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Microbial Diversity of the Estuarial James River

Microbial Diversity of the Estuarial James River. Jenifer Unruh VCU-HHMI Summer Scholars Program Mentor: Dr. Shozo Ozaki. Project. Hypothesis: The microbial communities in estuarine waters are distinct in relation to the salinity gradient.

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Microbial Diversity of the Estuarial James River

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  1. Microbial Diversity of the Estuarial James River Jenifer Unruh VCU-HHMI Summer Scholars Program Mentor: Dr. Shozo Ozaki

  2. Project • Hypothesis: The microbial communities in estuarine waters are distinct in relation to the salinity gradient. • Methodology: The structure of microbial communities along the estuarine waters of the James River was assessed using the small subunit ribosomal RNA gene sequences.

  3. Metagenomics • What is metagenomics? • Metagenomics is the study of microbial genomes collected from their natural environment. • Why is metagenomics important? • Less than 1% of organisms can be successfully cultured in a lab. • What could be expected of the other 99% of organisms?

  4. Map of the James River

  5. Organism Collection Location • The James River Estuary • Site 75 – freshwater, salinity >1 • Algal growth • Site 21 – estuarine, salinity = 16 ppt • Bay salinity is usually about 20-22 ppt • Collected 10 Liters of water from each site • The Interest of Estuaries • Blend of organism dynamics • Unique communities

  6. Harvesting the Microbes in Water Samples by Filtration • Serial Filtration • 2 micron filter • 0.45 micron filter Vacuum Filtration 10 Liters of sample water (1 site)

  7. Once the cells are harvested…

  8. Clones of Amplified DNA

  9. Data Analysis • Sequencher • Used this program to analyze and trim sequences from both sites using the same parameters. • NCBI BLAST • Obtained top 10 results, used to identify sequences • ClustalW • Used to form phylogenetic tree of identified sequences

  10. Observe • The Bacterial Diversity • Representatives • Proportions of differing bacteria • Think about • What traits may be evident in the bacterial species that reside in high and low saline environments?

  11. Site 21 Site 75 >UnculturedActinobacteria_75 >UnculturedActinobacteria_75 >UnculturedActinobacteria_75 >UnculturedActinobacteria_75 >UnculturedActinobacteria_75 >UnculturedActinobacteria_75 >UnculturedActinobacteria_75 >UnculturedActinobacteria_75 >UnculturedAcintobacteria_75 >UnculturedActinobacteria_75 >UnculturedActinobacteria_75 >UnculturedActinobacteria_75 >UnculturedActinobacteria_75 >UnculturedActinobacteria_75 >UnculturedBacteria_75 >UnculturedBacteria_75 >UnculturedBacteria_75 >UnculturedBacteria_75 >UnculturedBacteria_75 >BetaProteobacteria_75 >UnculturedBacteroidetes_75 >UnculturedBacteroidetes_75 >UnculturedBacteroidetes_75 >UnculturedBacteroidetesSphingobacteria_75 >UnculturedBacteroidetesSphingobacteria_75 >UnculturedBacteroidetesSphingobacteria_75 >>Bacteroidetes_75 UnculturedCyanobacteria_75 >UnculturedCyanobacteria_75 >UnculturedCyanobacteria_75 >Cyanobacteria_75 >UnculturedGammaProteobacteria_75 >UnculturedPlanctomycete_75 • >UnculturedActinobacteria_21 • >UnculturedActinobacteria_21 • >UnculturedActinobacteria_21 • >UnculturedActinobacteria_21 • >UnculturedActinobacteria_21 • >UnculturedActinobacteria_21 • >AlphaProteobacteria_21 • >AlphaProteobacteria_21 • >AlphaProteobacteria_21 • >AlphaProteobacteria_21 • >UnculturedAlphaProteobacteria_21 • >UnculturedAlphaProteobacteria_21 • >UnculturedAlphaProteobacteria_21 • >UnculturedAlphaProteobacteria_21 • >UnculturedAlphaProteobacteria_21 • >UnculturedBacteria_21 • >UnculturedBacteria_21 • >UnculturedBacteria_21 • >Uncultured Bacteroidetes_21 • >UnculturedBacteroidetes_21 • >UnculturedBacteroidetes_21 • >UnculturedBacteroidetes_21 • >UnculturedBacteroidetesCytophagales_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacterium_21 • >UnculturedFlavobacteriaceae_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >Flavobacteria_21 • >Flavobacteria_21 • >FlavobacteriaFormosa_21 • >FlavobacteriaFormosa_21 • >FlavobacteriaFormosa_21

  12. List of DNA Sequence Identities • >UnculturedActinobacteria_21 • >UnculturedActinobacteria_21 • >UnculturedActinobacteria_21 • >UnculturedActinobacteria_21 • >UnculturedActinobacteria_21 • >UnculturedActinobacteria_21 • >UnculturedActinobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedAcintobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedActinobacteria_75 • >UnculturedAlphaProteobacteria_21 • >UnculturedAlphaProteobacteria_21 • >UnculturedAlphaProteobacteria_21 • >UnculturedAlphaProteobacteria_21 • >UnculturedAlphaProteobacteria_21 • >AlphaProteobacteria_21 • >AlphaProteobacteria_21 • >AlphaProteobacteria_21 • >AlphaProteobacteria_21 • >UnculturedBacteria_21 • >UnculturedBacteria_21 • >UnculturedBacteria_21 • >UnculturedBacteria_75 • >UnculturedBacteria_75 • >UnculturedBacteria_75 • >UnculturedBacteria_75 • >UnculturedBacteria_75 • >Uncultured Bacteroidetes_21 • >UnculturedBacteroidetes_21 • >UnculturedBacteroidetes_21 • >UnculturedBacteroidetes_21 • >UnculturedBacteroidetes_75 • >UnculturedBacteroidetes_75 • >UnculturedBacteroidetes_75 • >>Bacteroidetes_75 • >UnculturedBacteroidetesCytophagales_21 • >UncultureBacteroidetesSphingobacteria_75 • >UncultureBacteroidetesSphingobacteria_75 • >UncultureBacteroidetesSphingobacteria_75 • >BetaProteobacteria_75 • UnculturedCyanobacteria_75 • >UnculturedCyanobacteria_75 • >UnculturedCyanobacteria_75 • >Cyanobacteria_75 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacteria_21 • >UnculturedFlavobacterium_21 • >UnculturedFlavobacteriaceae_21 • >Flavobacteria_21 • >Flavobacteria_21 • >FlavobacteriaFormosa_21 • >FlavobacteriaFormosa_21 • >FlavobacteriaFormosa_21 • >UnculturedGammaProteobacteria_75 • >UnculturedPlanctomycete_75

  13. DNA Sequence Identities • Actinobacteria • Alpha Proteobacteria • Bacteroidetes • Cytophagales • Sphingobacteria • Beta Proteobacteria • Cyanobacteria • Flavobacteria • Formosa • Gamma Proteobacteria • Plantomycete

  14. ClustalW

  15. The Newest Development • This is where it gets Fun • In order to validate the serial filtering system, we checked for the possibility of there being any 18S (eukaryotic) rRNA in the samples post 1 micron and post 2 micron filters. • Was there? • Yes! • What does that mean? Eukaryotes are typically about 5 microns in size, so what could pass through a 2 micron filter ?

  16. The Newest Development • Through some investigation we found that picoplankton was a likely candidate for the eukaryotic DNA found in our samples. • Why picoplankton • Size, picoplankton are typically about 1 micron in size and their genomes are 12.56 Mb (relatively small) • The 18S primers used to amplify the 18S rRNA matched the picoplankton genome using Sequencher • So, we prepared the 18S rRNA for sequencing. • Guess What! Picoplankton was the most reoccurring sequence hit, but there were also other interesting results.

  17. The Big Picture • My participation thus far is in the first step of DNA Characterization.

  18. Conclusion • There are strong correlations between some bacterial species to their environment, while less correlation exists between more adaptable bacteria and their environment. • There is a very diverse microbial community in the James River, but at this time we do not have enough quantitative data to assess the true dynamics of the microbial communities.

  19. Thank You • My Mentor, Dr. Shozo Ozaki • Dr. Paul Bukaveckas and his Graduate Students • Brent Lederer • Matthew Beckwith • Ph.D Student Jennifer Feittweis • Dr. Allison Johnson • Dr. Greg Buck

  20. References • Crump et al. 2004. Microbial Biogeography along an Estuarine Salinity Gradient. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2004, p. 1494–1505 • Mitchell L. Sogin et al. 2006. Microbial diversity in the deep sea and the underexplored ‘‘rare biosphere’’. Harvard University, MA, June 2006. • http://www.ncbi.nlm.nih.gov/ • http://workbench.sdsc.edu/ • <http://openwetware.org/images/thumb/d/db/Be109ligation.jpg/300px-Be109ligation.jpg> • <http://www.zum.de/Faecher/Materialien/beck/bilder/!transfo.gif>

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