Types of DNA found in the environment: Is Propidium monoazide an effective tool for reducing the amplification of extracellular DNA in soil? Patrick J. Marsden1,2, Tess Brewer1, Noah Fierer1 1Cooperative Institute for Research in Environmental Science, University of Colorado, Boulder,2Community College of Denver, CO. Make sure your title matches your abstract. Include your contact information somewhere on the poster. If you use figures make sure all the axes are labeled and easy to read from 6 feet away, and photos are high resolution because this will be printed big. I’ve set the page size to 3’ x 4’ which I think is manageable and I know can print from power point. Your board will let you go bigger if you want to, but much bigger is hard to transport etc. You can completely rearrange this however you like  Also feel free to change the background to whatever suits you. If you use a picture as the background, make sure it doesn’t make it hard to read the text – you may want white/pale text boxes if that is the case. Basically the design is up to you! I find writing the text in word then cutting and pasting helps a bit with typos – typing into a poster this size is hard to read. (You can zoom in though!). One final tip – if you print the poster shrunk to fit the whole page on normal letter head, you should be able to read it. If not, your text is too small! Types of DNA found in the environment: Extracellular DNA (DNA plays many ecological roles1 and comes from a wide range of sources2) DNA inside dead but intact cells DNA from living organisms The problem: DNA based analysis examines the total DNA in a sample regardless of its origin. This can cause inaccuracy when trying to depict a living microbial population. Like taking population density data of a forest by counting all the trees alive or dead The questions: What percentage of DNA in a soil sample comes from living viable organism? Can PMA be used to as a means to reduce the amplification of DNA that is not from live cells? Discussion These results are consistent with a similar experiment looking at the effects of TSS (total suspended solids) on PMA’s effectiveness in fermenter sludge. Bae and Wuertz6 found that higher TSS decreased the effects of PMA on a sample. Since PMA works by UV light activation it makes sense that a dense soil slurry would inhibit light activation. Conclusions: PMA was not effective in soil at the slurry ratio of 0.1 g to 1 mL. There are at least two possible explanations Light was not able to penetrate the slurry due to turbidity. Test for light activation by making serial dilution slurries and checking the over all effect of PMA Chemical interactions in the soil prevented PMA to bind to DNA \Test for chemical interference by making soils with specific properties and composition. Method To start to answer our questions we analyzed how PMA would effect DNA suppression with a straight live or dead bacteria culture. PMA was added to either live actively growing Escherichia coli culture or a heat killed culture (samples were taken and smeared on agar to confirm death), samples were then either exposed to light or kept in the dark until DNA extraction. We then applied the same treatment to two different soils that were sterilized. One set of samples were spiked with dead E. coli cells, while the other samples were treated with PMA with out the cell spike. How PMA selects for living organisms: Because PMA cannot cross a living cell membrane due to electrical charge differences, but can cross the compromised cell wall of a dead cell, PMA acts as a live/dead selection with respect to amplifiable DNA. PMA will bind to itself in the absence of DNA Figure 1 Figure 2 Figure 1 and 2 Mean number of DNA sequences per sample for each given condition, based on qPCR results using universal 16s rRNA (ribosomal gene sequence used to identify microorganisms) primers 515f 806r. All error bars represent +/- 1 S.E. Propidium Monoazide (PMA) Intercalates (chemically weaves) through exposed double stranded DNA Because of PMA’s positive charge it is unable to cross the intact cell membrane of a live viable cell. It will cross the damaged or compromised cell wall of a dead microbe Upon UV light exposure PMA covalently binds to DNA or to itself making it no longer effective. Has shown to be effective in clinical settings to differentiate between live and dead bacteria.2,3 PMA has recently been used in a few environmental studies.4,5 used in a few environmental studies.2,3,4 References 1. Pietramellara, G., et al., Extracellular DNA in soil and sediment: fate and ecological relevance. Biology and Fertility of Soils, 2009. 45(3): p. 219-235. 2. Nielsen, K.M., et al., Release and persistence of extracellular DNA in the environment. Environmental Biosafety Research, 2007. 6(1-2): p. 37-53. Nocker, A., et al., Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology. Journal of Microbiological M ethods, 2009. 76(3): p. 253-261. Nocker, A.R.-H., Tim; Montijn, Roy; Schuren, Frank; Kort, Remco, Discrimination between live and dead cellsin bacterial communities from environmental water samples analyzed by 454 pyrosequencing. International Microbiology, 2010. 13(2): p. 59-65. 5. Yergeau, E., et al., The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses. ISME J, 2010. 4(9): p. 1206-1214. 6. Bae, S. and S. Wuertz, Discrimination of Viable and Dead Fecal Bacteroidales Bacteria by Quantitative PCR with Propidium Monoazide. Applied and Environmental Microbiology, 2009. 75(9): p. 2940-2944. Figure 3 mean DNA copy number found in sterilized soil with out receiving the dead E. coli spike and then PMA treatment, two different soils types were used error bars represent +/- 1 S.E. Figure 4 mean DNA copy number found in sterilized soil after receiving the dead E. coli spike and indicated PMA treatment, two different soils types were used error bars represent +/- 1 S.E. Results Our initial test on live and dead E. coli cultures with out soil gave us an expectation for the effect PMA should have on soil spiked with dead E. coli. PMA Reduced dead E. coli copy numbers by 99.9% (fig 2). While PMA reduced live E. coli copy numbers by ~42% (fig 1) we can assume that some of that reduction came from dead cells in the culture and not PMA chemo-toxicity. PMA showed no effect on either soil A or soil B (fig 3 and 4). Contact information: Patrick Marsden pmarsden@msudenver.edu Acknowledgements The RECCS Program is managed collaboratively by CIRES Education and Outreach and the Boulder Critical Zone Observatory.  Funding has been provided by the Director’s Office of CIRES and an NSF supplemental grant to the Boulder Critical Zone Observatory.