixation. Parabasalians, such as Cristamonadea, exhibit morphological diversity, with some taxa being joeniids, calonymphids, or devescovinids, residing primarily in termite guts. To explore the coevolutionary history and morphological evolution, this study aims to describe devescovinid communities in P. simplicicornis and K. flavicollis using morphological and molecular approaches. Phylogenetic analysis reveals the relationships among Devescovina, Metadevescovina, Macrotrichomonas, and Calonympha. A misidentification of published sequence AB458854 Joenia annectens provides valuable insights into how species are classified, while the discovery of previously unknown symbionts demonstrates the extent of diversity within these ecosystems. Notably, Clade 2 was named Prototermanova, where novel Cristamonadea species were identified, exhibiting genetic and morphological similarities to Devescovina. Similarly, Clade 4 was labelled Trichoterm, where two novel Devescovina species challenged existing taxonomic classifications. DNA sequencing analyses provided additional validation, highlighting the genetic diversity and potential novelty of symbionts within the termite gut. Morphological examination aligns with previously identified genera, and BLAST analysis supports observations of potential novelty in certain symbionts. Protists from P. simplicicornis and K. flavicollis show close relation to Joenia and Devescovina, respectively. This study sheds light on the complexity of termite symbiotic relationships and underscores the need for continued research to fully comprehend protist diversity within termite guts.

The partitioning of photosynthates between their sites of production (source) and their sites of utilization (sink) is a major determinant of crop yield and the potential of regulating this translocation promises substantial opportunities for yield increases. Ubiquitous overexpression of the plant type I proton pyrophosphatase (H+-PPase) in crops improves several valuable traits including salt tolerance and drought resistance, nutrient and water use efficiencies, and increased root biomass and yield. Originally, type I H+-PPases were described as pyrophosphate (PPi)-dependent proton pumps localized exclusively in vacuoles of mesophyll and meristematic tissues. It has been proposed that in the meristematic tissues, the role of this enzyme would be hydrolyzing PPi originated in biosynthetic reactions and favoring sink strength. Interestingly, this enzyme has been also localized at the plasma membrane of companion cells in the phloem which load and transport photosynthates from source leaves to sinks. Of note, the plasma membrane-localized H+-PPase could only function as a PPi-synthase in these cells due to the steep proton gradient between the apoplast and cytosol. The generated PPi would favor active sucrose loading through the sucrose/proton symporter in the phloem by promoting sucrose hydrolysis through the Sucrose Synthase pathway and providing the ATP required to maintain the proton gradient. To better understand these two different roles of type I H+-PPases, a series of Arabidopsis thaliana transgenic plants were generated. By expressing soluble pyrophosphatases in companion cells of Col-0 ecotype and H+-PPase mutants, impaired photosynthates partitioning was observed, suggesting phloem-localized H+-PPase could generate the PPi required for sucrose loading. Col-0 plants expressed with either phloem- or meristem-specific AVP1 overexpression cassette and the cross between the two tissue specific lines (Cross) were generated. The results showed that the phloem-specific AVP1-overexpressing plants had increased root hair elongation under limited nutrient conditions and both phloem- and meristem-overexpression of AVP1 contributed to improved rhizosphere acidification and drought resistance. It was concluded that H+-PPases localized in both sink and source tissues regulate plant growth and performance under stress through its versatile enzymatic functions (PPi hydrolase and synthase).

To efficiently produce biofuels and meet the planet’s rising energy demands, different biofuel production methods need to be developed and improved. One of the ways is to produce fatty acid methyl esters (FAMEs) in Synechocystis sp. PCC 6803, a versatile strain of cyanobacteria. In this thesis, Synechocystis was engineered to produce and excrete methyl laurate. In this pathway, first, lauroyl-ACP from fatty acid biosynthesis is converted to laurate by a thioesterase (TE) from Umbellularia californica. Then, the laurate is methylated to methyl laurate by a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster. The TE/∆slr1609 strain of Synechocystis sp. PCC 6803 contains the TE gene and lacks the slr1609 gene encoding an acyl–acyl carrier protein synthetase, which functions in free fatty acid reuptake. The DmJHAMT gene was introduced into this strain for FAME production.
The DmJHAMT gene was cloned into a vector that contains neutral sites from the Synechocystis genome, making it suitable for homologous recombination, and a kanamycin resistance gene, for selection. The obtained plasmid was verified using restriction digests and Sanger sequencing. The sequence analysis and comparison of the cDNA in the obtained plasmid and the mRNA transcript of the same gene revealed three amino acid differences. Subsequent comparison with homologous genes in other Drosophila species revealed the differences in the cDNA match those of the other species, and thus, the gene most likely is functional.
The plasmid was transformed into Synechocystis, and PCRs were used to confirm proper integration and segregation. The TE/∆slr1609/DmJHAMT strain produced 62 mg/L methyl laurate in 12 days under a light intensity of 150 µmol photons m-2 s-1, bubbled with 0.5% CO2 at a rate of 30 mL/min, and supplemented with 0.5 mM methionine. The laurate levels did not decrease over time, but instead, remained stagnant after day 3. When the strain was grown in the same conditions without methionine, the laurate concentrations continued to increase above 400 µM, suggesting minimal methyl laurate production and thus a strong need for methionine supplementation. This work provides further evidence of the viability and success of the introduced FAME production pathway, and improved efficiency may be gained in the future.

The oxygen sensitivity of hydrogenase is a large barrier in maximizing the efficiency of algal hydrogen production, despite recent efforts aimed at rewiring photosynthesis. This project focuses on the role of photosystem II (PSII) in extended hydrogen production by cells expressing the PSI-HydA1 chimera, with the goal of optimizing continuous production of photobiohydrogen in the green alga, Chlamydomonas reinhardtii. Experiments utilizing an artificial PSII electron
Therefore, it can be concluded that downstream processes are limiting the electron flow to the hydrogenase. It was also shown that the use of a PSII inhibitor, 3-(3,4-dichlorophenyl)-1,1- dimethylurea (DCMU), at sub-saturating concentrations under light exposure during growth temporarily improves the duration of the H2 evolution phase. The maximal hydrogen production rate was found to be approximately 32 nmol h-1 (µg Chl)-1. Although downregulation of PSII activity with DCMU improves the long-term hydrogen production, future experiments must be focused on improving oxygen tolerance of the hydrogenase as a means for higher hydrogen yields.

Traditional methods of genetic engineering are often limited to relatively few rounds of gene additions, deletions, or alterations due to a lack of additional available antibiotic resistance markers. Counter-selection marker methods can be used to remove and reuse marker genes as desired, resulting in markerless engineered strains and allowing for theoretically unlimited rounds of genetic modifications. The development of suitable counter-selection markers is vital for the development of model organisms such as cyanobacteria as biotechnological platforms.
In the hopes of providing other researchers with a new tool for markerless genetic engineering of cyanobacteria, the toxin MazF from E. coli was developed as a counter-selection marker in the most widely used cyanobacterium, Synechocystis sp. PCC 6803. The mazF gene from E. coli was cloned and inserted into a plasmid vector for downstream transformation of Synechocystis. The plasmid construct also contained two homologous flanking regions for integration of the insert into the Synechocystis genome, a nickel-inducible response regulator and promoter to control MazF expression, and a kanamycin resistance gene to serve as the antibiotic marker. In order to ensure the mazF plasmids could be cloned in a MazF-sensitive E. coli host even with slight promoter leakage, MazF expression was toned down by decreasing the efficiency of translation initiation by inserting base pairs between the ribosome binding site and the start codon of the mazF gene. Following successful cloning by E. coli, the mazF plasmids were then used to transform Synechocystis to create mazF mutant strains. Genomic analysis confirmed the successful transformation and segregation of mazF mutant strains containing the desired marker cassette. Phenotypic analysis revealed both growth arrest and production of mazF transcripts in mazF mutant strains following the addition of nickel to the cell cultures, indicating successful nickel-induced MazF expression as desired.

There is an ever-increasing need in the world to develop a source of fuel that is clean, renewable and feasible in terms of production and implementation. Hydrogen gas presents a possible solution to these energy needs, particularly if given a way to produce hydrogen gas efficiently. Biological hydrogen (biohydrogen) production presents a potential way to do just this. It is known that hydrogenases are active in wild-type algal photosynthesis pathways but are only active in anoxic environments, where they serve as electron sinks and compete poorly for electrons from photosystem I. To circumvent these issues, a psaC-hydA1 fusion gene was designed and incorporated into a plasmid that was then used to transform hydrogenase-free Chlamydomonas reinhardtii mutants. Results obtained suggest that the psaC-hydA1 gene completely replaced the wild-type psaC gene in the chloroplast genome and the fusion was expressed in the algal cells. Western blotting verified the presence of the HydA1-PsaC fusion proteins in the transformed cells, P700 photobleaching suggested the normal assembly of FA/FB clusters in PsaC-HydA1, and PSII fluorescence data suggested that HydA1 protein limited photosynthetic electron transport flow in the fusion. Hydrogen production was measured in dark, high light, and under maximal reducing conditions. In all conditions, the wild-type algal strain (with a normal PsaC protein) exhibited higher rates of hydrogen production in the light over 2 hours than the WT strain, though both strains produced similar rates in the dark.

Acyl Carrier Protein (ACP) is a small, acidic protein that plays an essential role in fatty acid synthesis by elongating fatty acid chains. ACP was isolated from an extract of a modified strain of Synechocystis sp. PCC 6803 that contains a thioesterase and from which the acyl-ACP synthetase has been deleted. Using ammonium sulfate precipitation to isolate a crude protein fraction containing ACP, immunoblot analysis was performed to determine relative amounts of free and acylated-ACP in the cell. The nature of fatty acids attached to ACP was determined by creating butylamide derivatives that were analyzed using GC/MS. Immunoblot analysis showed a roughly 1:1 ratio of acylated ACP to free ACP in the cell depending on the nutritional state of the cell. From GC/MS data it was determined that palmitic acid was the predominate component of acyl groups attached to ACP. The results indicate that there is a significant amount of acyl-ACP, a feedback inhibitor of early steps in the fatty acid biosynthesis pathway, in the cell. Moreover, the availability of free ACP may also limit fatty acid biosynthesis. Most likely it is necessary for ACP to be overexpressed or to have the palmitic acid cleaved off in order to synthesize optimal amounts of lauric acid to be used for cyanobacterial biofuel production.

The basic scheme for photosynthesis suggests the two photosystems existing in parity with one another. However, cyanobacteria typically maintain significantly more photosystem I (PSI) than photosystem II (PSII) complexes. I set out to evaluate this disparity through development and analysis of multiple mutants of the genetically tractable cyanobacterium Synechocystis sp. PCC 6803 that exhibit a range of expression levels of the main proteins present in PSI (Chapter 2). One hypothesis was that the higher abundance of PSI in this organism is used to enable more cyclic electron flow (CEF) around PSI to contribute to greater ATP synthesis. Results of this study show that indeed CEF is enhanced by the high amount of PSI present in WT. On the other hand, mutants with less PSI and less cyclic electron flow appeared able to maintain healthy levels of ATP synthesis through other compensatory mechanisms. Reduction in PSI abundance is naturally associated with reduced chlorophyll content, and mutants with less PSI showed greater primary productivity as light intensity increased due to increased light penetration in the cultures. Another question addressed in this research project involved the effect of deletion of flavoprotein 3 (an electron sink for PSI-generated electrons) from mutant strains that produce and secrete a fatty acid (Chapter 3). Removing Flv3 increased fatty acid production, most likely due to increased abundance of reducing equivalents that are key to fatty acid biosynthesis. Additional components of my dissertation research included examination of alkane biosynthesis in Synechocystis (Chapter 4), and effects of attempting to overexpress fibrillin genes for enhancement of stored compounds (Chapter 5). Synechocystis is an excellent platform for metabolic engineering studies with its photosynthetic capability and ease of genetic alteration, and the presented research sheds light on multiple aspects of its fundamental biology.

ABSTRACT

Sustainable global energy production is one of the grand challenges of the 21st century. Next-generation renewable energy sources include using photosynthetic microbes such as cyanobacteria for efficient production of sustainable fuels from sunlight. The cyanobacterium Synechocystis PCC 6803 (Synechocystis) is a genetically tractable model organism for plant-like photosynthesis that is used to develop microbial biofuel technologies. However, outside of photosynthetic processes, relatively little is known about the biology of microbial phototrophs such as Synechocystis, which impairs their development into market-ready technologies. My research objective was to characterize strategic aspects of Synechocystis biology related to its use in biofuel production; specifically, how the cell surface modulates the interactions between Synechocystis cells and the environment. First, I documented extensive biofouling, or unwanted biofilm formation, in a 4,000-liter roof-top photobioreactor (PBR) used to cultivate Synechocystis, and correlated this cell-binding phenotype with changes in nutrient status by developing a bench-scale assay for axenic phototrophic biofilm formation. Second, I created a library of mutants that lack cell surface structures, and used this biofilm assay to show that mutants lacking the structures pili or S-layer have a non-biofouling phenotype. Third, I analyzed the transcriptomes of cultures showing aggregation, another cell-binding phenotype, and demonstrated that the cells were undergoing stringent response, a type of conserved stress response. Finally, I used contaminant Consortia and statistical modeling to test whether Synechocystis mutants lacking cell surface structures could reduce contaminant growth in mixed cultures. In summary, I have identified genetic and environmental means of manipulating Synechocystis strains for customized adhesion phenotypes, for more economical biomass harvesting and non-biofouling methods. Additionally, I developed a modified biofilm assay and demonstrated its utility in closing a key gap in the field of microbiology related to axenic phototrophic biofilm formation assays. Also, I demonstrated that statistical modeling of contaminant Consortia predicts contaminant growth across diverse species. Collectively, these findings serve as the basis for immediately lowering the cost barrier of Synechocystis biofuels via a more economical biomass-dewatering step, and provide new research tools for improving Synechocystis strains and culture ecology management for improved biofuel production.

Marine pico-cyanobacteria of the genera Synechococcus and Prochlorococcus carry out nearly two thirds of the primary production in oligotrophic oceans. These cyanobacteria are also considered an important constituent of the biological carbon pump, the photosynthetic fixation of CO2 to dissolved and particulate organic carbon and subsequent export to the ocean’s interior. But single cells of these cyanobacteria are too small to sink, so their carbon export has to be mediated by aggregate formation and/or consumption by zooplankton that produce sinking fecal pellets. In this dissertation, I investigated for the first time the aggregation of these cyanobacteria by studying the marine Synechococcus sp. strain WH8102 as a model organism. I first found in culture experiments that Synechococcus cells aggregated and that such aggregation of cells was related to the production of transparent exopolymeric particles (TEP), known to provide the main matrix of aggregates of eukaryotic phytoplankton. I also found that despite the lowered growth rates, cells in the nitrogen or phosphorus limited cultures had a higher cell-normalized TEP production and formed a greater total volume of aggregates with higher settling velocities compared to cells in the nutrient replete cultures. I further studied the Synechococcus aggregation in roller tanks that allow the simulation of aggregates settling in the water column, and investigated the effects of the clays kaolinite and bentonite that are commonly found in the ocean. In the roller tanks, Synechococcus cells formed aggregates with diameters of up to 1.4 mm and sinking velocities of up to 440 m/d, comparable to those of larger eukaryotic phytoplankton such as diatoms. In addition, the clay minerals increased the number but reduced the size of aggregates, and their ballasting effects increased the sinking velocity and the carbon export potential of the aggregates. Lastly, I investigated the effects of heterotrophic bacteria on the Synechococcus aggregation, and found that heterotrophic bacteria generally resulted in the formation of fewer, but larger and faster sinking aggregates, and eventually led to an enhanced aggregation of cells and particles. My study contributes to the understanding of the role of marine pico-cyanobacteria in the ecology and biogeochemistry of oligotrophic oceans.