A War of Tiny Giants - Do viruses impact Pelagibacterales genotype dynamics in the Western English Channel

Lead Research Organisation: University of Exeter
Department Name: Biosciences

Abstract

One often hears that the rainforests are the 'lungs of the Earth', producing the oxygen that we breathe through photosynthesis and drawing down atmospheric carbon dioxide. However, perhaps less well-known is the fact that the oceans, and in particular coastal regions, are responsible for about half of all global photosynthesis as marine bacteria and algae (known as phototrophs) capture sunlight to produce metabolites for growth. Through cell death and the leaky nature of cell walls, products of photosynthesis find their way into the water, where they are consumed by other bacteria (known as heterotrophs), releasing the captured carbon dioxide back to the atmosphere. Perhaps the most important group of heterotrophs is the Pelagibacterales. These tiny cells dominate global oceans (up to 500,000 in every mL of seawater) and are responsible for converting up to 40% of marine photosynthetic products back to atmospheric CO2. As a result, they have a major impact on global carbon cycling and can be considered global bioengineers. For over a decade we have been studying how nutrient availability in the oceans drive Pelagibacterales ecology and evolution, in a bid to build better models of future global carbon cycling under the influences of climate change. As oceans warm and nutrients become less available, Pelagibacterales abundance, and their importance in carbon cycling, is set to increase further. However, not every member of the Pelagibacterales is equal - they differentiate into distinct ecological niches (known as 'ecotypes') with different capacities to take up resources and release important climate-changing gases such as methane and dimethylsulfide. Therefore, understanding which conditions favour which type of Pelagibacterales is of major importance for climate modelling.

Perhaps the only organisms on Earth more important to global carbon cycles than the Pelagibacterales are the viruses that infect them. Based on their extraordinary abundance and diversity, J.B.S. Haldane once quipped that 'The Creator would appear as endowed with a passion for stars, on the one hand, and for beetles on the other'. In comparison, the Creator's zeal for viruses would make stars and beetles appear to be a side-project performed with perfunctory indifference. Virus numbers are staggering - they are by far the most abundant and diverse organisms on Earth. If one assumes that there are 10 trillion galaxies in the universe and each one is similar to our Milky Way and contains 100 billion stars, then the oceans are populated with a million viruses for every star in the universe. Of these, the vast majority are viruses that infect and kill bacteria (known as 'phages'), and of these, around a quarter are thought to infect Pelagibacterales. Yet, until 2013, the existence of viruses that infect Pelagibacterales was entirely unknown! Similar to interactions between lions and wildebeest in the Serengeti, predation is rather unfortunate for the host, but provides benefits to the scavengers of the ecosystem. Phage-induced cell death releases the contents of the host cell into the water column and this soup of dissolved organic matter provides nutrients to surviving cells. Furthermore, like the arms-race between lions and wildebeest (bigger claws, horns etc.), bacteria and viruses co-evolve to produce resistance and counter-resistance mechanisms. In some cases, this co-evolution can lead to the emergence of new types of host, resistant to viruses and capable of thriving despite high viral abundance. Therefore, both nutrients and viral predation can influence the abundance and diversity of marine bacteria. This project is the first attempt to evaluate the impact of the viruses infecting Pelagibacterales on their diversity and abundance over seasonal timescales. The findings will enable us to build better models of future carbon biogeochemistry by accurately incorporating viral predation of the Pelagibacterales in global carbon cycling.

Planned Impact

Over the last two decades, models of ecosystem function have matured into sound scientific tools for operational forecasting systems to inform policy and management on issues critical to society including climate change and food security. Interactions between microbes and abiotic factors are the first level in a complex strophic web and thus underpin community function. Much of the work in developing these models, such as the European Regional Seas Ecosystem Model (ERSEM), include parameters for capturing microbial interactions, but the role of viruses in these systems has, to date, been largely ignored. A recent study into how ecosystems differ with and without viruses showed that viral interactions promote community productivity, increasing primary production and reducing the amount of carbon transferred to higher trophic levels. Yet, understanding of how viruses should be incorporated into models such as ERSEM remains unknown. Understanding how host-virus interactions differ across evolutionary scales will determine the evolutionary scale at which we need to incorporate viruses into future models. If host-range is broad and impact of viral predation across is uniform, then parameterisation may be reduced to simple terms such as total viral abundance. However, if host-range and infection efficiency differ widely across evolutionary and/or temporal scales, then parameterisation will need to be more nuanced. The focus of this proposal, the Pelagibacterales and its viruses, are the most abundant organisms on Earth. The amount of carbon encapsulated within members of the Pelagibacterales is similar to that of all marine fish, and it is responsible for converting up to 20% of global primary production back to atmospheric carbon dioxide. As oceans warm and become more stratified, further limiting nutrient availability, the dominance of Pelagibacterales is set to increase further. Similarly, there is as much carbon within pelagiphages as 45 million blue whales, but as yet, we are ignorant as to whether interactions between these groups are significant in situ. Thus, it is difficult to imagine an organism for study that would that would have greater impact on the accuracy of our models. Therefore, we can say with confidence that this research is of significant societal important to the broadest range of international stakeholders, including the general public, academics and policy makers. These benefits will be provided over the longest timescales as incorporation of data from this proposal improves model accuracy.

Furthermore, development of novel methods to measure viral host range in situ in complex communities will be of significant benefit to those both directly and indirectly involved in developing phage therapy as an alternative means of controlling infection in the face of increasing antimicrobial resistance. The disease burden and economic costs of infectious diseases continue to increase, even in developed countries such as the UK, and the lack of new antibiotics to combat emerging resistance and the threat of a post-antibiotic world is well publicised and a major focus of targeted funding. Phage therapy is most effective when the phages used are broad host range phages, to limit the emergence of counter resistance by the bacterial pathogen. The development of epicPCR approaches as part of this proposal will facilitate identification of broad-host range viruses for use in phage therapy, as well as enable monitoring of coevolution over time so that we can better understand long-term effectiveness of phage therapy.

The Pathways to Impact section details some of the avenues that will be used to engage with the general public to promote the benefits of this work.
 
Description We have developed a new high throughput method to isolate viruses associated important marine taxa that have until now been unculturable. This is enabling us and collaborators to experimentally validate hypotheses about the importance of viral predation on marine carbon biogeochemistry.

We have isolated >100 phages infecting the ubiquitous SAR11 clade, as well as phages infecting OM43. Global distribution of these phages supports a hypothesis of a viral seed bank. Using these new models, we have shown that (contrary to initial hypotheses), SAR11 can become resistant to phages during co-culture.

We have also completed a +10 year timeseries analysis of SAR11 community dynamics in the Western English Channel and the Sargasso Sea. We show that warm water ecotypes of SAR11 are abundant even in winter time in the Western English Channel. We also show that very little of the variance of SAR11 is not explained by bottom up processes, indicating that viral predation of SAR11 is low in the oceans, despite their huge abundance. This is important for trying to predict what is going to happen when the oceans warm and different SAR11 populations (and their viruses) become more prevalent.
Exploitation Route Now that we have robust model systems for important marine methylotrophs, we submitted a recent NERC grant to better understand the role of viral predation on methylotrophy in the oceans. This was not funded. However a student submitted a similar idea to Simons Foundation and was awarded a fellowship to pursue the research at Oregon State University.

The taxa we have isolated are also being used as model systems for evaluating the performance of a new type of electron microscope in collaboration with the University of Cambridge.

Methods developed during this project to isolate viruses on fastidious microbes are now being used to isolate phages for clinical use against Coxiella spp, a major AMR risk and potential bioweapon.
Sectors Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://www.nature.com/articles/s41396-020-00872-z#Ack1
 
Description BIOS-SCOPE II
Amount $420,000 (USD)
Organisation Simons Foundation 
Sector Charity/Non Profit
Country United States
Start 11/2020 
End 10/2023
 
Title First host-virus model system for methylotrophic clade OM43 
Description OM43 is an important marine clade that blooms following phytoplankton blooms and plays a critical role in the remineralisation of methanol back to atmospheric CO2. Until now, no isolated viruses existed for this system. We have now developed a robust host-virus model system for investigating the influence of viral predation on methylotrophy. 
Type Of Material Cell line 
Year Produced 2021 
Provided To Others? Yes  
Impact OM43 are some of the smallest free-living cells. We are collaborating with colleagues at Cambridge University to use them as a model system for evaluating a new type of transmission electron microscope. The idea would be to then provide these as a standard for calibrating instrumentation. 
 
Title High throughput viral isolation for fastidious microbial taxa 
Description We have developed a novel high-throughput system for isolating ecologically important host-virus model systems that couples recent advances in dilution-to-extinction culturing of hosts with sequential enrichment and isolation of associated phages. Applied to five monthly samples from the Western English Channel, this method successfully isolated 117 new phage isolates including the first known siphovirus infecting SAR11 and the first known phages infecting members of the important methylotrophic bacterial clade OM43. Genomic analyses of 13 representatives suggests a plethora of viral diversity for these important taxa still remains to be discovered. Greater representation of viruses infecting fastidious hosts in culture collections and genomic databases will facilitate the experimental validation of host-virus interactions predicted from metagenomic data, and improve accuracy of computational approaches to evaluate ecological patterns from metagenomic data. 
Type Of Material Biological samples 
Year Produced 2019 
Provided To Others? No  
Impact Improved representation of important viral taxa in genomic databases and isolates for use in synthetic ecology. 
 
Title Long read viral metagenomics 
Description The purpose of this grant was to investigate the use of long read sequencing for better capture of viral metagenomes. As part of this grant, we have developed the MinION technology for accurately capturing viral genomes from environmental samples using long reads. This project has not only developed the bioinformatic analyses of long-read data, but has also delivered on its promise to use long reads for improving viral metagenomics. Using our new method, we show that short-read metagenomics currently used misses a significant and important component of the viral fraction. Our method successfully captures this diversity and improves the representation of viral diversity in metagenomes. We have continued to develop this method and have now optimised sequencing from 1 ng of input material and increased amplicon length to >7kbp. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? Yes  
Impact Genoscope are now planning to use our method for the next round of Tara Oceans viral sequencing. 
URL http://dx.doi.org/10.7717/peerj.6800
 
Title VirION 2 - improved long read viral metagenomics 
Description We have further developed our methods for long read viral metagenomics, decreasing input requirements down to 1 ng and developing a bioinformatic pipeline for robust analysis of the data. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? Yes  
Impact This method is going to be used to sequence the viromes from the next Tara Oceans cruise. We have had several international requests for developing this as a service in the University of Exeter Sequencing Centre. In addition, Exeter is aiming to become a centre of excellence for wastewater monitoring due to our enhanced capacity for capturing the viral fraction. 
URL https://www.biorxiv.org/content/10.1101/2020.10.28.359364v1.full.pdf
 
Title Viral CarrierSeq 
Description We have developed methods for capturing full length viral genomes on single reads from environmental viral populations. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? No  
Impact This new methodology enables high throughput single viral genomics at massive scale, as well as capturing DNA modifications within environmental viral populations for the first time 
 
Title First coupled long- and short-read viral metagenomic timeseries dataset 
Description This project has produced the first long-read viral metagenomic dataset to date, providing marine microbial ecologists with a powerful dataset to explore the population structure of viral communities. At present it comprises 13 samples and >1.4 M reads >15kbp in length, including >19,000 full viral genomes on single reads. 2022 update: This dataset now includes 36 short read cellular metagenomes 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? No  
Impact This project has produced the first long-read viral metagenomic dataset to date, providing marine microbial ecologists with a powerful dataset to explore the population structure of viral communities. At present it comprises 13 samples and >1.4 M reads >15kbp in length, including >19,000 full viral genomes on single reads. We have shown that long read sequencing captures far more important viral populations than short-read sequencing alone. 
 
Title Optimised assembly pipeline for long-read viral metagenomics 
Description We have developed a robust pipeline for long-read viral metagenomics that can recover full length genomes for viruses at >0.3% of the community, with 99.5% nucleotide accuracy. 
Type Of Material Data analysis technique 
Year Produced 2020 
Provided To Others? No  
Impact This will become the gold standard for long-read viral metagenomics data processing. 
 
Description Integrating Viruses into FluxNet 
Organisation Technical University Berlin
Country Germany 
Sector Academic/University 
PI Contribution The FluxNet model is used to predict interactions between taxa and environmental parameters in timeseries data. The outputs we are generating as part of this grant will be used directly to integrate viruses within the FluxNet model to better capture their influence on marine microbial communities.
Collaborator Contribution Ferdi Hellweiger is developing the FluxNet model to integrate our data into it.
Impact None yet - still early
Start Year 2020
 
Description Exeter Scholars Outreach Event July 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact My team was part of an outreach event that involved looking at microorganisms within pond water. We collected samples of ponds around the university and observed them under a light microscopy. After sequencing of these samples, school children participated in the usage of BLAST and using the NCBI database to establish the identity of organisms found within pond waters through their 16S rRNA.
Year(s) Of Engagement Activity 2019
 
Description Halstow Wassail November 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact My team participated in a workshop to identify the important microbial community associated with cidermaking, using long-read sequencing to perform community analyses. The product of the workshop was a wassail that was performed to 100 members of the general public.
Year(s) Of Engagement Activity 2019,2020
 
Description Microbiology Module for Canada Hill Primary School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We developed a curriculum for teaching kids about microbes in a local primary school. This included a video talking about microbes and an experiment where children tested the efficacy of hand sanitiser by licking their thumbs and sterilising one of them with hand sanitiser before making an imprint on agar plates. My GRA also gave a talk on what it's like being a marine scientist via Skype.
Year(s) Of Engagement Activity 2020
 
Description Pies, Pints and PhDs at the National Marine Aquarium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact As part of the adults only NMS lates series, Warwick-Dugdale presented to a general audience, explaining what her PhD is about and why it is important for them to know about it, including its wider implications.
Year(s) Of Engagement Activity 2020
URL https://www.visitplymouth.co.uk/whats-on/nma-lates-pints-pies-and-phds-p2808033
 
Description Skype a Scientist 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Skype a Scientist is a program which they match up to 5 classrooms with a Scientist. Then the Scientist and the teacher come up with what they want to be talked about whether it be the road to becoming a scientist, the science that scientist carries out, or a lesson plan based around the science. In the Fall 2019 session, two members of my team were matched up with two schools. The first school was a middle school in Dakar, Senegal where they discussed the impacts of ocean acidification and climate change. The second school was Rocky Mountain Deaf School in Denver, CO where they discussed their path to becoming a scientist, a bit about the research we conduct, and answered general science and life questions.
Year(s) Of Engagement Activity 2019,2020