Diatom-bacteria interactions: deciphering impacts on algal bloom regulation and toxicity

Primary supervisor: Dr Katherine Helliwell (Marine Biological Association)

Secondary supervisor: Dr Michael Cunliffe (University of Plymouth, Marine Biological Assocation)

Additional supervisor: Professor Willie Wilson, (University of Plymouth, Marine Biological Assocation). Email: wilwil@mba.ac.uk

Scientific background

Marine phytoplankton are vital in regulating our global climate, contributing almost half of the biosphere’s net primary production. Diatoms are one of the most important phytoplankton groups, generating as much organic carbon as all terrestrial rainforests combined. 

Diatoms form spatially extensive blooms that exert global-scale influences on biogeochemical cycles and underpin ecosystems. Due to the release of harmful toxins, some diatom blooms can also have a negative impact on marine ecosystems and fisheries.

The biotic interactions of diatoms with predators, parasites, competitors and symbionts, can profoundly influence natural diatom populations, and are an important factor regulating bloom dynamics and potential toxicity. 

Despite the clear importance of such interactions for diatom ecology and marine ecosystem functioning, little is known about the mechanisms diatoms employ to i) recognise and respond to other microbes, or ii) regulate the production of harmful toxins. 

These represent important knowledge gaps that need to be addressed, in order to better understand factors governing diatom bloom formation and toxicity.

Research methodology

This PhD will couple field sampling at the Western Channel Observatory coastal Station L4, which has regular diatom blooms, with state-of-the-art cell biological approaches in the laboratory. 

Field sampling will enable isolation of diatom-bloom associated microbes that will be brought into the laboratory for further experimentation. 

A combination of physiological (co-culturing), metabolomics and molecular approaches will be employed to examine the nature of such interactions, and their impact on diatom toxicity. 

A key aim will be to identify and characterise signalling pathways employed by diatoms to recognise and respond to their neighbours using live-cell imaging and CRISPR-Cas9 gene editing.  

Training

The successful candidate with gain training in cutting-edge cell and molecular biology approaches including live-cell imaging (e.g. confocal microscopy), CRISPR-Cas9 gene knock-out, genetic transformation, bioinformatics, and cloning, alongside microbial physiology and environmental microbiology techniques. Professional development, including training in core verbal and written communication, research and analytical skills will also be provided.

Person specification

An enthusiastic, motivated individual interested in how molecular mechanisms in the cell impact ecosystem level processes, with a relevant biological sciences degree (marine biology, microbiology, plant sciences).

Biotic interactions between phytoplankton and other microbes influence marine microbial community structure and function. Grazing activities of zooplankton, and viral infection are critical in controlling phytoplankton blooms. 

During the demise of phytoplankton blooms increases in bacterial abundance are also common. Certainly, bacteria can have a profound influence on phytoplankton metabolism. For instance, bacteria can stimulate production of the toxin domoic acid (DA) by the harmful diatom Pseudo-Nitzschia, suggestive of antagonistic interactions between diatoms and bacteria1. 

However, although pathogenic interactions between algicidal bacteria and diatoms have been reported in culture, knowledge of how such interactions are manifested, their prevalence in nature, and roles in regulating diatom blooms are largely unknown. 

Moreover, the signalling mechanisms mediating diatom responses to algicidal bacteria remain ill-resolved.

Evidence from the plant and animal fields indicate that Ca2+, the universal signalling molecule, is pivotal in mediating the molecular dialogue between plants and their pathogens. 

At the MBA, we have recently developed environmental biosensors that enables intracellular Ca2+ concentrations to be measured in diatom cells with high spatial and temporal resolution4. This PhD project will build on these recent advances to elucidate the role of Ca2+ signalling in mediating biotic interactions of diatoms. 

Sampling of the Western Channel Observatory station L4, for diatom-interacting bacteria, will elucidate the environmental relevance of algicidal bacteria in natural diatom communities.

Key Objectives:

1)  Isolate and identify diatom-interacting bacteria from diatom blooms: Environmental sampling at station L4 will enable isolation of bacteria frequently associated with diatom blooms. To screen for direct interactions an established plaque assay approach will be used. Lawns of three genetically tractable diatom species: Pseudo-Nitzchia multiseries, Thalassiosira pseudonana and Phaeodactylum tricornutum will be used as bait. Formation of clearance ‘plaques’ will enable identification of algicidal bacteria that negatively impact diatom growth.

2)  Examine impact of algicidal bacteria on diatom growth, physiology and toxicity (Pseudo-Nitzchia): Algicidal bacteria isolated in objective 1) will be taken forward for further examine the nature, specificity and mode of action of algilytic activity. Domoic acid production by Pseudo-Nitzchia will also be quantified in collaboration with Debbie Salmon (University of Exeter mass spectroscopy facility).

3)  Develop new Ca2+ imaging tools for toxic diatom Pseudo-Nitzchia-multiseries: Established genetic transformation approaches3 will be used to generate a Pseudo-Nitzchia strain encoding the Ca2+ indicator, RGECO.    

4)  Identify the signalling processes mediating diatom responses to bloom-associated bacteria: Epifluorescence microscopy and Ca2+ imaging will be used to examine early signalling responses of diatoms exposed to algicidal bacteria. Particular attention will also be paid to the role of bacterial metabolites. This will identify candidate signalling pathways for further characterisation. 

Risk mitigation

This project builds on a range of techniques already successfully developed in our laboratory. The physiological plaque assays to screen for algicidal bacteria is established. Additionally, strains of T. pseudonana and P. tricornutum encoding RGECO that successfully reports intracellular Ca2+ have been developed, providing low risk potential for immediate data collection. 

Moreover, we have also developed CRISPR-Cas9 genome editing approaches in P. tricornutum4. Alongside established approaches, the project presents exciting opportunities to apply these novel tools for a toxic diatom. 

References

1) Bates et al., Enhancement of Domoic Acid Production by Reintroducing Bacteria to Axenic Cultures of

the Diatom Pseudo-nitzschia multiseries. Nat. Toxins 3, 428–35 (1995)

2) Sabatino et al, Establishment of Genetic Transformation in the Sexually Reproducing Diatoms Pseudo-nitzschia multistriata and Pseudo-nitzschia arenysensis and Inheritance of the Transgene. Mar. Biotech. 17, 452–62 (2015)

3) Paul, Interactions of the Algicidal Bacterium Kordia algicida with Diatoms: Regulated Protease Excretion for Specific Algal Lysis. PLoS One 6, (2011)

4) Helliwell K. E., Chrachri A., Taylor A., Koester J., Wharam S., Wheeler G., and Brownlee C. Novel classes of voltage-gated Na+ and Ca2+ channels play important signalling roles in unicellular eukaryotes. In review.

Funding

This project has been shortlisted for funding by the ARIES NERC Doctoral Training Partnership. Undertaking a PhD with ARIES will involve attendance at training events.

ARIES is committed to equality & diversity, and inclusion of students of any and all backgrounds. All ARIES Universities have Athena Swan Bronze status as a minimum.  

Applicants from quantitative disciplines who may have limited environmental science experience may be considered for an additional 3-month stipend to take appropriate advanced-level courses.

Usually, only UK and EU nationals who have been resident in the UK for three years are eligible for a stipend. Shortlisted applicants will be interviewed on 26/27 February 2019.

For further information please see www.aries-dtp.ac.uk or contact us at aries.dtp@uea.ac.uk.