I have been thinking of how to measure data in our upcoming field tests for the soil regeneration project. We need to be able to monitor several environmental parameters during these tests, such as temperature, humidity, light intensity, soil moisture, etc. We also want to be able to do this remotely, either with a wireless setup, a gpms shield, or RFID sensors, and be able to store the data onsite for future use. I stated thinking of how to do this best, so I did some research and it seems that using an Arduino / Raspberry Pi using Dragino or XBee style setup will probably work. Stay tuned for progress.
I’ve always had a soft spot for detective novels. I even fancy myself a detective. Not like the ones you read about in pulp fiction made famous by Daly, Hamlet, and Spillane (to name a few), but a sleuth no less. I have spent the last couple of weeks playing detective in lab, you see. I am out on a hunt for molecules produced by these microbes I am studying. This type of investigative work is what attracted me to chemistry and biochemistry. At hand I have a set of known microbial metabolic pathways, a pretty good idea of what some of the intermediate and products are, a lot of chemistry knowledge, and a whole slew of really cool high tech tools.
The method is pretty straightforward. I feed my favorite microbe a known carbon source; it helps if I can radio-label a carbon or another atom on the substrate. I wait for a defined period of time, collect the media, and start the analysis. Step one: spin down cells, take off the supernatant, extract with dichloromethane, …
Yes, it is quite tedious, but many steps later, I have a small sample with metabolites extracted from the experimental microbial growth sample. Now the fun begins. First, what technique do I start with? The simplest is ultraviolet/visible (UV/vis) light spectra. What does the absorption spectra of your samples look like? Can you see something that resembles a conjugated backbone? How about an aromatic ring? Hints of structure here and there. But then you ask, does the sample contain one or many kinds of molecules? Can I somehow separate these molecules?
In this next step I might choose liquid chromatography (LC) or gas chromatography (GC). Which technique I use depends on certain properties of the molecules I am looking for, are they water-soluble? Are they volatile? For water-soluble molecules I select LC and for volatile molecules I select GC. Both of these separation techniques have a mass spectrometry instrument (MS) attached, allowing me to get a sense of the molecular mass of each separated compound. I inject and watch the molecules fly. Every peak I see reveals a wealth of information about the compounds ‘vitals’. What is the mass of the molecule? Does it fractionate into smaller defined compounds with a known mass? Do the fractionation patters I see match a compound previously characterized?
Next, I isolate and purify the compound using LC, collecting the fractions that have my compound of interest and use Nuclear Magnetic Resonance (NMR) spectroscopy to try to resolve ambiguities due to configuration around enantiomeric carbon atoms. I use proton NMR and carbon NMR and study the chemical shifts I see in my purified sample. From these set of data, I can deduce whether I have a ‘R’ or ‘S’ configuration around my enantiomeric carbon and a host of other structural relationships.
In the end, I have a larges set of ‘clues’ from which I will build a chemical structure of the compounds isolated from the microbial growth media. But that is not enough. In order to stake a claim to a new molecule and possibly a whole new molecular pathway, I have to present my findings to other chemists and microbiologists, convincing them that I have done my sleuthing well. It is very tedious work and most of the time you do not find a new molecule, but every once in a while, you isolate something, a molecule never seen before. The thrill of finding what has not been observed before, drives me to explore the secret metabolic pathways hidden in these microbial species.
Stay tuned to see how this tale turns out. HHH
It is 7:24 on the day before Thanksgiving. AC/DC is cranked up so loud my ears are starting to bleed and I have at least four more hours to go before I go home tonight. There are papers and three lab notebooks strewn all over my bench top. This is my fourth 18-hour day in a row. A crazy look in my eyes and a five-day growth on my face. A marathon set of experiments trying to decipher the growth curve of the community of deep earth microbes that I am trying to identify and characterize.
I started this morning over thirteen hours ago by coming in, turning on the gas to exchange the atmosphere in the anaerobic tent loading chamber, and promptly blowing out the seals in the CO2 regulator. You know that when this happens at 6:30 in the morning, it is not going to be a good day. I couldn’t find another one in the building (nor in friends labs in a couple of other buildings) so I settled in and waited until AirGas opened later that morning. Needless to say, it was closer to 1:00 in the afternoon before I got the tent back operational, putting me at least five hours behind schedule for an already jam packed day. So this is how I find myself with one more hour to wait while my microbial cells sit in the first incubation of many in a long protocol with which I will fix them in paraformaldehyde for later DAPI stain and FISH analysis.
Why do I do this? Why do I put in the long hours crazy hours? Because I love it. Plain and simple, that is the only answer I can give. I delve into the unseen, the unreal, the unknown world of extreme microbes, and try to make it visible, real, solving the mystery of who lives where and how the hell they do what they do in those most inhospitable places in which they thrive. I have to design an experiment with the full understanding that the equipment does not exist for me to do these experiments. I cannot go to a shelf and just pick up one of these and three of those and have some technician come over and set it all up for me. This is truly science driven by your capacity to design, invent, and assemble the equipment that you will need, while doing the experiments at the same time.
Sometimes I forget how incredibly crazy and out of this world what I do is. I get reminded of this when I try to explain what it is that I do to friends and family. I get this look of fear and awe when I explain that the things I study grow at 100-200 atmospheres, at temperatures between 50-75 degreed centigrade, and under acidic conditions so extreme it would peel the skin off your bones. Like I said, this is pretty cool stuff.
Well, I got to go now, the next cell wash and incubation is about to begin. While all this is extremely exciting, getting to that final answer is laborious, painstaking, and tedious. Still, I would not trade this life for any other one. This is what I am thankful for on this week. I get to do what I love (and sometimes get a little frustrated at) every day. Take care and have a great Thanksgiving.
Microbes require iron (Fe) as an essential element for growth and development. It has two environmentally stable oxidation states (II and III) readily participating in redox reactions covering a wide magnitude of biological electron transport and redox reactions including respiration, oxygen activation and binding, degradation of peroxides and superoxides, synthesis of DNA, proteins, and other organic molecules, and energy fixation pathways. At the same time, unregulated iron uptake can lead to toxicity, reactive oxygen species (ROS) and to inhibition of growth.
Microbes have evolved an iron storage mechanism used to store iron under limiting or environmental stress. The most studied system is that in Escherichia coli which produces three structurally and chemically related storage proteins; ferritins, bacterioferritins, and Dps (DNA-binding proteins during stationary phase). Ferritin and bacterioferritin are a tetracosameric structures capable of storing 2,500 and 1,800 iron atoms respectively. Bacterioferritin differs from ferritins in that they have an iron protoporphyrin IX (heme) at the interphase of each subunit. Dps is a dodecameric ferritin, which is induced under stationary phase of growth or by oxidative stress. These supramolecular structures help sequester iron in the cytoplasm and prevent toxicity of free iron in the cytoplasm.
Microbes living in low pH environments are subject to high concentrations of metals, in particular iron. How these microbes respond to metal stress is key to understanding how organisms control energy producing metabolic reactions in the cell. Recently, the genome of T. acidophilum was completed affording us a glimpse of the possible biochemical pathways responsible for the survival of this organism in an acidic environment, but analysis of the T. acidophilum genome did not reveal any molecular pathways for the production of known iron storage proteins ferritin, bacterioferritin, or Dps.
How does T. acidophilum manage the high concentrations of soluble iron, or other metals, liberated by its acidic environment? Does this organism have a novel iron storage mechanism? What are the cellular responses to stress to T. acidophilum cause by high metal concentration in the cytoplasm or by ROS? This is one of the questions that I am trying to answer using transcriptomics to look at differential gene expression of this Archeaon under varying iron and other environmental stress conditions.