Marine viruses and microbes
Photos courtesy of Viral Ecology and Informatics Lab January 31, 2022
With UD’s research ship, team uses revolutionary technique to study oxygen-producing bacteria
If a person wanted to study gazelles in Africa, the last thing they would want to do is put a fence around them. For one thing, they wouldn’t be able to observe their natural behavior and, for another, the lions would have a field day.
According to University of Delaware Professor Eric Wommack, the same can be said for microbes in the ocean. Usually, when researchers study ocean microbes, they collect water samples in containers and bring the samples back to the lab. Just like fenced-in gazelles, this doesn’t allow them the chance to observe the microbes’ behavior in their natural habitat.
“If you take the water and you put it in a big container, you change it,” said Wommack, deputy dean of the College of Agriculture and Natural Resources (CANR) and professor of environmental microbiology. “There are actually protists, things that eat bacteria, and when you put everything in a bottle, the protists go crazy. It’s a real challenge to study processes in the ocean when you can’t watch the same environment over time.”
In an attempt to study microbes in their natural environment, co-chief scientists Wommack and Shawn Polson, an associate professor of computer and information sciences and director of the Bioinformatics Core Facility in the Center for Bioinformatics and Computational Biology (CBCB), led other members of the Viral Ecology and Informatics Lab (VEIL) in a National Science Foundation EPSCoR-funded (NSF grant no. 1736030) mission on the R/V Hugh R. Sharp, UD’s 146-foot ocean-going research vessel. They spent eight days at sea to conduct what is known as a Lagrangian Experiment, monitoring a single water mass over time. (Joseph-Louis Lagrange, who died in 1813, was an Italian-born French mathematician who excelled in all fields of analysis and number theory and analytical and celestial mechanics.)
Viral Ecology and Informatics Lab
The members of the VEIL involved in the experiment included Wommack; Polson; Barbra Ferrell, VEIL lab coordinator; Rachel Keown, doctoral student in biological sciences; Amanda Zahorik, doctoral student in biological sciences; and Amelia Harrison, VEIL technician and master’s degree graduate in marine biosciences. In addition, they were joined by Bruce Kingham, director of the UD Sequencing and Genotyping Center.
During their time on the Sharp, they monitored a single water mass and sampled the viral and bacterial host populations found in that water mass. In doing this, they looked to determine how virus and bacteria populations interact, by observing which groups became more or less abundant and how quickly those changes occurred.
They were specifically interested in looking at two photosynthetic bacteria that are abundant in the world’s oceans and key players in the world’s carbon cycle.
Known as Prochlorococcus (PRO) and Synechococcus (SYN), these two photosynthetic bacteria are responsible for the production of roughly 25% of the oxygen on earth that exists in the atmosphere. While PRO tends to be dominant in the deepest parts of the ocean, SYN tends to be dominant closer to the shore.
Because they provide 25% of atmospheric oxygen, they are important players in the global carbon cycle and many other nutrient cycles so anything that impacts the ecology of the two groups of organisms — such as the viruses — has an impact on these nutrient cycles.
Viruses and microbes in the ocean
The lab worked with MetOcean Telematics, a company that develops and manufactures state-of-the-art data acquisition equipment. Their current-monitoring buoys are typically used for remote oceanographic and meteorological research, but the buoy was also ideal for VEIL’s Lagrangian Experiment. The buoy was equipped with Iridium satellite telemetry, GPS and a holey sock drogue, a type of sea anchor, that stayed 15 meters down in the water. As long as the researchers followed the buoy, they knew they would be sampling the same water.
Usually, when research like this is conducted, researchers will go to the same spot in the ocean over time. When they do that, however, they are not sampling the same water because currents are constantly moving water around the ocean. In addition, viruses are produced and disappear quickly so by sampling one spot and not following the same patch of water, means that changes in viral communities could simply be a result of water movement and not changes in viral-host interactions.
“This type of experiment was really different,” said Ferrell. “We were able to follow a specific body of water around. Instead of collecting samples at the same place and watching currents bring different communities into our location, we were following and sampling the same water over several days. Hopefully, that will tell us more about the infection dynamics going on in that water body.”
Because the viruses in the water turn over once a day, in order to study them effectively, the researchers took samples every six hours around the clock. They did this for two and a half days in one location close to the shore to sample for SYN and one location on the far side of the Gulf stream, over 250 miles out to sea, to look for PRO.
Once they retrieved their samples, the researchers worked constantly, performing microbial DNA extraction on board the ship. They used PCR to produce multiple copies of specific DNA sequences that provide insight into the biology of viruses, and sequenced the DNA while at sea. The MinION device, a portable sequencer from Oxford Nanopore Technologies, is about the size of a tablet and generates data within hours. The group also brought along two high performance computational nodes that allowed them to begin the bioinformatics analysis of the data while at sea.
The researchers will now look at DNA from the bacteria and the viruses they collected as well as the RNA from bacteria.
“DNA tells us something about which bacteria and viruses are there,” Ferrell said. “RNA tells us something about proteins that are being produced, so it helps us understand what bacteria and viruses might be doing. It’s exciting to see viral proteins being produced, because that tells us about current viral infections in our samples.”
The research team credited the crew of the Sharp with helping to make the study so successful, especially when it came to locating the floating buoy. This allowed the team to focus on collecting and sampling.
Usually, this type of research could take months. By using the capabilities of the R/V Sharp, a regional class vessel in the University-National Oceanographic Laboratory System (UNOLS), the team was able to collect, filter and process the samples on the ship, retrieving sequenced data and processing it before they got back to land.
“We sped up our process from months to hours,” said Wommack. “It’s something that is pretty cutting edge for microbial oceanography, and I think it will be quite revolutionary. It was an amazing feeling as a scientist and as a microbial oceanographer to be able to leave the ship with sequence data.”
Master’s thesis gene identification
From that sequenced data, the researchers can identify the viral populations and determine if a gene they are looking at is specific to Cyanophages — viruses that infect cyanobacteria such as PRO and SYN.
The idea to look for a specific gene from a Cyanophage sprung from Harrison’s master’s thesis.
Harrison, who received a master’s degree from the College of Earth, Ocean and Environment (CEOE) and a bachelor’s degree from UD’s College of Agriculture and Natural Resources (CANR), has worked with Wommack and Polson since 2014, when she was a first year undergraduate student. She recently started a new position at UD as a bioinformatics trainer in the CBCB Bioinformatics Core.
Harrison’s master’s thesis looked at Ribonucleotide reductase (RNR), an ancient gene that has different requirements for survival and can be used as an environmental indicator. For instance, there are RNRs that like oxygen and ones that are totally inactivated by oxygen, so having a specific RNR present in an environment indicates whether that is an oxygen-rich or oxygen-depleted environment.
“We figured out that we could get an idea about a viral population, at least in the marine environment, by looking just at these RNRs,” said Harrison. “This is pretty incredible because viruses as a whole, there’s no gene that all of them encode. There’s no universal marker, which there is for a lot of other organisms.”
It’s known that moving from the coast to the open ocean means moving from SYN populations to PRO populations, but by using this gene as a marker, the researchers could learn whether moving from SYN to PRO also meant a change in the viruses themselves and whether those viruses were more present at certain times of the day — from high light to low light.
“We were interested in seeing: are the viruses so generalized that there is always going to be something that they can infect or do they need specific conditions?” said Harrison. “The advantage to this RNR is that, since it’s a protein-coding gene, it also tells us something about the virus itself.”