1. 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 10 Liters of sample water (1 site) Vacuum Filtration Serial Filtration 2 micron filter 0.45 micron filter

7. Why use ribosomal RNA? all cellular organisms have ribosomes and therefore ribosomal RNA **ssu lsu define + pics Why is ssu rDNA prefered for analysis? The ssu gene is conserved Universality of the ssu gene evolutionary changes become apparent through mutations

8. Ribosomal RNA Ssu – small subunit Lsu – large subunit 16S – Prokaryotic 18S – Eukaryotic

9. Once the cells are harvested… DNA purification Amplification of 16S gene by PCR Cloning amplified DNA Sequencing of cloned DNA fragments

10. Clones of Amplified DNA

11. 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

12. 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?

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

14. List of DNA Sequence Identities 1. >UnculturedActinobacteria_21 2. >UnculturedActinobacteria_21 3. >UnculturedActinobacteria_21 4. >UnculturedActinobacteria_21 5. >UnculturedActinobacteria_21 6. >UnculturedActinobacteria_21 7. >UnculturedActinobacteria_75 8. >UnculturedActinobacteria_75 9. >UnculturedActinobacteria_75 10. >UnculturedActinobacteria_75 11. >UnculturedActinobacteria_75 12. >UnculturedActinobacteria_75 13. >UnculturedActinobacteria_75 14. >UnculturedAcintobacteria_75 15. >UnculturedActinobacteria_75 16. >UnculturedActinobacteria_75 17. >UnculturedActinobacteria_75 18. >UnculturedActinobacteria_75 19. >UnculturedActinobacteria_75 20. >UnculturedActinobacteria_75 21. >UnculturedAlphaProteobacteria_21 22. >UnculturedAlphaProteobacteria_21 23. >UnculturedAlphaProteobacteria_21 24. >UnculturedAlphaProteobacteria_21 25. >UnculturedAlphaProteobacteria_21 26. >AlphaProteobacteria_21 27. >AlphaProteobacteria_21 28. >AlphaProteobacteria_21 29. >AlphaProteobacteria_21 30. >UnculturedBacteria_21 31. >UnculturedBacteria_21 32. >UnculturedBacteria_21 33. >UnculturedBacteria_75 34. >UnculturedBacteria_75 35. >UnculturedBacteria_75 36. >UnculturedBacteria_75 37. >UnculturedBacteria_75 38. >Uncultured Bacteroidetes_21 39. >UnculturedBacteroidetes_21 40. >UnculturedBacteroidetes_21 41. >UnculturedBacteroidetes_21 42. >UnculturedBacteroidetes_75 43. >UnculturedBacteroidetes_75 44. >UnculturedBacteroidetes_75 45. >>Bacteroidetes_75 46. >UnculturedBacteroidetesCytophagales_21 47. >UncultureBacteroidetesSphingobacteria_75 48. >UncultureBacteroidetesSphingobacteria_75 49. >UncultureBacteroidetesSphingobacteria_75 50. >BetaProteobacteria_75 51. UnculturedCyanobacteria_75 52. >UnculturedCyanobacteria_75 53. >UnculturedCyanobacteria_75 54. >Cyanobacteria_75 55. >UnculturedFlavobacteria_21 56. >UnculturedFlavobacteria_21 57. >UnculturedFlavobacteria_21 58. >UnculturedFlavobacteria_21 59. >UnculturedFlavobacteria_21 60. >UnculturedFlavobacteria_21 61. >UnculturedFlavobacteria_21 62. >UnculturedFlavobacteria_21 63. >UnculturedFlavobacteria_21 64. >UnculturedFlavobacterium_21 65. >UnculturedFlavobacteriaceae_21 66. >Flavobacteria_21 67. >Flavobacteria_21 68. >FlavobacteriaFormosa_21 69. >FlavobacteriaFormosa_21 70. >FlavobacteriaFormosa_21 71. >UnculturedGammaProteobacteria_75 72. >UnculturedPlanctomycete_75

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

16. ClustalW

17. 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 ?

18. 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.

19. The Big Picture My participation thus far is in the first step of DNA Characterization. DNA Characterization Library Formation Sequencing of Library

20. 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.

21. 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

22. 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/Be109ligati on.jpg/300px-Be109ligation.jpg http://www.zum.de/Faecher/Materialien/beck/bilder/!tra nsfo.gif