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

Sampling Methods

PathPond

Site 2Site 3

Site 1Site 4

Site 5

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

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?

Preliminary Data

Source: Rachel Bigley

Preliminary Data (Cont)

Source: Rachel Bigley

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)

Questions???