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- All Subjects: Cyanobacteria--Genetics.
- Creators: Chandler, Douglas
Description
Synechocystis sp PCC 6803 is a photosynthetic cyanobacterium that can be easily transformed to produce molecules of interest; this has increased Synechocystis’ popularity as a clean energy platform. Synechocystis has been shown to produce and excrete molecules such as fatty acids, isoprene, etc. after appropriate genetic modification. Challenges faced for large–scale growth of modified Synechocystis include abiotic stress, microbial contamination and high processing costs of product and cell material. Research reported in this dissertation contributes to solutions to these challenges. First, abiotic stress was addressed by overexpression of the heat shock protein ClpB1. In contrast to the wild type, the ClpB1 overexpression mutant (Slr1641+) tolerated rapid temperature changes, but no difference was found between the strains when temperature shifts were slower. Combination of ClpB1 overexpression with DnaK2 overexpression (Slr1641+/Sll0170+) further increased thermotolerance. Next, we used a Synechocystis strain that carries an introduced isoprene synthase gene (IspS+) and that therefore produces isoprene. We attempted to increase isoprene yields by overexpression of key enzymes in the methyl erythritol phosphate (MEP) pathway that leads to synthesis of the isoprene precursor. Isoprene production was not increased greatly by MEP pathway induction, likely because of limitations in the affinity of the isoprene synthase for the substrate. Finally, two extraction principles, two–phase liquid extraction (e.g., with an organic and aqueous phase) and solid–liquid extraction (e.g., with a resin) were tested. Two–phase liquid extraction is suitable for separating isoprene but not fatty acids from the culture medium. Fatty acid removal required acidification or surfactant addition, which affected biocompatibility. Therefore, improvements of both the organism and product–harvesting methods can contribute to enhancing the potential of cyanobacteria as solar–powered biocatalysts for the production of petroleum substitutes.
ContributorsGonzalez Esquer, Cesar Raul (Author) / Vermaas, Willem (Thesis advisor) / Chandler, Douglas (Committee member) / Bingham, Scott (Committee member) / Nielsen, David (Committee member) / Arizona State University (Publisher)
Created2013
Description
Euendolithic cyanobacteria have the remarkable ability to actively excavate and grow within certain minerals. Their activity leads to increased erosion of marine and terrestrial carbonates, negatively affecting coral reef and bivalve ecology. Despite their environmental relevance, the boring mechanism has remained elusive and paradoxical, in that cyanobacteria alkalinize their surroundings, typically leading to carbonate precipitation, not dissolution. Thus, euendoliths must rely on unique adaptations to bore. Recent work using the filamentous model euendolith Mastigocoleus testarum strain BC008 indicated that excavation relied on transcellular calcium transport mediated by P-type ATPases, but the phenomenon remained unclear. Here I present evidence that excavation in M. testarum involves an unprecedented set of adaptations. Long-range calcium transport is achieved through the coordinated pumping of multiple cells, orchestrated by the localization of calcium ATPases in a repeating annular pattern, positioned at a single cell pole, adjacent to each cell septum along the filament. Additionally, specialized chlorotic cells that I named calcicytes, differentiate and accumulate calcium at concentrations more than 500 fold those of canonical cells, likely allowing for fast calcium flow at non-toxic concentrations through undifferentiated cells. I also show, using 13C stable isotope tracers and NanoSIMS imaging, that endolithic M. testarum derives most of its carbon from the mineral carbonates it dissolves, the first autotroph ever shown to fix mineral carbon, confirming the existence of a direct link between oxidized solid carbon pools and reduced organic pools in the biosphere. Finally, using genomic and transcriptomic approaches, I analyze gene expression searching for additional adaptations related to the endolithic lifestyle. A large and diverse set of genes (24% of 6917 genes) were significantly differentially regulated while boring, including several master regulators and genes expectedly needed under this condition (such as transport, nutrient scavenging, oxidative stress, and calcium-binding protein genes). However, I also discovered the up-regulation of several puzzling gene sets involved in alternative carbon fixation pathways, anaerobic metabolism, and some related to photosynthesis and respiration. This transcriptomic data provides us with several new, readily testable hypotheses regarding adaptations to the endolithic lifestyle. In all, my data clearly show that boring organisms show extraordinarily interesting adaptations.
ContributorsGuida, Brandon Scott (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Chandler, Douglas (Committee member) / Bingham, Scott (Committee member) / Roberson, Robert (Committee member) / Arizona State University (Publisher)
Created2016
Description
Some cyanobacteria, referred to as boring or euendolithic, are capable of excavating tunnels into calcareous substrates, both mineral and biogenic. The erosive activity of these cyanobacteria results in the destruction of coastal limestones and dead corals, the reworking of carbonate sands, and the cementation of microbialites. They thus link the biological and mineral parts of the global carbon cycle directly. They are also relevant for marine aquaculture as pests of mollusk populations. In spite of their importance, the mechanism by which these cyanobacteria bore remains unknown. In fact, boring by phototrophs is geochemically paradoxical, in that they should promote precipitation of carbonates, not dissolution. To approach this paradox experimentally, I developed an empirical model based on a newly isolated euendolith, which I characterized physiologically, ultrastructurally and phylogenetically (Mastigocoleus testarum BC008); it bores on pure calcite in the laboratory under controlled conditions. Mechanistic hypotheses suggesting the aid of accompanying heterotrophic bacteria, or the spatial/temporal separation of photosynthesis and boring could be readily rejected. Real-time Ca2+ mapping by laser scanning confocal microscopy of boring BC008 cells showed that boring resulted in undersaturation at the boring front and supersaturation in and around boreholes. This is consistent with a process of uptake of Ca2+ from the boring front, trans-cellular mobilization, and extrusion at the distal end of the filaments (borehole entrance). Ca2+ disequilibrium could be inhibited by ceasing illumination, preventing ATP generation, and, more specifically, by blocking P-type Ca2+ ATPase transporters. This demonstrates that BC008 bores by promoting calcite dissolution locally at the boring front through Ca2+ uptake, an unprecedented capacity among living organisms. Parallel studies using mixed microbial assemblages of euendoliths boring into Caribbean, Mediterranean, North and South Pacific marine carbonates, demonstrate that the mechanism operating in BC008 is widespread, but perhaps not universal.
ContributorsRamírez-Reinat, Edgardo L (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Chandler, Douglas (Committee member) / Farmer, Jack (Committee member) / Neuer, Susanne (Committee member) / Arizona State University (Publisher)
Created2010