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 20-22 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
•Serial Filtration
•2 micron filter
•0.45 micron filter
Vacuum Filtration
10 Liters of sample
water (1 site)
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 21
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>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
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
List of DNA Sequence Identities
1.
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>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
32.
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>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
63.
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>UnculturedFlavobacteria_21
>UnculturedFlavobacterium_21
>UnculturedFlavobacteriaceae_21
>Flavobacteria_21
>Flavobacteria_21
>FlavobacteriaFormosa_21
>FlavobacteriaFormosa_21
>FlavobacteriaFormosa_21
>UnculturedGammaProteobacteria_75
>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?
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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

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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.
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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. 2004. Microbial Biogeography along an
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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/
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