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. Methodology: The structure of microbial communities along the estuarine waters of the James River was assessed using the small subunit ribosomal RNA gene sequences.

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?

Map of the James River

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 ppt Collected 10 Liters of water from each site The Interest of Estuaries Blend of organism dynamics Unique communities

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

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

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

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

Clones of Amplified DNA

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

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?

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

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_ >UnculturedActinobacteria_ >UnculturedActinobacteria_ >UnculturedActinobacteria_ >UnculturedActinobacteria_ >UnculturedAcintobacteria_ >UnculturedActinobacteria_ >UnculturedActinobacteria_ >UnculturedActinobacteria_ >UnculturedActinobacteria_ >UnculturedActinobacteria_ >UnculturedActinobacteria_ >UnculturedAlphaProteobacteria_ >UnculturedAlphaProteobacteria_ >UnculturedAlphaProteobacteria_ >UnculturedAlphaProteobacteria_ >UnculturedAlphaProteobacteria_ >AlphaProteobacteria_ >AlphaProteobacteria_ >AlphaProteobacteria_ >AlphaProteobacteria_ >UnculturedBacteria_ >UnculturedBacteria_ >UnculturedBacteria_ >UnculturedBacteria_ >UnculturedBacteria_ >UnculturedBacteria_ >UnculturedBacteria_ >UnculturedBacteria_ >Uncultured Bacteroidetes_ >UnculturedBacteroidetes_ >UnculturedBacteroidetes_ >UnculturedBacteroidetes_ >UnculturedBacteroidetes_ >UnculturedBacteroidetes_ >UnculturedBacteroidetes_ >>Bacteroidetes_ >UnculturedBacteroidetesCytophagales_ >UncultureBacteroidetesSphingobacteria_ >UncultureBacteroidetesSphingobacteria_ >UncultureBacteroidetesSphingobacteria_ >BetaProteobacteria_ UnculturedCyanobacteria_ >UnculturedCyanobacteria_ >UnculturedCyanobacteria_ >Cyanobacteria_ >UnculturedFlavobacteria_ >UnculturedFlavobacteria_ >UnculturedFlavobacteria_ >UnculturedFlavobacteria_ >UnculturedFlavobacteria_ >UnculturedFlavobacteria_ >UnculturedFlavobacteria_ >UnculturedFlavobacteria_ >UnculturedFlavobacteria_ >UnculturedFlavobacterium_ >UnculturedFlavobacteriaceae_ >Flavobacteria_ >Flavobacteria_ >FlavobacteriaFormosa_ >FlavobacteriaFormosa_ >FlavobacteriaFormosa_ >UnculturedGammaProteobacteria_ >UnculturedPlanctomycete_75

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

ClustalW

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 ?

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 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.

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

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.

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

References Crump et al Microbial Biogeography along an Estuarine Salinity Gradient. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2004, p. 1494– 1505 Mitchell L. Sogin et al Microbial diversity in the deep sea and the underexplored ‘‘rare biosphere’’. Harvard University, MA, June on.jpg/300px-Be109ligation.jpg nsfo.gif