Performing Department
(N/A)
Non Technical Summary
Algae, including cyanobacteria, are an emerging crop for generating carbon neutral renewable commodities like biofuels, foods, and pharmaceuticals. But water sustainability is a prominent concern in agricultural algal pond cultivation both ecologically and financially, with water separation alone accounting for 20-30% of current algal production costs. Immobilized biofilm cultures address this challenge by reducing separation costs and water suspension needs. Biofilms are sticky aggregates of bacterial cells that are often studied in the context of bacterial pathogens, as they facilitate bacterial resilience to harsh environments like human and other animal bodies. Cyanobacterial biofilms are not pathogenic but possess dynamic, stress-adaptive compositions - holding constituents that protect both the cyanobacteria and neighboring plants and soils. However, little is known about how these biofilms, or what in them, protects the cyanobacteria from harsh abiotic or environmental climate-relevant stresses, like drought, temperature, salt, and pH. Therefore, I am genetically reprogramming or engineering native biofilm matrix modulators in a model industrial cyanobacterium to determine mechanisms conferring abiotic stress resilience in a strain with inherently poor biofilm formation and stress tolerance. Genetic mutant biofilm formation and viability under abiotic stress will be compared with un-engineered or "wild type" cyanobacterial cultures. Initial mutants exhibit ~10x increased viability under drought stress compared to wild type, presenting compelling preliminary evidence that abiotic stress tolerance can be engineered into a non-tolerant cyanobacterium. Next, mutants will be exposed to different stresses and profiles of their biofilm contents and gene expression will be compared under ideal conditions vs stress to ascertain what biologically contributes to stress adaptation.Overall, this fundamental research aims to generate more stress-robust industrial cyanobacterial strains, reducing economic and ecological burdens in the algal cultivation industry and helping facilitate the expansion and accessibility of algal crops. Uncovering biological mechanisms of stress-tolerance in cyanobacteria is translatable towards improvement of climate resilience within the ag sector across soil and crop health. The project will impact the public community by generating open-access peer-reviewed journal article(s), numerous public research presentations to diverse audiences, training and mentorship for underrepresented and early-career scientists and facilitating engaging public scientific outreach around topics like climate change and adaptation in agriculture. This research is relevant to the AFRI Plant Health and Production and Plant Products priority by targeting tunable stress resilience phenotypes in model cyanobacteria with translational applications towards terrestrial crop and soil health in the face of increasing climate instability. This study also advances AFRI priorities in Bioenergy, Natural Resources, and Environment by enhancing environmental stress tolerance in an emerging biofuel crop and improving water sustainability.
Animal Health Component
0%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
The major goal of this project is to better understand the biological underpinnings of abiotic stresstolerancerelatedtosecreted extracellularpolymeric substances (EPS) in cyanobacteria. EPS are positively implicated in protective biofilm formation and stress response, but their modulation or compositional variance related to stress has not been evaluated. To best assess this, we will modulate EPS production and composition by genetic engineering of a model cyanobacterium, Synechocystis PCC 6803 (S. 6803), that natively produces little EPS or biofilm and exhibits poor baseline abiotic stress tolerance. Mechanisms of abiotic stress tolerance are important to study towards developing adaptive, resilient agricultural solutions to safeguard both crops and soils in the face of global climate change related stresses, such as drought and temperature.The first objective of this study is to generate unique, targeted S. 6803 genetic mutants with altered EPS and improved biofilm tendency. We will pursue five identified genetic targets likely to yield phenotypes of interest. We will then compare mutant viability under abiotic stress, such as drought, temperature, pH, high UV, or high salt with the unaltered S. 6803 wild type (WT).The second objective of this study will elucidate biological mechanisms of stress resilience in EPS and biofilm modulated mutants. To ascertain how stress resilience is conferred to mutants, the top three genetic targets that show enhanced tolerance under a stress condition from the first objective will be combined and reassessed for stress tolerance both separately and in combination and compared with WT. Stressed vs unstressed mutants as well as mutant vs WT under stress are likely to show different EPS and gene expression profiles, so we use these methods to uncover potential different mechanisms of abiotic stress tolerance.
Project Methods
General MethodsCloning: Gene overexpression mutants will be generated by insertion of genes of interest fused to a fluorescent reporter gene on a self-replicating broad host range plasmid backbone. Knockout mutants will be generated by insertion of an antibiotic resistance cassette and counter-selection marker into the genomic region of interest using homologous recombination via suicide vector. Cloning vectors will be generated by Gibson assembly and introduced into cells by electroporation. Overexpression mutant protein production will be validated by flow cytometry. Mutants will be confirmed by colony polymerase chain reaction (cPCR) and gene expression validated by real-time quantitative PCR (RT-qPCR).Characterization:Baseline cyanobacterial growth will be measured in suspended shake flask cultures. Optimal biofilm formation will be assessed on varied surfaces possessing diverse electrostatic properties using crystal violet - a dye that sticks to biofilms. Mild nutrient shifts (1.2x or 0.8x) within S. 6803 culture medium increases biofilm formation so cultures will be grown in both standard and mildly shifted nutrient conditions. Mutants that show changes in biofilm formation will be analyzed further. EPS production will be assessed by estimating total carbohydrate content and released polysaccharides (RPS), the sugar component of extracellular polymeric substances (EPS). If a change in either sugar fraction is observed in mutants vs wild type, further compositional analysis of RPS and lipopolysaccarides (LPS) will be conducted using qualitative and quantitative gas and liquid chromatography. Sulfated polysaccharide content will be assessed by anion exchange column chromatography. Viable biomass will be quantified by phospholipid fatty acid analysis (PLFA) and biofilm culture growth by oxygen evolution. Cell surface charge will be estimated by zeta potential (ζp). Biofilm gene expression profiles will be measured by comparative RNA-seq with data analysis in R studio.Abiotic Stress: Biofilms will be generated under optimal conditions on substrates determined above. Triplicate biofilms will be exposed to a stress regime for a single stress, with other variables held constant: dehydration-rehydration, high light, increase or decrease in temperature, high salinity, and high or low pH. Stress response will be compared to wild type and analyzed as above for viability via PLFA analysis, and differences in EPS and transcriptional profiles. As PLFAs rapidly degrade upon cell death, they provide a representative proxy of viable culture correlated with cell abundance.Evaluation: All cloning validation, characterization and abiotic stress data will be analyzed statistically and visualized as appropriate using R Studio and Microsoft Excel. Data will be collected using a minimum of n = 3 biological replicates. Types of data include growth or viability metrics (optical density, oxygen evolution, or PFLA), gene expression (relative transcript abundance, tag fluorescence), EPS composition (RPS or LPS composition, zeta potential), and comparison of these metrics between typical culture and abiotic stress conditions. Success is measured by 1) generation of proposed mutants and 2) analysis of potential differences between wild type and mutant a) EPS composition and b) abiotic stress tolerance, as measured by cell biomass viability.Potential pitfalls and alternative approaches:Excess EPS production can contribute to clumping associated with biofilm formation leading to cell aggregates that impede accurate flow cytometry or culture density measurements. Methods for non-lethal biofilm dispersion, like incubation with nitrate, DNAse I, TWEEN, or alginate lyase are promising in other gram-negative bacteria. These are likely to also disrupt cyanobacterial biofilms and facilitate accurate analysis. Further, if potential gene knockout targets are lethal to cells, CRISPRi can be used instead for gene knock down. Additionally, chlorophyll a (chl a) content - the primary photosynthetic pigment - is an alternative to PLFA analysis as an estimator of cell viability in cyanobacteria, as chl a is also rapidly degraded upon cell death. Chl a is useful for relative viability, but fails to estimate true culture density as chl content fluctuates by cell state and chl a autofluorescence is not a reliable measure of biofilm cultures as only the top cells are read. Finally, this study focuses on bulk biofilm component, EPS, as the major driver of changes in viability outcomes. Transcriptomic data provides valuable clues to this, but transcript abundance does not always correspond to protein or metabolite contents - say, if a rapidly damaged protein is involved. Should targeted mutants not yield expected susceptibilities, follow-up proteomics and eDNA assays - as eDNA is involved in biofilm scaffold stability - will likely uncover any other underlying mechanisms.