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

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Cells and Instruments, but no Folsom Prison Blues

Brian Wuertz, Warren Wilson College

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In my previous post, “Hiding in plain sight”, I introduced DOSS, a compound that has been recently identified as a probable obesogen. We are especially concerned about the potential of this compound to cause obesity symptoms in developing children through exposure from their mothers. While DOSS is in many products we use daily, such as homogenized milk and makeup products, it is commonly prescribed to pregnant women in the form of Colace stool softener. I am investigating both how much DOSS is in certain places in the body and how it may promote obesity.

One of the main concerns about obesity is that it elevates the risk of developing other diseases such as diabetes or cancer by causing a state of chronic inflammation (Bianchini 2002).  Chronic inflammation in  adipose tissue is regulated by immune cells, including macrophages. Macrophages are immune cells found throughout the body that help to fight against infection by recognizing invading bacteria and engulfing them in a process called phagocytosis, literally meaning to eat the other cells. In addition to phagocytosis macrophages are important regulators of the larger inflammatory response by secreting proteins that tell other cells to initiate or maintain a state of inflammation (Fujiwara 2005). This inflammatory reaction may be induced by DOSS. We have seen evidence of increased inflammation and obesity in mice treated with DOSS, so in order to figure out what causes that I am focusing on macrophages because of the way they regulate inflammation.

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I am isolating macrophages from breast milk samples under this hood in a sterile environment to make sure they are not contaminated with bacteria.

One way to study the inflammatory response of macrophages is to expose them to DOSS and then see if they produce the inflammatory proteins. Instead of trying to measure the secreted proteins, we can measure how much RNA is made in the cell. The RNA is the translator molecule that takes the plan for the protein from the DNA and makes it available for the cell to read and make the right protein. I identified genes for four different inflammatory proteins to measure the RNA so we can test if DOSS causes the macrophages to make more of any of them. I am testing macrophages that I am isolating from human placenta and breast milk tissue because the developing child is influenced by inflammation in the placenta and breast milk. Macrophages in these tissues could be the source of inflammation that influences how the child develops.

Okay so we have talked about cells, but what about the instruments? In my last post I introduced my instrument of choice, but did not call it that. It is not a guitar or a saxophone, but the HPLC, or high performance liquid chromatograph. This is simply a fancy instrument used to separate chemical compounds by forcing them through a tiny filter column filled with tiny beads. Some compounds stick more to the beads than others, so when you flow a liquid through the column the compounds come out of the column at different times. It is essential to separate the compounds in a sample because then you can measure the amounts of individual compounds.

We want to know where DOSS goes in the body, so we need to be able to measure how much of it is in a sample. I am working to get a system up and running to measure the amounts of DOSS in samples from different cells and tissues. We want to be able to measure DOSS in humans and in marine mammals such as dolphins. Dolphins are exposed to DOSS in the COREXIT oil spill dispersal agent that is applied to large and small scale oil spill issues along coastlines and in harbors. Dolphins are an important sentinel species, meaning that they can provide insight into human health issues.

I have to prepare a column and get the right mixture of solvents to make DOSS come off of the column in a timely fashion and in a way that we can measure it. The measurement is actually done with a mass spectrometer, which measures allows us to identify the compound based on how much it weighs. The number of atoms and types of atoms in the compound determine the mass of the compound. This mass is how the instrument measures the compound. The technique I am using is therefore called liquid chromatography mass spectrometry or LC-MS and the instrument is also referred to by LC-MS. Hopefully by the end of the summer I will be able to find beautiful data with this instrument that will make a coherent tune rather than a jumble of notes.

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This is the MS part. It measures the mass of the compound and then breaks it apart and measures the mass of the pieces of the compounds and the amount of the compounds.

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This is the LC or liquid chromatography part of the LC-MS instrument. Most of the work is figuring out the best solvent system to the sample through the small column with the red tag on it.

Funding for this REU program is generously provided by the National Science Foundation and hosted by the College of Charleston. Dr Demetri Spyropoulos at the Medical University of South Carolina is graciously hosting my research project and providing mentorship.

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References:

Bianchini, F., Kaaks, R., and Vainio, H. (2002). Overweight, obesity, and cancer risk. The Lancet Oncology 3, 565–574.
Fujiwara, N., and Kobayashi, K. (2005). Macrophages in Inflammation. Current Drug Target -Inflammation & Allergy 4, 281–286.