Fresh stacks of muddy bacteria

Lilia Garcia, Illinois Wesleyan University

The Approach: In my last post, I wrote about Gracilaria, an invasive red seaweed on the coast of South Carolina, and its effect on Vibrio bacteria. My project aims to record the number and strains, or types, of Vibrio growing around Gracilaria and compare it to seaweed-free areas. I will also compare the Vibrio count residing on Gracilaria versus the Vibrio residing on a native seaweed called Ulva to see how an invasive species changes the bacterial community. Lastly, I want to understand how Gracilaria stops the growth of specific Vibrio strains by producing chemical compounds.

Mud samples under Gracilaria, taken by K. Coates

To begin solving my questions, I will go out to collect samples in the mudflats outside of the Grice Laboratory. I will collect tubes of water, clumps of Gracilaria and Ulva, and mud from underneath and 1.5 feet away from Gracilaria. Afterwards, I’ll spread all the samples onto dishes with nutrients specifically used to grow Vibrio. The bacteria grow in spots called colonies, and I will count each spot to see how much Vibrio there is in each sample. I am looking for a different amount of colonies in mud samples collected within or away from Gracilaria patches, and a difference in colony numbers between the Ulva and Gracilaria.

Dishes of unique Vibrio, taken by L. Garcia

A single dish from a mud sample can contain hundreds of colonies, differing in color, shape, size, and texture. Each of these colonies represent a different strain of Vibrio, uncovering the diversity of bacteria at different distances from Gracilaria. I will characterize which unique colonies are dangerous to human health, and whether they are found near or away from Gracilaria.

Zones of inhibition against Vibrio strain, taken by L. Garcia

As previously mentioned, I will also test Vibrio strains against chemical compounds made on the surface of Gracilaria. These compounds are able to control the kind of bacteria that grow around seaweed, changing the microscopic habitat. I will mix Gracilaria with chemicals to remove its surface chemistry, then spot the compounds onto dishes growing Vibrio from my mud samples. I am looking for large clear circles, called zone of inhibitions, that tell me the specific strain of Vibrio cannot grow due to the compound.

Nearly all we know about the ecological and economic impact of Gracilaria focuses on large animals, such as fish. My project zooms in on micro-organisms that have been overlooked. The information I collect will help us understand how invasive Gracilaria is changing bacterial communities not only in the Charleston Harbor, but potentially the entire coast.  Although invisible, bacteria make up the foundation of ecosystems and high Vibrio levels may be dangerous for our health. I look forward to finding the answers to my questions hiding quietly in the mud.

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

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Cloning our way to a perfect sequence

Kelsey Coates, Duquesne University

The Approach: In my first blog post, “FROM FEMALE TO MALE – MUD SNAILS TELL ALL!,” I described the goal of my research, to sequence isoforms of a hormone receptor called the Retinoid X Receptor (RXR) in the eastern mud snail.  

Mud snails all over a beach at Fort Johnson, SC.

These isoforms have yet to be sequenced in the mud snail! But what exactly is a DNA sequence? DNA is made of building blocks called nucleotides. A DNA sequence is the order of the nucleotides. A sequence like ACG could tell the organisms’ body to do one thing while a sequence like AGC could tell the organisms’ body to do another. A bit of the sequence has already been identified, but there is a gap in the sequence we are still trying to figure out.  

Theoretically, different chemicals or different concentrations of the same chemical can change the relative levels of the RXR isoforms. If this hypothesis is confirmed, mud snails can be used in the future to detect contaminants that affect marine organisms in the Charleston Harbor. Their patterns of isoform expression might suggest which seasonal contaminants are present in the environment where they live. For example, chemical one may trigger isoform A which has sequence ACG while chemical two may trigger isoform B which has sequence AGC.            

So how will we get these sequences? It starts with amplifying the known sequence of the mud snail that surrounds the isoform, including the mysterious gap. Amplification will be done by polymerase chain reaction (PCR) to ensure there are thousands of copies of the DNA to work with. After purification, the sequence is ready to be incorporated into a plasmid along with an antibiotic resistance component. Bacteria, like E. Coli, store their DNA in plasmid form compared to the double-helix form of humans.

Plates of E.Coli in the presence of Ampicillin set in the incubator.

Luckily for us, plasmids are easily manipulated and are reproduced rapidly in bacteria. E. Coli will be grown in the presence of the antibiotic ampicillin with the sequence we cloned into its DNA.  If the sequence is incorporated into the plasmid, the bacteria will have anti-biotic resistance and be able to grow on the ampicillin plates. The bacterial colonies with our plasmid will be PCR amplified. Then, after a final plasmid preparation, the samples from E. Coli can be sent to a lab that specializes in sequencing. Hopefully the lab will identify the gap and we will achieve our goal!

ACKNOWLEDGEMENTS

I would like to acknowledge Dr. Demetri Spyropoulos, Edwina Mathis, Dr. Bob Podolsky, The Fort Johnson REU Program, The Hollings Marine Lab, NOAA, and The Grice Marine Lab. This research was supported by the Fort Johnson REU Program, NSF DBI-1757899.

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.

Meddling with Mysterious Macroalgae

Pressley Wilson, University of South Carolina Aiken

The problem: If you have ever been in the ocean, you have probably come across a piece of seaweed, which is a type of macroalgae. Macroalgae are simply algae that can be seen without a microscope. These organisms undergo photosynthesis, produce carbon, and can reduce the levels of phosphates and nitrates in water (Champagne et al. 2015).

Although algae are one of the most important parts of marine ecosystems, the algae microbiome (the bacteria that live in and around algae) is highly unknown and further research is needed to uncover this critical macroalgae information. Is there a relationship between bacteria and algal species? Is there a relationship between the algae’s physical features and the bacteria? Or is the microbiome the same throughout the algae, regardless of variation in species and physical features?

In order to answer these questions, I am conducting a research project this summer looking at the bacteria that are associated with intertidal macroalgae from One’ula Beach, Hawai’i.

Intertidal region of One’ula Beach, Hawaii

The five species below were chosen from the intertidal region of One’ula: Asparagopsis taxiformis (1), Avrainvillea sp (2), Halimeda discoidea (3), Padina sanctae-crucis (4), and Dictyota sandviscensis (5). These species were chosen because they range from red, brown, and green algae; have varying physical features; and all currently grow on the intertidal region of One’ula Beach.

Macroalgae from One’ula Beach, Hawaii (Photo credit: Dr. Heather Spalding)

Asparagopsis, Avrainvillea, and Dictyota are uncalcified, Halimeda is calcified, and Padina is lightly calcified. Asparagopsis has fluffy upright filaments, Avrainvillea and Padina have a fan-shaped thallus, Halimeda has flattened segments, and Dictyota has dichotomous branches. All species are native to One’ula Beach, Hawaii, except Avrainvillea which is an invasive species, meaning it is not native to the area. These species represent a diverse array of brown algae (Padina and Dictoyta), green algae (Avrainvillea and Halimeda) and one red alga (Asparagopsis).

This research will lead to a better understanding of algae, which could lead to a better understanding of all photosynthetic marine organisms. Furthermore, this research will be used as preliminary results for Dr. Heather Spalding’s work in the Northwestern Hawaiian Islands determining if there is a relationship between spatial patterns and the algae microbiome, beginning in August.

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. 

References

Champagne P, Hall G, Liu X, Wallace J, Yin Z. 2015. Determination of Algae and Macrophyte Species Distribution in Three Wastewater Stabilization Ponds Using Metagenomics Analysis. MDPI – Water. 7(7): 3225-3242.

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).

Expect the Unexpected in Science

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Alessandra Jimenez, Whitworth University

As this internship has recently come to an end, I now begin to reflect on the wonderful yet challenging experience I had conducting observational research on Atlantic brown shrimp (Farfantepenaeus aztecus). In the last few weeks of this 10-week summer program, there was a fascinating yet unexpected turn of events. In particular, results of the experiment pointed to conclusions that I initially found myself unprepared for!

In summary, the focus of this experiment was to test effects of immune response on the ability to escape predators in shrimp. The escape mechanism, called tail-flipping (see video below) is actually powered anaerobically. However, recovery from this energetic behavior absolutely requires oxygen (is aerobic). As further explained in previous blog posts (click here and here), a recently discovered consequence of mounting an immune response against bacterial infection involves depression of aerobic metabolism. So, my mentor and I decided to focus on the recovery aspect (aerobic) of the escape response and predicted that this aerobic process would be impaired in shrimp injected with bacteria. At the same time, we predicted that the anaerobic part of this mechanism would be significantly impacted.

A slow-motion video of an Atlantic brown shrimp juvenile tail-flipping in an experimental tank (c) Alessandra Jimenez

The last few weeks of the internship mainly consisted of analysis, arriving at conclusions, and publicly reporting results. After testing tail-flipping ability (click here for an explanation of how this was tested) in a total of 42 shrimp juveniles, 30 of these were chosen for final analysis. Using a statistics software called Sigmaplot (version 12.5), I conducted tests that basically compared experimental groups based on the two variables I investigated: treatment type (bacteria or saline) and time given after injection (4 or 24 hours). Afterwards, results were deemed important based on significance values assigned by these Sigmaplot tests.

Significant results were very surprising!  Overall, results suggested that metabolic depression (indirectly caused by the immune response) did not have an impact on recovery (aerobic). At the same time, the most unexpected finding of all suggested that bacterial exposure actually increased anaerobic tail-flipping activity in Atlantic brown shrimp juveniles! Thus, this result called for a complete change in focus from the aerobic part to the anaerobic part of this particular escape response.

So, how could I possibly explain the increase in anaerobic processes found through this experiment? After much pondering and going through scientific literature, I formulated a new hypothesis. An important enzyme in crustaceans called arginine kinase is involved in the storage and creation of anaerobic energy that can be used for tail-flipping. Recent studies involved injecting bacteria into live crustacean tissue and comparing arginine kinase expression levels with controls. Results indicated a significant increase in expression in bacteria-injected tissue, especially in abdominal muscle (important for tail-flipping!). Based on these investigations, I now think that there may be a link between immune response and levels of anaerobic metabolism. Further research is required to explore this.

The final stages of the internship included creating and presenting a Powerpoint presentation of our work, and submitting a manuscript of my summer investigation. Overall, this REU internship experience has been challenging yet exciting, and has confirmed my love for marine biological research. As I mentioned at the end of my presentation, “expect the unexpected in science”.

powerpoint presentation - REU 2015

Picture of me right before giving my Powerpoint presentation (c) Alessandra Jimenez

References:

Burnett, L. E., Holman, J. D., Jorgensen, D. D., Ikerd, J. L., & Burnett, K. G. (2006). Immune defense reduces respiratory fitness in Callinectes sapidus, the Atlantic blue crab. Biological Bulletin, 211(1), 50-57.

Gruschczyk, B., Kamp, G., 1990. The shift from glycogenolysis to glycogen resynthesis after escape swimming: studies on the abdominal muscle of the shrimp, Crangon crangon. J Comp Physiol B, 753-760.

Scholnick, D. A., Burnett, K. G., & Burnett, L. E. (2006). Impact of exposure to bacteria on metabolism in the penaeid shrimp Litopenaeus vannamei. Biological Bulletin, 211(1), 44-49.

Yao, C., Ji, P., Kong, P., Wang, Z., Xiang, J., 2009. Arginine kinase from Litopenaeus vannemai: Cloning, expression, and catalytic properties. Fish Shellfish Immunol 26, 553-558.

Many thanks to College of Charleston for hosting my project, Dr. Karen Burnett and Hollings Marine Laboratory for guidance and work space, and NSF for funding the REU program.

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