Grantee Research Project Results
Final Report: Molecular Tools to Predict Cyanobacteria Toxin Production
EPA Grant Number: SU839455Title: Molecular Tools to Predict Cyanobacteria Toxin Production
Investigators: Kapoor, Vikram , Estrada, Fabiola , Mertins, Andrea , Gupta, Indrani
Institution: The University of Texas at San Antonio
EPA Project Officer: Callan, Richard
Phase: I
Project Period: December 1, 2018 through November 30, 2019
Project Amount: $15,000
RFA: P3 Awards: A National Student Design Competition Focusing on People, Prosperity and the Planet (2018) RFA Text | Recipients Lists
Research Category: P3 Challenge Area - Air Quality , P3 Challenge Area - Sustainable and Healthy Communities , P3 Challenge Area - Chemical Safety , P3 Awards , P3 Challenge Area - Safe and Sustainable Water Resources
Objective:
The main goal of our research is to develop and demonstrate a robust molecular method for the prediction and monitoring of Harmful Algal Blooms (HABs) in freshwater systems, based on gene expression of a model cyanobacterium (Microcystis aeruginosa) under different environmental factors such as changes in temperature and nutrient (N, P) loadings. To meet this overall goal, the following three objectives are proposed: 1) determine the effect of different environmental factors on the physiological and metabolic responses ofM. aeruginosa, which leads to toxin production; 2) investigate the impact of different environmental factors on the functional gene expression ofM. aeruginosaand evaluate how toxin production is regulated at molecular level; and 3) integrate the results of molecular biology tools with biochemical and analytical methods to elucidate the mechanism of toxin production by M. aeruginosa.The principal hypothesis, which was evaluated as part of this project is, ‘Can specific gene expression measurements be used as predictive and quantitative indicators of toxin production kinetics?’ We hypothesize that any changes in toxin production pathways in M. aeruginosa can be inferred from the expression of genes coding for these pathways and that the transcriptional responses will differ with different environmental factors.
The physiological and metabolic responses were monitored during the course of the experimental run including growth rate, microcystin production, surface zeta potential, and cell morphology. We employ reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assays to measure the fold changes in transcript levels of microcystin synthesis genes in M. aeruginosa cultures exposed to different environmental conditions. Integrating the use of molecular biology tools with conventional biochemical methods will enable us to describe the mechanistic model of cyanobacterial toxin production under different environmental factors.
Summary/Accomplishments (Outputs/Outcomes):
The toxic strain M. aeruginosa PCC 7806, was used in our study since this strain is the most studied with completely sequenced genome and well identified toxin-synthesis genes, providing the best understood background for our in-depth study. The axenic cultures of M. aeruginosa PCC 7806 were maintained and grown in our laboratory using the BG11 medium. A combination of several different experimental treatments is being tested to mimic different environmental factors. Temperature and nutrients were considered as primary environmental factors in exposure experiments. Three temperature regimes (20, 25, or 30 °C) and five nutrient loadings of different N: P (5, 25, 76.86, 100, and 150) were selected to be tested. Light intensity is kept constant with 12h:12h light/dark cycles. For the first set of experiments, triplicate flasks were incubated for each N: P loading at room temperature (25 °C) for 30 days. The physiological growth was monitored daily by measuring absorbance at 750 nm, and cell counting was conducted using Countess II Automated cell counter. At predetermined intervals, aliquots were withdrawn from the treatment vessels and immediately centrifuged at 21,000 g for 3 min. The supernatant was removed, and the pellets were stored at -80 °C for molecular assays. RNA and DNA were extracted from the stored pellets and cDNA was synthesized from the purified RNA. The relative expression of the functional genes mcyA, mcyD, mcyE was quantified by RT-qPCR.
Although the microcystin-producing genes were quantified at the rate lower than the lowest calibration standard, slight stimulation in gene expression was observed as cyanobacterial cells reached exponential phase. Upregulation in the expression of microcystin-producing genes was observed with an increase in N:P loading. For instance, there was an increase in the relative expression of mcyA gene for N:P = 150 from day 4 to day 12. Similarly, the relative expression of mcyE increased for N:P = 150 from day 4 to day 12. On the contrary, no expression was observed for mcyD for N:P = 5. For N:P = 76, there was slight stimulation in the expression of all genes. In general, an increase in gene expression happened in early to mid-exponential phase of growth. As the cyanobacteria grew during 12 days, an increase in Microcystis 16S rRNA gene content was observed with an increase in N: P ratios.
Conclusions:
Microcystin production in water supply reservoirs is a global public health problem. Understanding the physiology and genetic activities of toxic cyanobacteria, including their responses to the presence of anthropogenic nutrients such as nitrogen, is central to managing harmful blooms. To our knowledge, this will be the first study to examine transcriptional and physiological changes occurring in M. aeruginosa cultures under different environmental conditions at different growth rates. With a comprehensive examination of the role of nutrients, with respect to other environmental factors such as temperature, on M. aeruginosa, a clearer understanding of the growth, toxicity, and genetic responses of this cyanobacterium may offer valuable insight into effective strategies for controlling these potentially devastating blooms.
This work will provide valuable insight into the mechanistic interaction of toxin production in cyanobacteria with different environmental factors, and provide fundamental physiological and transcriptional information to further explore and predict the behavior and impacts of cyanobacterial blooms in freshwater systems. The results of this study will offer an opportunity to develop approaches for the prediction and monitoring of HABs as well provide insight to the molecular level approaches for mitigation of HABs. While the scope of the proposed project is limited to few environmental factors, we must consider that different environmental conditions are expected to occur with many other nutrients present in any given water system. Consequently, future studies will benefit from examining HABs in freshwaters using a more holistic approach employing a combination of emerging (gene transcription) and conventional (growth rate) technologies.
Journal Articles:
No journal articles submitted with this report: View all 2 publications for this projectSupplemental Keywords:
drinking water, decision making, cyanotoxinThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.