Classification of a Novel Marine Classification of a Novel Marine Bacterial Species:Bacterial Species:

HTCC 2207HTCC 2207

Brett Mellbye

Mentors: Dr. Stephen Giovannoni, Kevin Vergin

Giovannoni Lab, Department of Microbiology

SamplingSampling 16S rRNA 16S rRNA

Environmental Environmental Sampling -> Sampling ->

Phylogenetics Phylogenetics before before culturing and other culturing and other characterizationcharacterization

SAR92 cladeSAR92 clade Class: Class:

GammaproteobacteriaGammaproteobacteria Abundance & Importance Abundance & Importance

in biogeochemical cyclesin biogeochemical cycles

IsolationIsolationHTC = High-throughput culturingHTC = 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 ClassificationTaxonomical Classification Growth ConditionsGrowth Conditions

Variety of carbon concentrations and Variety of carbon concentrations and temperaturestemperatures

MicroscopyMicroscopy Electron MicroscopyElectron Microscopy

Genome SequencingGenome Sequencing Sequencing through Moore Sequencing through Moore

Foundation Marine Microbe Genome Foundation Marine Microbe Genome ProjectProject

Analysis of entire genome sequenceAnalysis of entire genome sequence ProteomicsProteomics

Identify proteins of importanceIdentify proteins of importance Nif, Puf operonsNif, Puf operons

Growth CurvesGrowth Curves Cells grown in LNHM:Cells grown in LNHM:

Low-Nutrient Heterotrophic Low-Nutrient Heterotrophic Medium: 0.2 µm filtered, Medium: 0.2 µm filtered, autoclaved seawater amended autoclaved seawater amended with NHwith NH44Cl & KHCl & KH22POPO44

Unknown Carbon Concentration: Unknown Carbon Concentration: dissolved organic carbon = dissolved organic carbon = ~80 µM~80 µM

Cultures of 2207 amended with Cultures of 2207 amended with Mixed Carbon (MC): 1X equal to Mixed Carbon (MC): 1X equal to 0.001% [wt/vol] D-glucose, D-0.001% [wt/vol] D-glucose, D-ribose, succinic acid, pyruvic acid, ribose, succinic acid, pyruvic acid, glycerol, N-acetyl D-glucosamine, glycerol, N-acetyl D-glucosamine, and 0.002% [vol/vol] ethanoland 0.002% [vol/vol] ethanol

Mixed Carbon (MC) Concentration Mixed Carbon (MC) Concentration Growth CurveGrowth Curve

Cultures incubated at 16 degrees C in the dark

How does carbon concentration affect growth?

Oligotrophic response1000

10000

100000

1000000

10000000

0 1 2 3 4 5 6

Cells per ml

Days

0XMC

0.1XMC

0.5XMC

1XMC

5XMC

10XMC

Error in experimental procedure

Morphology Changes: Based on Morphology Changes: Based on carbon concentration?carbon concentration?

Differences in cell Differences in cell morphology observed morphology observed during mixed carbon during mixed carbon concentration growth concentration growth curvecurve

0X MC

Morphology Changes: Based on Morphology Changes: Based on carbon concentration?carbon concentration?

0.5X MC0.5X MC

Morphology Changes: Based on Morphology Changes: Based on carbon concentration?carbon concentration?

1X MC1X MC

Morphology Changes: Based on Morphology Changes: Based on carbon concentration?carbon concentration?

5X MC5X MC

Morphology Changes: Based on Morphology Changes: Based on carbon concentration?carbon concentration?

10X MC10X MC

Morphology Changes: Based on Morphology Changes: Based on carbon concentration?carbon concentration?

10X MC10X MC 0X MC

Temperature Conditions Growth Temperature Conditions Growth CurveCurve Cultures (1X MC) Cultures (1X MC)

grown at 4, 10, 16, grown at 4, 10, 16, 23, & 30 degrees C23, & 30 degrees C

No growth at 4 and No growth at 4 and 30 degrees C30 degrees C

How does temp. How does temp. affect growth?affect growth?

No treatment reached No treatment reached maximum cell densitymaximum cell density

1000

10000

100000

1000000

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

Cells per ml

Days

10C

16C

23C

Electron MicroscopyElectron Microscopy

Cells fixed with Cells fixed with glutaraldehyde glutaraldehyde and negatively and negatively stainedstained

Examined using Examined using a transmission a transmission electron electron microscopemicroscope

Coming Soon..Coming Soon..

Repeat growth experiments in 50ml Repeat growth experiments in 50ml cultures of amended seawater to verify cultures of amended seawater to verify effects of excess carbon and extreme effects of excess carbon and extreme temperaturestemperatures

20L culture for proteomics20L culture for proteomics Genome sequence analysisGenome sequence analysis

AcknowledgementsAcknowledgements The Gordon & Betty Moore FoundationThe Gordon & Betty Moore Foundation Dr. Stephen GiovannoniDr. Stephen Giovannoni Dr. Douglas F. BarofskyDr. Douglas F. Barofsky Kevin VerginKevin Vergin Sarah SowellSarah Sowell Giovannoni LabGiovannoni Lab American Society for MicrobiologyAmerican Society for Microbiology HHMI and Dr. Kevin AhernHHMI and Dr. Kevin Ahern