'Omics' Tools Decoding the Genetic Seascape

Senior Research Scientist José A. Fernández Robledo has studied the oyster parasite Perkinsus for decades. He has grown generations of Perkinsus in his laboratory, delved into the parasite’s historical impact on Damariscotta River oyster populations, and is currently evaluating the potential of engineering it for a malaria vaccine. And yet, Perkinsus can still surprise him.

One day, when he and Senior Research Scientist Joaquín Martínez Martínez were browsing old Perkinsus images taken with an electron microscope, they saw a few cells dotted with something unexpected: viruses.

The scientists set out to study the relationship between virus and host, but they quickly ran into limits of traditional techniques and could not isolate the virus from Perkinsus’ cells.

“Using the classical microbiological tools was tough and only partially successful,” Martínez Martínez said. “Viruses don’t look like much more than dots when viewed through an electron microscope, so we needed really clear samples to identify them.”

These challenges led Martínez Martínez and Fernández Robledo to try a new approach: a state-of-the-art suite of technologies frequently referred to as “omics.” This rapidly evolving methodology includes molecular techniques such as genomics, transcriptomics, and proteomics.

Omics allow scientists to characterize and quantify pools of genetic data — from all the genes of a single organism to an entire community of organisms.

Studying specific genes reveals fundamental information about any organism. An oyster's genes can show what shape its shell will be or how it regulates its body temperature. Studying all of the genetic information of that same oyster can help scientists answer much more complex questions — what the oyster eats, how it interacts with its environment, and what other species it is related to.

The power of omics lies in the capacity to analyze these large sets of genetic information, and to do so quickly. This enables researchers to pinpoint issues that would have been impossible two decades ago and explore two of the most complex questions in microbiology: what microbes exist in the environment, and how do they function?

“Omics are some of the most powerful tools we have to understand life in its many forms,” Martínez Martínez said. “In this project, they let us read the parasite’s genetic activity and pinpoint the presence and impact of the virus.”

Traditionally, microbiology has relied on growing samples in the laboratory, but this process has limitations. Microbes grown in the laboratory may act differently than they would in nature. Certain environments, like the deep sea, are so extreme that they cannot be replicated.

Omics tools give scientists the option to bypass cultivation and go straight to the source — microbes grown in their natural environment. This analysis can take place on many scales: from a single cell to an entire microbial community. Certain omics techniques pinpoint only the active genes in a microbe, and others reveal all the genetic information an organism possesses.

Bigelow Laboratory researchers are utilizing these technologies to answer many complex questions. Several senior research scientists use omics to explore questions at scales that range in magnitude from microscopic to global — from identifying how specific bacteria are related, to cataloging every microbial species in the ocean.

“Omics tools replaced techniques that had been the core of microbiology for over a century, becoming our main toolset,” said Senior Research Scientist Ramunas Stepanauskas, who directs the Single Cell Genomics Center, a hub of omics work at Bigelow Laboratory. “They tell us about environments from the ocean, to soils, to human guts — anywhere you find microbes.”

Across these varied microbial environments, omics techniques allow Bigelow Laboratory researchers to analyze how a given ecosystem functions and unravel the relationships between the microbes that live there. These lines of questioning have the potential to resolve fundamental mysteries and generate revolutionary applications.

“Omics technologies are allowing us to study these specific, nuanced questions that are essential for our understanding of how the planet works,” said Senior Research Scientist Peter Countway.

He is using omics techniques to interrogate an ecosystem that is still full of microbial mysteries: the Southern Ocean surrounding Antarctica. Certain phytoplankton produce a compound called dimethylsulfoniopropionate (DMSP) that provides essential nutrients to marine bacteria. The consumption of DMSP can lead to the release of dimethyl sulfide (DMS), a gas that helps form clouds, thereby influencing global climate.

In the spring of 2018, Countway, Senior Research Scientist Paty Matrai, and Senior Research Associate Carlton Rauschenberg returned to Antarctica to continue their study of how marine microbes respond to different concentrations of DMSP. Omics techniques allow them to examine this system at multiple scales: from the specific ways that individual microbes are processing DMSP, to the way the entire community functions and changes.

“This process takes place on a molecular level, but it has a huge potential impact on atmospheric chemistry and global climate,” Countway said. “It’s a process we’d struggle to understand without the power of omics.”

These rapidly evolving technologies also have the potential to change how we look at life itself. The ability to examine entire microbial communities has revealed a level of diversity that biologists have long suspected but been unable to prove with traditional techniques.

"Omics technologies have opened our eyes to the true diversity of the world," said Senior Research Scientist Dave Emerson. "That’s their real power."

Marine Virus Investigation

The oyster parasite Perkinsus spreads disease that causes significant losses to the United States shellfish aquaculture industry — all while sustaining a viral infection. This apparent paradox led Senior Research Scientists José Fernández Robledo and Joaquín Martínez Martínez to ask an interesting question in their virus-parasite study: what if this virus wasn’t actually bad, but somehow beneficial to the parasite?

Investigating that question requires unprecedented research. Martínez Martínez and Fernández Robledo are undertaking the first coordinated effort to isolate and characterize a marine virus, as well as the first research into the virus of a marine parasite. Access to modern omics technologies means that Martínez Martínez and Fernández Robledo will be able to take further strides than researchers studying viruses ever have.

“Virus research used to be mostly descriptive,” Fernández Robledo said. “Now we have tools that allow us to interrogate the virus and answer questions, so our research will be proactive.”

In tandem with omics approaches, Fernández Robledo plans to use the powerful gene-editing tool CRISPR-Cas9 to label the virus so that it will fluoresce under the microscope, making it easier for Martínez Martínez to perform experiments on the virus itself.

This research has the potential to clarify the complex interactions between virus, parasite, and oyster, and it could shed light on other parasitic relationships as well. Perkinsus can serve as a model of how a pathogen is infected by a virus — which is by definition a parasite itself.

“We’re investigating the parasite of a parasite,” Martínez Martínez said. “This project is difficult, but omics tools are putting us on the right path.”

Understanding this relationship could have impacts far beyond biology, as well. Developing a robust, sustainable aquaculture industry is key to growing Maine’s coastal economy — but the disease caused by Perkinsus, which has increased in oysters by 65 percent over the last decade, is a severe threat. Preventing and treating oyster disease would benefit Maine and other states whose economies rely on shellfish aquaculture.

“There’s a lot of research into preventing this parasite from spreading on oyster farms,” Martínez Martínez said. “Adding an understanding of viruses to the mix makes it possible to get the biological and ecological information that will let us cure this parasite once and for all.”

In Maine’s aquaculture industry and beyond, Martínez Martínez and Fernández Robledo believe this project could cause a paradigm shift. Using omics tools to pinpoint how the virus shapes Perkinsus’ impact on its host has the potential to change the archetype of how biologists think about parasitic infections — with repercussions for human, as well as shellfish, health.

“Everyone in the world has been infected by a virus,” Fernández Robledo said. “Sometimes the clue to help solve such a major problem is in a marine organism, and omics approaches make this important research possible.”

Creating a Genomic Database

In 2000, the Human Genome Project forever changed the field of biology when it published the first genetic blueprint of a human being. This genome provided a crucial reference point for future studies and enabled the development of omics technologies.

“Marine microbiologists took these new omics techniques and started using them on microbes in the ocean, but without the genomic blueprints needed to understand the results,” Senior Research Scientist Ramunas Stepanauskas said. “Now the field is upside-down. Scientists are producing tons of data, but we can only interpret a small fraction of it without reference genomes.”

Stepanauskas has an ambitious goal: to create a marine parallel to the Human Genome Project — with one big difference. Instead of mapping the genetic code of one organism, he aims to create a comprehensive global database that contains the genomes of all marine bacteria and phytoplankton on the planet.

This database will provide the reference necessary for the marine microbiology research community to take full advantage of the opportunities omics offer. The hub of this work is the Single Cell Genomics Center, the research and service center Stepanauskas directs at Bigelow Laboratory. It is the first facility of its kind, and uses techniques pioneered at Bigelow Laboratory to read the genomic blueprints of individual cells. In the decade since the facility opened, Stepanauskas’ team has analyzed tens of thousands of cells from environments as diverse as soils, seawater, the International Space Station, and gut contents of bees and mice.

Their ocean microbe database project was launched through funding by the Simons Foundation, and Stepanauskas’ team is currently working to sequence the surface layer of the tropical and subtropical ocean.

Though the aim of this effort is large, the samples needed to complete it are surprisingly small. Omics methods allow Stepanauskas to extract genomic information for thousands of organisms from a single drop of seawater.

The first sample came from Bermuda. About 15,000 genomes were produced from that one drop, equal to the number of microbial genomes sequenced by the entire world in 2014.

“That’s an indication of how fast technology is moving forward in this field,” Stepanauskas said. “We can do things now that were completely impossible just a few years ago.”

The ocean microbe database is already more extensive than that of the human genome. Stepanauskas' team has the capacity to complete it in two years, creating a global reference database from tens of thousands of genomes. This speed is especially staggering when compared with the traditional laboratory cultivation techniques that were the bread and butter of marine microbiology before omics existed.

“The sun will turn into a red giant before we can do this with cultivation techniques,” Stepanauskas said. “Omics is what makes this challenge possible.”

Stepanauskas also hopes that this project will clarify fundamental questions about how microbes evolve. In addition to sharing genes “vertically,” the way human parents transfer genes to their children, microbes also transfer genes “horizontally” between one another in the environment.

This process creates complex family trees and drives microbial evolution. Understanding horizontal gene transfer will shed light on microbial diversity, help redraw the lines between species, and could even redefine what a species is.

“In a given drop of water, does horizontal gene transfer happen once in a million years, or every minute?” Stepanauskas asked. “We can’t answer these relatively simple questions yet, but single cell genomics gives us the tools to do so.”

Unlocking the Iron Microbiome

Senior Research Scientist Dave Emerson can learn a lot from a dab of mud or a teaspoon of water — with potentially enormous impacts for oceanography, industry, and human health.

Complex ecosystems exist at scales large and small, and that scale determines the tools at a scientist’s disposal. A forester can visit a stand of trees and count each pine, ash, and beech. Microbial ecosystems, where Emerson’s research takes place, do not afford the luxury of being able to see your study subject.

Emerson studies the iron microbiome, the world of microbes that process iron for energy. Just as a forest is a community of species, the iron microbiome is a complex community of iron-processing microbes that interact with one another and their environment. These microbes are crucial to chemical processes in places as diverse as underground aquifers and deep-sea hydrothermal vents.

Emerson uses omics techniques to study how these microbes are related to one another and understand the roles they play in their environment. From a small water sample, he can extract and analyze the genetic information of all the microbial species in a community.

“The beauty is that it’s easy to go out and take a sample of water,” Emerson said. “These techniques give you an inexpensive, broad snapshot of the community.”

This work is at the core of omics: studying the link between a microbe’s physiology and potential function in its ecosystem. Emerson essentially asks two questions of every sample: who lives in the environment, and what do they do there?

One of the prime tools at his disposal is a gene referred to as “16S.” All microbes — and humans — have this gene, but the exact code is unique to each species.

Identifying the 16S genes in a sample is like taking attendance in a classroom. Finding all versions of 16S present tells Emerson which microbes live in the sampled environment. To determine their roles, Emerson uses multiple omics techniques, including sequencing individual cells in Bigelow’s Single Cell Genomics Center.

Together, these approaches are revealing new microbial processes that exist on Earth — and possibly Mars, which is known as the “red planet” for its high iron content.

“Omics are incredibly powerful for answering a whole range of questions, on topics from ecology to physiology to biochemistry to evolution,” Emerson said.

Answering such questions about the iron microbiome will have far-reaching implications, including clarifying large-scale ocean and global climate processes. The amount of iron available in the ocean controls the growth of phytoplankton, microscopic plants that fuel marine food webs and influence gas exchange with the atmosphere.

“The iron cycle is incredibly important globally, and we know very little about it,” Emerson said. “Omics tools are letting us delve into the functional relationships that regulate it.”

Microbes are also essential to life outside of the ocean, and communities of microbes exist almost everywhere on Earth — including inside animals. All complex animals have microbiomes, where microbes living in the gut interact with one another and with the animal’s cells.

Learning how the relatively simple iron microbiome works can teach scientists about much more complex systems. Environmental omics studies have already helped to identify the human microbiome, one of the biggest advances in biomedicine in the last decade.

“Not only are omics revolutionizing our understanding of how our planet works,” Emerson said, “they are changing our whole view of what it is to be human.”