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Classification of a Novel Marine Bacterial Species: HTCC 2207

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 ->

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Classification of a Novel Marine Bacterial Species: HTCC 2207

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  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 -> 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? • Differences in cell morphology observed during mixed carbon concentration growth curve 0X MC

  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

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