How to Train Your Shewanella

Katherine Mateos, Carleton College

The Approach: In my previous post, I introduced my project, investigating the role of Antarctic bacterium, Shewanella BF02, in the cycling of volatile organic sulfur compounds (VOSCs). 

Sterile technique in action Photo Credit: Peter Lee

The first order of business in this effort is keeping the Shewanella alive and happy. In order to do this in the lab, I make a liquid (known in the biology world as “medium”) for the Shewanella to live in. Our medium is designed to resemble Blood Falls in chemical makeup. In particular, it is very salty, and contains iron and sulfate. I am also careful to remove all the dissolved oxygen in the medium, since the Blood Falls water has very little oxygen. In my medium, I am also careful to keep out any bacteria other than my Shewanella. Since microbes are everywhere, including in the air, on my skin, and on the lab bench, I  use a special set of techniques to avoid unwanted bacteria from infecting my samples. 

Membrane Inlet Mass Spectrometer

Once we have a perfect mix of chemicals for Shewanella, I also add my target organic sulfur compounds. Because I want to see if Shewanella changes these added compounds, I keep track of them using a technique called isotope labeling. Isotope labeling is a clever trick, where the target compounds are tagged with atoms that are the tiniest bit heavier than the ones that we usually see. If Shewanella make the labeled compounds into the VOSC products that I am interested in, those products will also have the same tag, making it easy to identify them.

To identify the tiny differences in mass between tagged and untagged molecules, I use a piece of equipment called a mass spectrometer. A mass spectrometer works kind of like a scale and can determine the mass of each molecule. This allows me to detect isotopically labeled VOSC products. If I see isotopically labeled products, I can be pretty sure that the Shewanella are cycling the labeled compound that I added to their medium. 

Thank you to my mentor, Dr. Peter A. Lee, and our collaborators, Dr. Jill Mikucki and Abigail Jarratt, for their guidance in the research process. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.

Advertisements

Shewanella: Sneaky Sulfur Cyclers?

Katherine Mateos, Carleton College

Like Shewanella, I too thrive at cold temperatures!

Life can survive almost anywhere! From hot pools on volcanoes to beneath ice-cold glaciers, pretty much all of the inhabitants in these hostile environments are so small that you cannot see them with your bare eye. These extremophiles, as they are often called, include tiny single-celled microbes—bacteria and archaea. By studying tiny microbes we can answer big questions: How did life begin on Earth?  How can we find life on other planets? How will our planet respond to its changing climate?

Outflow of Blood Falls on the Taylor Glacier. Image credit: Dr. Jill Mikucki.

This summer, I am working with one of these extremophiles, a type of bacteria separated out from a sample from Blood Falls, Antarctica. This lake is a pool of brine (very salty water) covered by more than 150 feet of ice from the Taylor Glacier. Blood Falls gets its name from the bright red stain that the brine leaves on the Taylor glacier as it leaks out from beneath the glacier. As you would expect, this location is cold and dark, but the chemicals in the brine are what truly make this ecosystem extreme. For one, Blood Falls is super salty, over twice as salty as the ocean. Most water has oxygen trapped within it, but Blood Falls has very little. Two important chemicals are also found in unusually high quantities: iron and sulfur.

Electron Microscope image of Shewanella BF02. Image credit: Bruce Boles.

The bacteria that I am studying makes good use of the iron in this environment. Like a battery produces energy from a variety of chemical reactions, Shewanella (strain BF02) gets most of its energy by harnessing the energy that is released when one chemical form of iron changes to another. However, there might be another source of energy Shewanella can live off of—perhaps a chemical that contains sulfur. Sulfur is one of the most common elements on earth, found in pesticides, foods, and in humans. Sulfur can form compounds with other common elements including hydrogen, carbon, and oxygen. Some of these chemicals, known as volatile organic sulfur compounds (VOSCs), easily evaporate into our atmosphere and affect our environment. We want to know if the Shewanella are creating these VOSCs, and if they do, what chemicals the Shewanella turn into VOSCs.

The strain of Shewanella that I am studying is from an extreme ecosystem but similar Shewanella are found throughout many ocean ecosystems. We can treat Blood Falls as a model to learn about the way that our oceans will affect our environment.  Even though Shewanella are too small to see with your bare eyes, figuring out what compounds they break down can help us understand the future of the environment around the world.

Thank you to my mentor, Dr. Peter A. Lee, and our collaborators, Dr. Jill Mikucki and Abigail Jarratt, for their guidance in the research process. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899.

References

Mikucki, J. A. et al. A contemporary microbially maintained subglacial ferrous ‘ocean’. Science 324, 397–400 (2009).

Sievert, S. M., Kiene, R. P. & Schulz-Vogt, H. N. The sulfur cycle. Oceanography 20, 117–123 (2007).

Zetaproteobacteria: The Journey Continues

Lauren Rodgers, Rutgers University

20180724_141800

Findings: This past summer, we have been working furiously with one goal in mind: measuring the concentrations of iron in the sediments around Charleston to identify if they would be a good habitat for Zetaproteobacteria. We scoured Charleston for the perfect muddy sampling sights and battled the pluff, almost losing a few boots in the process, while also gaining lots of bug bites. We then spent hours in the lab, making solutions, extracting iron, running the spectrophotometer, and collecting data.  And now, after 10 long weeks, it is coming to an end.

This summer of researching Zetaproteobacteria with Heather, Alejandra, and Sarg has taught me so many things. One of which is that science does not happen overnight. You may have an idea of how you are going to do something, but when it comes to actually carrying it out, odds are that it will not go as you think. Science is a dynamic process. You are trying things out, failing, brainstorming for other ways to do things, revising methods, and most of all learning. And this part of the process is what makes it exciting. I experienced this first hand when it came to conducting the ferrozine assay. Much of the research detailing methods for a ferrozine assay were only written for liquid samples, not for sediment samples, so we had to come up with methodology for extracting iron from the sediments. As you can imagine, this took a lot of trial and error. Trial and error such as figuring out the hard way that the water you are using to make up the solutions is actually contaminated with iron, or that glass cuvettes tend to contain their own concentrations of iron in them as well. It was frustrating at times,  but this process was so important for me because it was one of the first times that I was able to take in ideas from many different sources and develop something of my own. It was hard work, but it was all worth it in the end. We were able to collect samples, successfully extract the iron, and measure the iron concentrations, gaining some very exciting results in the end.

What’s next for Zetaproteobacteria?

Now that we have optimized the ferrozine assay for measuring the concentrations of iron in the sediments, we can continue our research on Zetaproteobacteria. The first objective that we will work towards is identifying if Zetaproteobacteria are actually present in the sediments. If they are found, we will then quantify how many Zetaproteobacteria are actually present.

If Zetaproteobacteria are found in the sediments around Charleston, it could have many implications. The first implication is that they could be affecting the local iron cycle around Charleston through their transformations of Fe(II). Zetaproteobacteria have also been shown to be able to live on solid metal and use the Fe(II) present in it, quickening the metal’s rate of rusting. If they are found in Charleston, they could be speeding up the rusting of ships or even metal pipes. Lastly, their presence in Charleston would add evidence to their potential worldwide distribution.

The project from this summer may be finished, but the Zetaproteobacteria journey has just begun!

IMG_3784

The Fullerton Lab Team stopping to pose while sampling at Kearns Park on the Wando River. (p.c. Heather Fullerton)


I would like to thank my mentor, Dr. Heather Fullerton, for guiding me through this research, and my Fullerton Lab members for assisting me in the field and in the lab throughout the summer. I would also like to thank the National Science Foundation for funding this research as well as the College of Charleston and Grice Marine Lab for their support.


 

To B12 or not to B12, that is the question…

Bryce Penta, University of Notre Dame

As the summer draws to an end, so too does this segment of my research with phytoplankton and vitamin B12. After completing three separate experiments, my project has finally reached its end.

One experimental design encompassed the first two experiments and used a mixed community of phytoplankton straight from the ocean while aboard the R/V Savannah, while the other relied on a culture of Phaeodactylum tricornutum, a phytoplankton species, that had all bacteria removed in laboratory settings. The first pair of experiments were conducted with an addition of both vitamin B12 and nitrate and the final experiment implemented a limitation of the same two nutrients. I specifically looked at the effect of varying availability of these nutrients on the photosynthetic efficiency and growth of the cultures.

IMG_5851

Photo confirming the lack of bacteria in the phytoplankton cultures in lab. The picture also shows that the cultures contained two body forms of Phaeodactylum tricornutum. Photo credit: Lena Pound

This study proposed that an increase in the availability of these nutrients would lead to an increase in efficiency and growth, as well as a decrease leading to a lower efficiency and growth. While we expected an effect of vitamin B12 on the phytoplankton functioning, in all three experiments B12 lacked any significant effect; however, nitrate showed a strong effect on the photosynthetic efficiency and growth in all experiments except the deep sea boat experiment.

While only nitrate exhibited a significant effect on the phytoplankton, this could be due to an alternate metabolic pathway that can bypass the need for vitamin B12. Using methionine synthase E (MetE) rather than the more efficient methionine synthase H (MetH) that requires vitamin B12, the Phaeodactylum tricornutum cultures functioned properly in the absence of vitamin B12 (Helliwell et al. 2011). Unlike the laboratory experiment, the ocean experiments may have lacked a B12 response due to microbes in the water already producing more than enough of the nutrient. Vitamin B12 lacked significant response in our experiments, but other experiments with species that lack the MetE synthase that allows for proper functioning without vitamin B12. Possible B12 effects on phytoplankton could lead to better climate modeling as phytoplankton form the basis of one of the world’s largest ecosystems.

These past ten weeks have culminated in a project that I am proud to have worked on this summer. Though my time here has ended, the people I have met here and the relationships formed over the summer will continue on in the future.

IMG_5608

Lee lab on the R/V Savannah showing off the catch of the trip, a 55 inch wahoo. Photo credit: Bryce Penta

Acknowledgements

This project is possible due to funding from the NSF College of Charleston Summer REU program and the Grice Marine Laboratory. Project ideation and collaboration with Dr. Peter Lee and the Di Tullio lab from the College of Charleston. Lab space and facilities provided by the Hollings Marine Laboratory.

References

Helliwell, K.E., Wheeler, G.L., Leptos, K.C., Goldstein, R.E., Smith, A.G. (2011) Insights into the Evolution of Vitamin B12 Auxotrophy from Sequenced Algal Communities. Molecular Biology and Evolution 28 (10): 2921-2933.

Unknown-5Unknown-1Unknown-4