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

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

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

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

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

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

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

Morphology Changes: Based on carbon concentration? 0.5X MC

Morphology Changes: Based on carbon concentration? 1X MC

Morphology Changes: Based on carbon concentration? 5X MC

Morphology Changes: Based on carbon concentration? 10X MC

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

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

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

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

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