What a Small World: The Microbial Environment Experienced by Wild Caenorhabditids Christopher Abin...
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Transcript of What a Small World: The Microbial Environment Experienced by Wild Caenorhabditids Christopher Abin...
What a Small World:The Microbial Environment Experienced by Wild Caenorhabditids
Christopher Abin UO SPUR 2010
Florida Int’l University
PI: Patrick Phillips, PhD Brendan Bohannan, PhD
Mentors: Michelle Parmenter Keaton Stagaman
What are nematodes?
• Slender, worm-like
• Typically ≤ 2.5 mm long
• Ubiquitous
• ≥ 28,000 species described (~16,000 parasitic)
The Genus Caenorhabditis
• Bacteria-rich environments
+
Animal vectors
• True soil nematodes?
• Noted model organism: C. elegans
Limitations in Caenorhabditis Research
• Ecological data is lacking
• No information on natural diet
or bacterial associations
• E. coli strain OP50 is an unnatural food source
Objectives
• Immediate: – Which bacteria are Caenorhabditis species
encountering in the wild?
• Future:– How do differences in the source of nutrition
influence the demography of populations?
Soil Collection
• Koffler Scientific Reserve at Jokers Hill (KSR)
• King Township, Ontario
• 348 hectares:– Fields– Wetlands– Grasslands– Forest
Isolation of Nematodes
Spread ~1–2 grams of soil around the E. coli OP50 lawn of a standard NGM petri dish and moistened with 1 mL S basal buffer (Cholesterol, NaCl, KH2PO4, and K2HPO4)
Barrière & Felix (2006)
Nematode Identification
• Molecular methods:
Soil DNA extraction
PCR amplification of 28S
(LSU) ribosomal RNA gene
~ 1,500 bp
Site 1
Site 2
Site 3
Site 4
Site 5
Pos. (N
2)
Ladder
Isolation of Nematode-Associated Bacteria1-2 grams of soil plated onto non-seeded NGM agar plates
Picked colonies of bacteria in close association with nematodes onto LB plates
Dilution streaking to obtain pure cultures
Picked individual worms onto Luria-Bertani (LB) plates
Picked individual colonies of bacteria
1 2
Identification of Bacterial Isolates
1. Genomic DNA extraction
2. PCR amplification of 16S rDNA
Ladder
1A 1B 1D1C 1E 2A 2B
500 bp
500 bp
Ladder
2C 3D3C3B3A2E2D
Identification of Bacterial Isolates
1. Genomic DNA extraction
2. PCR amplification of 16S rDNA
3. Clone PCR product into a plasmid
Identification of Bacterial Isolates
1. Genomic DNA extraction
2. PCR amplification of 16S rDNA
3. Clone PCR product into a plasmid
4. Transform into E. coli
Identification of Bacterial Isolates
1. Genomic DNA extraction
2. PCR amplification of 16S rDNA
3. Clone PCR product into a plasmid
4. Transform into E. coli
5. Purification of plasmid DNA
Source: Promega Corporation
Identification of Bacterial Isolates
1. Genomic DNA extraction
2. PCR amplification of 16S rDNA
3. Clone PCR product into a plasmid
4. Transform into E. coli
5. Purification of plasmid DNA
6. Restriction digest with EcoRI
4,000 bp
500 bp
4,000 bp
500 bp
Identification of Bacterial Isolates
1. Genomic DNA extraction
2. PCR amplification of 16S rDNA
3. Clone PCR product into a plasmid
4. Transform into E. coli
5. Purification of plasmid DNA
6. Restriction digest with EcoRI
7. Sanger sequencing
Identification of Bacterial Isolates
1. Genomic DNA extraction
2. PCR amplification of 16S rDNA
3. Clone PCR product into a plasmid
4. Transform into E. coli
5. Purification of plasmid DNA
6. Restriction digest with EcoRI
7. Sanger sequencing
8. NCBI BLAST sequence alignment
Species Identification
Site Isolate Species Sequence Similarity* Gram (+) or (-)
1
A Pseudomonas putida 99.0% Neg.
B Comomonas testosteroni 100% Neg.
C Pseudomonas entomophila 99.0% Neg.
D Mesorhizobium sp. 97.0% Neg.
E Ochrobactrum sp. 89.7% Neg.
2
A Acinetobacter haemolyticus 99.0% Neg.
B Enterobacter sp. 99.0% Neg.
C Acinetobacter sp. 98.0% Neg.
D Serratia proteamaculans 100% Neg.
E Serratia proteamaculans 99.0% Neg.
3
A Acinetobacter iwoffii 98.0% Neg.
B Serratia proteamaculans 99.0% Neg.
C Stenotrophomonas maltophilia 100% Neg.
D Stenotrophomonas maltophilia 100% Neg.
*Nucleotide sequence similarity to known prokaryotic 16S rDNA sequences in the NCBI Database
Species Overlap
P. putidaC. testosteroniP. entomophilia
Mesorhiuzobium sp.Ocrobactrum sp.
A.haemolyticusEnterobacter sp.Acinetobacter sp.
A. iwoffiiS. maltophilia
S. proteamaculans
N/A
N/A
N/A
Site 1
Site 3
Site 2
Analysis of Gut Microbiota
PCR amplification of 16S rDNA
Surface sterilization of worms via 25% glycerol wash protocol
Supernatants spread onto LB plates to check for residual bacteria
1 2
3
Cloning and sequencing
5
3,000 rpm x 8 mins x 8 washes
Freeze crack with liquid N2
4
Glycerol Wash Results
• Surface sterilization could not be achieved
• Protocol repeated for E. coli strain OP50 with same result
• Centrifugation speed too high?
• Addition of a lysozyme preparatory step?
Conclusions
• Presence of Caenorhabditis species confirmed using
molecular methods
• Collected 11 different bacterial species belonging to 8
different genera from three sampling sites
• Gut microbial community analysis was unsuccessful
• Glycerol wash protocol requires further optimization
Future Directions
• Identify Caenorhabditids present in soil samples
• Isolate and characterize more bacteria from all sites
• Optimize glycerol wash
• How do different food sources affect fecundity and lifespan?
Acknowledgments
• Patrick Phillips• Brendan Bohannan• Michelle Parmenter• Keaton Stagaman
• Phillips Lab:Rose Reynolds
Jenni Anderson
Bryn Gaertner
Tim Ahearne
Lauren Noll
Emily Ebel
Anna Crist
Alecia Stewart-Malone
Rachel Bigley
• U of O SPUR Program– Peter O’Day– Blakely Strand– SPUR Interns
• National Science Foundation (NSF)