Scientific Sleuthing

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

Thermoplasma acidophilum -a model organism to study iron stress in microbes?

Thermoplasma acidophilum. Linda Stannard, UCT/Photo Science Library.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.

Darwin’s 200th Annivesary

Picture of Charles DarwinToday is the birthday of Charles Darwin.  To me, Charles Darwin embodies the ideals of a scientist and a humanist.  I grew up in a household which did not believe in the evolution of life forms over time through natural selection.  The world was close to 6000 years old and we were approaching the end of the world.  The eminent destruction of the world as I knew it had a troubling effect on my being.

I was 13 years old when I first heard of the Theory of Natural Selection and was amazed and excited by this new finding.  When I asked about it, the answers I received were unsatisfactory.  As I learned about chemistry, radiation energy, and the structure of atoms I began to realize that the stories I grew up with had some serious holes.  After a while, I stopped asking those around me and took to reading textbooks I checked out of the public library near my house.  In those teenage years, I learned not to believe in an idea just because someone said it was true, but to investigate, analyze, hypothesize, and interrogate life until I was satisfied that the data explained my observations through an order set of laws.  These principles were what drove me to return to school to pursue science as a life long passion.

To negate the existence of evolutionary forces which drive the selection of traits which allow a species to survive environmental stresses is a self defeating position to hold.  To deny the environmental forces which drive the theory of evolution is akin to denying the forces driving the theory of gravity.  We see the results of evolution every time we ride a horse, take our dogs for walks in the part, and read about how a pathogenic organism has developed resistance to the most current drug regimens.

I hope that you celebrate Darwin Day with me and stop for a moment sometime during the day to appreciate the life and contributions of this great renaissance man.

Professor John M. Essigmann – Mentor and Friend – Wins MIT’s Dr. Martin Luther King Jr. Leadership Award

Professor John Essigmann was awarded the Dr. Martin Luther King Leadership Award tonight for his work over his tenure at MIT as an advocate for the minority community making students, faculty, and all other members of MIT feel welcome at MIT.  John is one of those people who you meet in life and are immediately comfortable with him.  I first met John when I was visiting MIT in the spring of 2000.  I had just been accepted to the Chemistry PhD program and was in Cambridge on the prospective student visiting weekend.  I sat at the dinner table with Professors John Essigmann and Cathy Drennan and had a great time at dinner.  John and Cathy made everyone feel comfortable and welcome to MIT.

The next time I met John was when I was a Teaching Assistant for 5.07, the Chemistry version of Biological Chemistry.  I got to know John and eventually asked him to be the chair of my Thesis Committee.  As time passed and I got to know John better, I realized what an amazing person he is.  He and Ellen, his wife, at Simmons Hall, a really cool undergraduate dormitory at MIT.  The things John does around MIT are just too numerous to list here.

John also works to educate students who suffer from economic necessity worldwide. He has worked as an educator in Thailand for over two decades, dedicating his time to teach students in Thailand on how to design and develop drug research programs that investigate and provide relief to diseases which affect  third world countries.

I can’t think of a better person to receive this prestigious award than John.  Kudos to you!

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