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Effect of environmental toxins on GATC methylation in E. coli May 3, 2011

Posted by ljsteele in Biology, Chemistry, Ecology, Environment/Conservation, Evolution, Genetics, Health, Marian University curriculum, Physiology.

With the end of the semester drawing near it is becoming that time again when the results are piling in from research you have been working on all semester. As we speak, the final data collection and analysis is taking place in biochemistry, a team of student researchers are exploring of environmental toxins of DNA methylation  in the bacterium E. coli. 

The Bacterial Genome

Bacteria exist throughout the world and can survive in almost any climate . Bacteria are unicellular and can consist in a wide range of environments such as a pond all the way to soil.  One unique attribute of the bacterial genome is that it contains adenine methylation , opposed to mammalian organisms which contain cytosine methylation at GpC islands.  Adenine methylation is when a methyl group becomes attached to the adenine nucleotide on the DNA. When a methyl group is donated from SAM to form a covalent attachment, it is made on the adenine which can cause steric hindrance of transcription factors and differential effects of DNA binding proteins, which can contribute to a change in gene expression.  In previous studies  it has been shown when E. coli is exposed to different carbon sources (ie glycerol or glucose).  Some areas of the genome become demethylated.  In the bacteria E. coli almost every adenine (A) in the GATC sequence is methylated.  To block the methyation at the GATC sequence, a protein must be present to inhibit the DAM methyltransferase from depositing a methyl group on the adenine.

What does Methylation do?

Adenine methylation has many roles in bacteria. Methylation can effect gene expression, cell cycle, virulence, and how proteins interact with the DNA. For the research we are performing, we are concerned with what effect the environment has on changing adenine methylation on the GATC repeats. There are about 20,000 GATC repeats in the E. coli genome and under normal log growth conditions almost every single repeat is methylated. It has been found that when bacterial cells are in a log growth phase there are 6-10 sites which are not methylated. These nonmethylated sites lie up and down stream of promoters of different genes. The lack of methylation may allow DNA binding proteins to modulate their function to allow a functional change in gene expression.

Pollutants and the Genome

In the study we are performing we wanted to see how three classes of chemicals pollutants commonly found in the Midwest affect adenine methylation at the GATC site. We choose three pollutants to represent chemicals that fit into the families of common water pollutants, which are heavy metals, chlorinated compounds and nitrogen rich compounds.Gel Electrophoresis

The above families of compounds will be compared to samples collected from different areas around the campus of Marian University, Indianapolis, IN. Supplements will be added to all the samples to generate a rich liquid media that will facilitate bacterial growth.  With 6 different test groups and 2 controls we are going to seek to determine if any of our known compounds or a compound present in our environmental sample has an effect on the methylation.  The determination of methylation can be done by using restriction enzyme digest with endonuclease selecting specifically for the nonmethylated site.  The enzyme we have chosen was MBO and AVI.  When all the genomic DNA from the bacteria is extracted and digested, then it will be ran on a gel to be imaged to determine if the bands of digested DNA differ depending on the chemicals present during growth.  This is a time efficient way to examine if any changes in methylation levels have occurred.

What Does It All Mean?

For conclusion, the relevance of this study includes a few things.  This study will provide evidence to show if environmental toxins have an effect on bacterial DAM methylation. One role bacteria play in an ecosystem is influencing the flow of nutrients which support plant and algae growth. The results of our proposed study may display that toxins have an effect on methylation patterns which could lead to an increase the mutation rate of the bacteria genome itself.   Destructive mutations may decrease bacterial populations leading to a disruption in the ecosystems nutrient flow, hence disruptions in plant and algae growth with effect additional aquatic and terrestrial organisms.



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