Award: OCE-1657898

Award Title: Collaborative Research: Building a framework for the role of bacterial-derived chemical signals in mediating phytoplankton population dynamics
Funding Source: NSF Division of Ocean Sciences (NSF OCE)
Program Manager: David L. Garrison

Outcomes Report

Phytoplankton and bacteria are important influencers of biogeochemical cycling in the oceans and are principal contributors to the marine food web. Therefore, microbial interactions are key factors in regulating and understanding these critical processes. Mutually beneficial interactions can include recycling of nutrients and production of defensive compounds against competitors and pathogens. Antagonistic relationships are also possible, such as bacterial predation on phytoplankton and release of anti-algal toxins. Since many bacteria-phytoplankton interactions are likely mediated by the release and response to chemical signals, identification of these compounds can enhance our understanding of microbial chemical communications and how they may influence population dynamics, including phytoplankton blooms. Pseudoalteromonas is a genus of Gram-negative bacteria found in diverse marine environments. Pseudoalteromonads produce a diverse array of specialized metabolites, including alkylquinolones, hydroxyquinolones, and N-acyl homoserine lactones for cell-to-cell signaling. We previously found that Pseudoalteromonas galatheae A757 produces the signaling molecule 2-heptyl-4-quinolone (HHQ), and that this compound further demonstrates selective and potent anti-algal toxicity to the bloom forming phytoplankton Emiliania huxleyi. This suggests that alkylquinolones such as HHQ may serve multiple roles for microbial interactions, both as quorum sensing signals to coordinate gene expression within a bacteria population and as potential toxins for attacking prey. This was a collaborative project with three principal investigators. We sought to use our newly discovered HHQ-phytoplankton relationship as a model system to investigate the potential role that bacteria quorum-sensing signals could have in driving phytoplankton population dynamics. Within that framework, three aims were pursued: (1) conduct laboratory experiments to investigate factors that promote HHQ production, (2) establish a mechanistic understanding of how HHQ induces phytoplankton mortality, and (3) use field-based experiments to understand how HHQ influences natural microbial assemblages. Using laboratory-based co-culturing methods, P. galatheae A757 was shown to be detrimental to the survival of E. huxleyi once the phytoplankton had reached a stationary growth phase. A highly sensitive and selective mass spectrometry method was developed to accurately measure HHQ concentrations in seawater samples. We found that HHQ was indeed produced by A757 during the co-culture experiments, but the concentrations were variable and did not achieve thresholds associated with killing E. huxleyi. This suggests that HHQ secretion was not solely responsible for the observed antagonistic effects. Stability studies showed that HHQ is highly stable in seawater and in the presence of E. huxleyi. We further used our mass spectrometry method to detect a broader range of signaling molecules produced by P. galatheae A757 and found this bacterium can produce an array of alkylquinolone and acyl homoserine lactones, indicating the presence of multiple quorum sensing networks. These findings suggest that HHQ may contribute to the toxic interactions between P. galatheae A757 and E. huxleyi CCMP2090, but likely acts in concert with other factors, and encourage further investigations into the production and regulation of bacterial virulence factors that target E. huxleyi. Using a multipronged approach which included both transcriptomic and proteomic analyses, HHQ exposure to E. huxleyi was found to prolong S-phase arrest in phytoplankton cells. This was coincident with the accumulation of DNA damage and a lack of repair, despite the induction of the DNA damage response. The growth inhibition was reversible for cells moved to HHQ-free media. Surprisingly, HHQ-exposed phytoplankton were also protected from viral mortality, suggesting a new role of quorum-sensing signals in regulating multitrophic interactions. Field-based incubation experiments with HHQ were conducted over the course of a stimulated phytoplankton bloom to probe how natural communities of phytoplankton and bacteria respond. HHQ treatments caused nanoplankton and prokaryotic cell abundances to decrease, and further altered the composition of particle-associated and free-living microbiota. Pseudoalteromonas species increased in relative abundance following HHQ exposure. Bacteria taxa that increased in abundance when exposed to HHQ likely possess the genetic potential to bind HHQ. In summary, these incubation studies found that HHQ has the capacity to influence microbial community organization, suggesting alkylquinolones have functions beyond bacterial communication and may influence microbial community structures. In total, the cooperating laboratories measured HHQ production and its potential toxic effects in bacterial co-cultures with E. huxleyi, studied the stability of HHQ in seawater and in the presence of E. huxleyi, examined the mechanism of action of HHQ toxicity against E. huxleyi, and investigated the influences of HHQ on the composition of microbial communities mesocosm experiments. These in-depth studies of a bacteria-plankton relationship have contributed to our understanding of how chemically mediated interactions may influence microbial populations in marine ecosystems. Furthermore, this project was conducted with participation of scientists at multiple career levels, including undergraduate and graduate student researchers, and therefore has advanced the mission of developing new experts to further pursue scientific inquiries. Last Modified: 03/30/2022 Submitted by: David C Rowley

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Principal Investigator: David C. Rowley (University of Rhode Island)