1. Classification of a Novel Marine Bacterial Species: HTCC 2207
Brett Mellbye
Mentors: Dr. Stephen Giovannoni, Kevin Vergin
Giovannoni Lab, Department of Microbiology

2. Sampling 16S rRNA Environmental Sampling -> SAR92 clade
Phylogenetics before culturing and other characterization
SAR92 clade
Class: Gammaproteobacteria
Abundance & Importance in biogeochemical cycles

3. Isolation HTC = High-throughput culturing
High-throughput culturing by Dilution to Extinction:
1. Collect seawater
2. Dilute to 10 cells/mL
3. Dispense into 48-well microtiter plates
4. Incubate 3 weeks
5. Screen for growth

4. Taxonomical Classification
Growth Conditions
Variety of carbon concentrations and temperatures
Microscopy
Electron Microscopy
Genome Sequencing
Sequencing through Moore Foundation Marine Microbe Genome Project
Analysis of entire genome sequence
Proteomics
Identify proteins of importance
Nif, Puf operons

5. Growth Curves Cells grown in LNHM:
Low-Nutrient Heterotrophic Medium: 0.2 µm filtered, autoclaved seawater amended with NH4Cl & KH2PO4
Unknown Carbon Concentration: dissolved organic carbon =
~80 µM
Cultures of 2207 amended with Mixed Carbon (MC): 1X equal to 0.001% [wt/vol] D-glucose, D-ribose, succinic acid, pyruvic acid, glycerol, N-acetyl D-glucosamine, and 0.002% [vol/vol] ethanol

6. Mixed Carbon (MC) Concentration Growth Curve
Cultures incubated at 16 degrees C in the dark
How does carbon concentration affect growth?
Oligotrophic response
Error in experimental procedure

7. Morphology Changes: Based on carbon concentration?
0X MC
Differences in cell morphology observed during mixed carbon concentration growth curve

8. Morphology Changes: Based on carbon concentration?
0.5X MC

9. Morphology Changes: Based on carbon concentration?
1X MC

10. Morphology Changes: Based on carbon concentration?
5X MC

11. Morphology Changes: Based on carbon concentration?
10X MC

12. Morphology Changes: Based on carbon concentration?
10X MC
0X MC

13. Temperature Conditions Growth Curve
Cultures (1X MC) grown at 4, 10, 16, 23, & 30 degrees C
No growth at 4 and 30 degrees C
How does temp. affect growth?
No treatment reached maximum cell density

14. Electron Microscopy
Cells fixed with glutaraldehyde and negatively stained
Examined using a transmission electron microscope

15. Coming Soon..
Repeat growth experiments in 50ml cultures of amended seawater to verify effects of excess carbon and extreme temperatures
20L culture for proteomics
Genome sequence analysis

16. Acknowledgements The Gordon & Betty Moore Foundation
Dr. Stephen Giovannoni
Dr. Douglas F. Barofsky
Kevin Vergin
Sarah Sowell
Giovannoni Lab
American Society for Microbiology
HHMI and Dr. Kevin Ahern