Haloarchaea: Getting molecular

Ben Farmer, University of Kentucky

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The Approach: In my last post, I introduced the concept of extremophiles. My study organisms this summer, the haloarchaea, are extremophiles adapted to thrive in the most hypersaline environments on Earth. My goal this summer in Dr. Rhodes’ lab is to examine what makes these adaptations possible.

We know that the surface of proteins found in haloarchaea are not only acidic, but they are also negatively charged. Haloarchaea proteins likely use this negative charge to better compete with salt ions for water molecules and keep from unfolding in hypersaline water. Changes in the type of amino acids that make up haloarchaea proteins explains why their proteins are able to become abnormally acidic. To add another layer of molecular complexity, there are a set of molecules at play here called tRNA: tRNA mediate the transition from genetic code to proteins in the cell. We are investigating whether expression of certain tRNA corresponds with the change in overall acidity of haloarchaea proteins.

To test this hypothesis, first we needed to obtain haloarchaea species. The particular species I am analyzing is Haloferax sulfurifontis (or just Haloferax), which was isolated from a sulfur-rich spring in Oklahoma, USA (Elshahed et al. 2004). We created hypersaline solutions (called media) to grow the cells in, which in this case was tailored to support Haloferax. The most important part was providing enough salt to make the media hypersaline, which meant nearly 10x more concentrated than seawater. When the media was ready, we introduced cells of Haloferax in small quantities in a process called inoculation. Now that we had cells growing, we next determined which tRNA were present in their genomes with the help of the Genomic tRNA database (Lowe and Eddy 1996).

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Falcon tubes containing cultures of several archaea species. Growing archaea cells turn the media orange/red as a result of carotenoid pigments.

To begin tRNA analysis, we filled the wells of a plastic plate with an oligonucleotide (oligo, or short DNA sequence) for each tRNA gene we found in Haloferax. Readying the well plate involved suspending the dry oligos in water and then making sure that the concentration of DNA was the same for each mixture. A microarray was created by randomly placing 8 samples of each of the 40+ oligos on a larger plate, and then printing the results. Using 8 random samples ensured higher accuracy. The tRNA was then “labelled,” by incorporating a radioactive phosphate (32P) into the living Haloferax cells. Each tRNA oligo produced signals corresponding to how much they reacted to the radioactivity. These signals showed up as spots on the array, and the spots were directly proportional to abundance (expression) of tRNA. Through image analysis of the spot intensity, we determined the expression of each type of tRNA.

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Machine used to produce microarray results, courtesy of Dr. Renaud Geslain’s lab

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An example of the spots produced by radioactivity. Each black spot indicates a specific tRNA, and the intensity of the spot tells us the abundance of that tRNA in Haloferax. I aligned the yellow circles to the spots using imageJ software, which allowed me to quantify tRNA abundance.

Organisms require physiological adaptations to cope with environmental disturbances, and this often is apparent on the level of proteins. Amino acids are the building blocks of proteins, and mRNA provides the code for these building blocks. We are assuming that mRNA expression is changing in Haloferax to cope with extreme salinity, and it would follow that tRNA expression is changing in tandem. So, analyzing tRNA in haloarchaea provides us with a better of how organisms like extremophiles manage to adapt to the outer limits of environmental conditions on Earth.


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

Elshahed MS, Savage KN, Oren A, Gutierrez MC, Ventosa A, Krumholz LR (2004) Haloferax sulfurifontis sp. nov., a halophilic archaeon isolated from a sulfide- and sulfur-rich spring. Int J Syst Evol Microbiol 54:2275–2279

Grelet S, McShane A, Hok E, Tomberlin J, Howe PH, Geslain R (2017) SPOt: A novel and streamlined microarray platform for observing cellular tRNA levels. PLoS One 1–17

Lowe TM, Eddy SR (1996) TRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25:955–964

Reed CJ, Lewis H, Trejo E, Winston V, Evilia C (2013) Protein Adaptations in Archael Extremophiles. Archaea 2013:1–14

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