Classification of a Novel Marine Bacterial Species: HTCC 2207
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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
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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
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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
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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
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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
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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
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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
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Morphology Changes: Based on Morphology Changes: Based on carbon concentration?carbon concentration?
0.5X MC0.5X MC
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Morphology Changes: Based on Morphology Changes: Based on carbon concentration?carbon concentration?
1X MC1X MC
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Morphology Changes: Based on Morphology Changes: Based on carbon concentration?carbon concentration?
5X MC5X MC
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Morphology Changes: Based on Morphology Changes: Based on carbon concentration?carbon concentration?
10X MC10X MC
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Morphology Changes: Based on Morphology Changes: Based on carbon concentration?carbon concentration?
10X MC10X MC 0X MC
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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
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0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Cells per ml
Days
10C
16C
23C
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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
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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
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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