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.

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Algae Microbiome: The Hidden World

Pressley Wilson, University of South Carolina Aiken

The Approach: In my previous post, I discussed (1) the importance of an organism’s microbiome in relation to its health and (2) the importance of algae in marine ecosystems due to their ability in producing oxygen, removing nitrogen and phosphorus from water, and exchanging inorganic carbon.

Considering the importance of these two variables, this summer I am researching the relationship between the algae microbiome and algae species in One’ula Beach, Honolulu, Hawai’i.  

DNA extraction in progress. Photo credit: Dr. Heather Fullerton.

The objectives of my research project are:

  • Identify relationship between algae microbiome and algae species
  • Identify relationship between the microbiome with algae’s morphology

Prediction

This study will predict there is a variation in the microbiome between algae species, due to the different species characteristics, such as calcification.

Sample Collection

The five algal species of different morphologies were hand-sampled by Dr. Heather Spalding at the intertidal region of One’ula Beach. The algae samples were rinsed with artificial saltwater to remove dirt and loosely associated bacteria. After cleaning, each species were placed into (1) a micro-centrifuge tube with 0.5 mL of RNA-later or (2) a 15 milliliter conical tube with 1.5 mL RNA-later. The RNA-later is a DNA preservation agent, was used to stabilize the algae DNA. The samples were stored at 4°C overnight. This overnight incubation allowed the RNA-later to penetrate the bacterial and algal cells to the DNA. After this incubation all tubes were frozen at -20°C and shipped overnight on dry ice to the College of Charleston, South Carolina, where they were stored in  -80°C freezer until DNA extraction.

Sample Analysis a

A completed gel electrophoresis.

A MoBio Fast DNA Spin Kit was used to extract the DNA from the algal samples. This DNA is then tested by PCR to determine if bacteria are present on the algae. To determine the number of bacteria present, qPCR is used. This molecular biology technique is used to quantify specific genes in a sample.

The PCR samples will be analyzed using gel electrophoresis, a molecular biology procedure that uses an electrical current to separate the components of the sample DNA by size. The qPCR data will be compared to a known DNA standard to determine the number of bacteria in our samples and calculations will be performed using excel.

Acknowledgements

I would like to thank Dr. Heather Fullerton for her guidance and support with this project and Dr. Heather Spalding for her sample collection. This project is supported by the Fort Johnson REU Program, NSF DBI-1757899. 

Invisible Neighbors: How Gracilaria Changes Bacterial Communities

Lilia Garcia, Illinois Wesleyan University

The Problem: It only takes a walk along the mudflats to notice large patches of wiry, red seaweed. The seaweed is called Gracilaria vermiculophylla, an invasive organisms that is native to East Asia (SERC, 2019)  The seaweed is hard to miss, but its effects on the ecosystem are not easily seen. This summer I will be studying how Gracilaria affects a bacterial community invisible to the naked eye.

Mudflat with Gracilaria, taken by L. Garcia

According to previous studies, Gracilaria is found to increase the amount of a bacteria called Vibrio (Gonzalez, et al., 2014). This may not mean much at first, since most of us don’t think about microscopic interactions. Bacteria, however, are important in maintaining the health of complex environments like estuaries. They cycle and break down nutrients and organic matter, influencing oxygen, carbon, and nitrogen levels. An increase in one group of bacteria, such as Vibrio, can change these patterns. And like most of us know, bacteria tends to spread easily. There are a few strains, or types, of Vibrio, such as V. vulnificus, V. parahaemolyticus, and V. cholera, that are dangerous to human health. An increase in these strains may cause an increase in disease from swimming or eating infected food.†

Vibrio growing on petri dish, taken by L. Garcia

We known Vibrio levels increase with Gracilaria, but we do not know how this happens. We also don’t know if all Vibrio strains increase together, or if only a few strains grow. To understanding the relationship between Gracilaria and Vibrio, I will record how much total Vibrio and how many strains of Vibrio grow in and away from patches of Gracilaria. In order to preserve its own health, Gracilaria produces compounds that promote or stop organisms from growing around it (Assaw et al., 2018). These are compounds I will test against different strains to study the mechanism Gracilaria uses affect specific Vibrio levels. I want to see how the growth of each strain is affected by different extracts. Will the strains further away from the Gracilaria be unable to grow when exposed to a certain type of extract? Will other strains grow better with the extract?

We tend to think about invasive species on a large scale, assessing the damage it causes to other familiar animals and plants. The ecosystem relies on tiny, cellular organism and studying how bacteria changes leads to a deeper understanding of environmental health. An invisible community is changing as Gracilaria flourishes, and there is a lot left to learn about it. 

Acknowledgements

Thank you to my mentor Dr. Erik Sotka, and our collaborator Dr. Erin Lipp. I would also like to thank Dr. Alan Strand and Kristy Hill-Spanik for their supporting guidance. Lastly, thank you to Dr. Loralyn Cozy (IWU) for preparing me to succeed in the lab. All research is funded by Grice Marine Lab and College of Charleston through the Fort Johnson REU Program, NSF DBI-1757899

References

Assaw S, Rosli N, Adilah N, Azmi M, Mazlan N, Ismail N. 2018. Antioxidant and Antibacterial Activities of Polysaccharides and Methanolic Crude Extracts of Local Edible Red Seaweed Gracilaria sp. Malays Appl Biol. 47(4): 135-144. 

Fofonoff PW, Ruiz GM, Steves B, Simkanin C, & Carlton JT. 2019. National Exotic Marine and Estuarine Species Information System. 

Gonzalez D, Gonzalez R, Froelich B, Oliver J, Noble R, McGlathery K. 2014. Non-native macroalga may increase concentrations of Vibrio bacteria on intertidal mudflats. Mar Ecol Prog Ser. 505: 29-36.

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).

Haloarchaea: Life at the Brink

Ben Farmer, University of Kentucky

IMG_20180705_150556What comes to mind when you think of extreme environments? The freezing tundra of Antarctica, or maybe the fiery lava flows of a Hawaiian volcanic zone? Those particularly interested in marine science may think of the deep ocean, perhaps the Marianas Trench. Whichever drastic environment you think of, one fascinating thing ties all of these extremes together: life finds a way to thrive in each of them.

Earth is home to as many as 1 trillion species, and the bulk of them are microbes (Locey and Lennon 2016). Microbes that are adapted to live in conditions that are inhospitable to most life on Earth are called extremophiles. Archaea and bacteria, the two domains of life aside from eukaryotes, represent the majority of extremophiles. While archaea were long thought to be a type of bacteria since the two appear very similar, archaea are more closely related to humans. Archaea are an important model organism because they have forged a niche in just about every habitat imaginable. Hot springs in Yellowstone National Park were among the first locations where archaea were discovered and owe their vibrant colors to these microbes (Oren and Rodriguez-Valera 2001).

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Man-made salt pans of Bonaire, Dutch Caribbean, tinged pink by archaea. Halophiles dominate these artificial habitats. Credit: Benjamin van de Water, Flickr, 2009.

Haloarchaea are what I am studying this summer at the College of Charleston. Halo– is a prefix meaning “salt,” and haloarchaea are halophilic, or salt-loving. Perhaps the most famous location that haloarchaea have been found is in the Dead Sea – evidently not so dead after all. Haloarchaea are commonly found in water 10 times as salty as the ocean, in conditions known as hypersaline. Our goal is to investigate what adaptations have made that possible.

We know that the amino acid composition of halophiles is unusually acidic (Martin et al. 1999). Proteins of halophiles are therefore also unusually acidic, which allows their proteins to properly fold in hypersaline conditions. What we do not know is whether the expression of proteins can change at different salinities. Better understanding how proteins are adapted in haloarchaea lends itself to understanding extremophiles on a broader scale.

Mechanisms that allowed microbes to function in seemingly inhospitable environments were likely responsible for evolution of life on Earth (Rampelotto 2010). There are many habitats today that mimic extreme environments from both ancient history and current conditions on other planets, such as Mars. Martian soil is incredibly salty, a result of surface water that evaporated long ago (https://dornsife.usc.edu/labs/laketyrrell/life-in-hypersaline-environments/). Halophiles may have once lived in those hypersaline Martian waters. Therefore, knowledge that we gain about haloarchaea adaptations is valuable to our understanding of life both on Earth and elsewhere.


Acknowledgements

Many thanks to my mentor, Dr. Matthew Rhodes, who has introduced me to everything from cell culturing to python. This project is funded through the National Science Foundation and supported by the Fort Johnson REU Program, NSF DBI- 1757899.



References

Locey KJ, Lennon JT (2016) Scaling laws predict global microbial diversity. Proc Natl Acad Sci 113:5970–5975

Martin DD, Ciulla R a, Roberts MF (1999) Osmoadaptation in Archaea. Appied Enviromental Microbiol 65:1815–1825

Oren A, Rodriguez-Valera F (2001) The contribution of halophilic Bacteria to the red coloration of saltern crystallizer ponds. FEMS Microbiol Ecol 36:123–130

Rampelotto PH (2010) Resistance of microorganisms to extreme environmental conditions and its contribution to astrobiology. Sustainability 2:1602–1623

 

Bacteria in the Ocean? That Eat Iron??

Lauren Rodgers, Rutgers University

Version 2The problem: Have you ever asked yourself, what is iron? It is an element? A rock? Some weird orange-ish substance? Is it the tool that you use to get the wrinkles out of clothes? And what does iron even do? Does it just sit there? Does anything eat it? Can we make things out of it? Iron is one of the most abundant elements on earth, yet not many people know much about the important role it plays in our lives.

Iron is more than just an element, or something found within a rock. It’s a nutrient, something necessary for the growth and metabolism of almost every living organism on Earth (Hedrich & Johnson, 2011). In the ocean, iron is found in two different forms, ferrous iron or Fe(II), which is soluble in water, and ferric iron or Fe(III), which is insoluble in water (Hedrich & Johnson, 2011). Because ferrous iron is soluble it is the form of iron that can be used by most organisms in the water (Hedrich & Johnson, 2011). This ferrous iron, however, is limited in the ocean despite its abundance in the Earth’s crust. In fact, Fe(II) is present only in incredibly small concentrations, making it a major limiting factor of growth for all of the plants and algae in the ocean. This is important because these plants and algae serve as the base of many food chains, so if there is a limitation on the growth of these organisms, it affects every other organism throughout the food chain. Though iron is an extremely important nutrient for many living organisms, it is still not well understood. One of the least understood aspects is how iron specifically cycles through different marine environments. Does it ever change form? Does anything add iron to the ocean? Does anything take iron out of the ocean? These questions bring us to Zetaproteobacteria.

Zetaproteobacteria is a recently discovered class of iron-oxidizing microbes. This just means that the bacteria eat iron in the form of Fe(II) and produce Fe(III) as a waste product (Emerson et al., 2007; Chiu et al., 2017). In fact, these waste products can take on the form of hollow tubes, also called tubular sheaths, or twisted stalks that you can see under the microscope!

 

Zetaproteobacteria were initially described in 2007 near hydrothermal vents, utilizing the large concentrations of Fe(II) that were present in the fluid that spewed from the vents (Emerson et al., 2007).

Iron Mat

Iron mat composed of Zetaproteobacteria on a lava rock near the submarine Loihi volcano. (A. Malahoff, Hawaii, Loihi Volcano, July 1988)

How do Zetaproteobacteria relate to the cycling of iron? 

Zetaproteobacteria, with their role in eating iron and transforming it from its soluble Fe(II) state into its insoluble Fe(III) form may have an important role in the cycling of iron through the environment, functioning as an important source of iron removal.

Since their discovery, Zetaproteobacteria have also been observed in many other habitats, including coastal estuarine habitats with lower levels of iron, similar to that of Charleston, SC. (Laufer et al., 2017; Chiu et al., 2017). Our study will try to identify if these Zetaproteobacteria are present in the muddy soils around Charleston, as well as measure the levels of Fe(II) and Fe(III) in the rivers where these bacteria may be found.

 

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Hopefully, through the study of the distribution of Zetaproteobacteria across the globe, including the chemical characteristics of the different environments that they inhabit, we may get a clearer picture of how iron cycles in aquatic environments and the role that these Zetaproteobacteria play.


I would like to thank my mentor, Dr. Heather Fullerton, for guiding me through this research. 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.


References 

Chiu, B. K., Kato, S., McAllister, S. M., Field, E. K., & Chan, C. S. (2017). Novel pelagic iron-oxidizing Zetaproteobacteria from the Chesapeake Bay oxic-anoxic transition zone. Frontiers in Microbiology, 8(JUL), 1–16. https://doi.org/10.3389/fmicb.2017.01280

Emerson, D., Rentz, J. A., Lilburn, T. G., Davis, R. E., Aldrich, H., Chan, C. S., & Moyer, C. L. (2007). A novel lineage of proteobacteria involved in formation of marine Fe-oxidizing microbial mat communities. PLoS ONE, 2(8), e667. https://doi.org/10.1371/journal.pone.0000667

Hedrich, S., Schlömann, M., & Johnson, D. B. (2011). The iron-oxidizing proteobacteria. Microbiology,157(6), 1551–1564.

Laufer, K., Nordhoff, M., Halama, M., Martinez, R. ., Obst, M., Nowak, M., … Kappler, A. (2017). Microaerophilic Fe(II)-oxidizing Zetaproteobacteriaisolated from low-Fe marine coastal sediments – physiology and characterization of their twisted stalks. Applied and Environmental Microbiology, 83(February), AEM.03118-16. https://doi.org/10.1128/AEM.03118-16

Mori, J. F., Scott, J. J., Hager, K. W., Moyer, C. L., Küsel, K., & Emerson, D. (2017). Physiological and ecological implications of an iron- or hydrogen-oxidizing member of the Zetaproteobacteria, Ghiorsea bivora, gen. nov., sp. Nov. ISME Journal, 11(11), 2624–2636. https://doi.org/10.1038/ismej.2017.132

The BMA of Today

Christine Hart, Clemson University

2017-06-22 10.29.36

In previous blog posts I described the sand-dwelling microalgae, also known as benthic microalgae (BMA), which are essential to estuary ecosystems. Not only do they produce the air we breathe and food we eat, they also inform us about the subtle changes that are occurring in our environment. Changes that otherwise may go unnoticed.

How do BMA show these environmental changes? By forming the foundation of estuarine energy, they provide a snapshot of how the estuary is functioning as a whole. If changes occur in BMA patterns, this may indicate changes in the overall ecosystem. BMA are also easily characterized and compared using modern molecular approaches. These qualities make BMA living indicators, or bioindicators, that are important in monitoring future ecosystem health.

BMA become visible in the upper layers of sediment at low tide. Later, they decrease in density—or biomass—as the tide rises. Our project studied the mechanism for the increase of biomass during low tide. Previous studies suggested that the mechanism for biomass increase is vertical migration of BMA from lower layers to upper layers of sediment. We also tested whether BMA growth due to high light exposure contributes to the biomass increase.

Our results indicated that both vertical migration and growth due to sunlight exposure were important to the increase in biomass. This is the first contribution to literature that recognizes a multifaceted approach to BMA biomass changes.

Additionally, we studied in how the biomass increase was connected to patterns in the type of BMA in Charleston Harbor. Previous studies suggested that increasing biomass was connected to changes in the abundance of BMA species; therefore, we expected to see the amount of certain BMA species change based on their exposure to migration and sunlight.

We were surprised by our findings. In this study, we found that BMA did not vary over short time periods (by tidal stage or by exposure to migration and sunlight). Instead, we found that BMA varied spatially and over a period of 6 years. In fact, only one of the dominant species of BMA remained the same from 2011 to 2017 (Figure 1).  The long-term change in community coincides with geological changes in the sampling site (Figure 2).

QualitativeLvM-MS

Figure 1. The relative abundance of each dominant BMA species from 2011 to 2017 is shown immediately after sediment exposure (T0) and 3 hours later (TF). Only one species—Halamphora coffeaeformis—remains dominant in 2017. This is evidence of a dramatic change in the dominant type of BMA in Grice Cove.

These are positive results for the use of BMA as bioindicators. If types of BMA are invariable over short periods of time, measurements of BMA will be more precise. Bioindicators must be capable of showing changes that are occurring on a larger environmental scale; therefore, it would be a good sign if the change in BMA community reflects the changing geological environment (Figure 2). Still, more studies on the temporal and spatial patterns of BMA communities should be conducted before BMA can be used as bioindicators.

Changes in Grice Cove

Figure 2. Aerial view of Grice Cove sampling site over time. The approximate location of the sampling site is shown by the white line. Sampling sandbar has changed over time, possibly contributing to community changes. Source: “Grice Cove” 32 degrees 44’58”N 79 degrees 53’45”W. Google Earth. January 2012 to March 2014. June 20, 2017.

This study contributed new information to the studies of BMA biomass during low tide, and showed that the BMA of today in Grice Cove are significantly different than in previous years.

 

Thank you to my mentor, Dr. Craig Plante, and my co-advisor, Kristina Hill-Spanik, for their support and guidance. This project is funded through the National Science Foundation and supported by College of Charleston’s Grice Marine Laboratory.

 

Literature Cited:

Holt, E. A. & Miller, S. W. (2010) Bioindicators: Using Organisms to Measure Environmental Impacts. Nature Education Knowledge 3(10):8.

Lobo, E. A., Heinrich, C. G., Schuch, M., Wetzel, C. E., & Ector, L. (n.d.). Diatoms as Bioindicators in Rivers. In River Algae (pp. 245-271). Springer International Publishing. doi:10.1007/978-3-319-31984-.

MacIntyre, H.L., R.J. Geider, and D.C. Miller. 1996. Microphytobenthos: the ecological role of
 the “Secret Garden” of unvegetated, shallow-water marine habitats. I. Distribution, abundance and primary production. Estuaries 19:186-201.

Rivera-Garcia, L.G., Hill-Spanik, K.M., Berthrong, S.T., and Plante, C. J. Tidal Stage Changes in Structure and Diversity of Intertidal Benthic Diatom Assemblages: A Case Study from Two Contrasting Charleston Harbor Flats. Estuaries and Coasts. In review.