Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Jacob Spriester - CSU Bakersfield
Co-Author(s): Martha Peraza, California State University, Bakersfield, Bakersfield, CA; Jeroen Gillard, California State University, Bakersfield, Bakersfield, CA
Diatoms are an abundant group of eukaryotic unicellular algae contributing up to 25% of global primary production. Diatoms are also attractive candidates for biotechnological applications, such as biofuel production or water remediation. The model species Phaeodactylum tricornutum is a good candidate for these applications due to its extensive genomic resources and its broad salinity tolerance, which allows P. tricornutum to grow in marine and brackish water. In natural environments, diatoms rely mainly on the availability of nitrate (NO3-), which they reduce to ammonia with the help of the enzyme nitrate reductase. However, a few diatoms, like P. tricornutum, are known to be able to grow heterotrophically on various nitrogenous carbon compounds, such as amino acids. This is important to water remediation applications, because P. tricornutum could potentially treat wastewater by eliminating organic contaminants. However, initial experiments aiming to characterize the growth on such organic compounds showed a strong negative response to amino acid L-asparagine (Asn) and eventually led to cell death throughout the entire culture.
We sought to test the hypothesis that P. tricornutum produces a toxic exudate that is mediated by the diatom stress signaling molecule nitric oxide (NO) when grown in saltwater with Asn as the sole nitrogen source. Along with microscopy, growth of P. tricornutum was monitored in triplicate using a fluorescence multiwell plate reader equipped with excitation and emission filters for the measurement of cell chlorophyll autofluorescence as a proxy for cell density (Ex: 395-445; Em: 661-681 nm). Cultures that were grown with NO3- as their sole nitrogen source were used as the negative control for the bioassays.
We found that when spent media from cultures that experienced cell death was added to healthy cultures growing in NO3-, cell growth was affected and ultimately led to the collapse of the culture. We further found that none of the cultures that grew solely on Asn experienced culture-wide cell death when they were grown in freshwater instead of saltwater. Finally, applying NMMA, a NOS inhibitor, and PTIO, a NO scavenger, led to the rescue of cell death, thereby indicating that NO is involved in the downstream response to this unknown natural product.
Together, these experiments supported our hypothesis that P. tricornutum cells grown on Asn produce a toxic exudate solely in saltwater that causes cell death and leads to the collapse of a culture through NO signaling pathways. Ongoing research aims to characterize the toxic exudate and the investigating of the involved molecular pathways using transcriptome sequencing and reverse-genetics approaches.Not Submitted
Funder Acknowledgement(s): Funder acknowledgements: CSU Council on Ocean Affairs, Science & Technology; CSU Bakersfield Student Research Scholars (SRS) Program; CSU Louis Stokes Alliance for Minority Participation
Faculty Advisor: Jeroen Gillard, email@example.com
Role: I am now the main researcher of this project. I have been working on this since April 2016. The initial experiment that led to our observation and questions were done by my coauthor, Martha Peraza. Since then, I have conducted experiments on the effects of the toxin produced by Phaeodactylum tricornutum on healthy P. tricornutum cultures, comparing the production of the toxin in saltwater and freshwater, nitric oxide signaling related experiments, comparing different strains of P. tricornutum to test their sensitivity to the toxin, and testing if other diatom species produce a toxic exudate.