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

• Slender, worm-like • ≥ 28,000 species described (~16,000 parasitic)
• Typically ≤ 2.5 mm long
• Ubiquitous

• Bacteria-rich environments
+
Animal vectors
• True soil nematodes?
• Noted model organism: C. elegans

• Ecological data is lacking
• No information on natural diet or bacterial associations
• E. coli strain OP50 is an unnatural food source

• Immediate:
– Which bacteria are Caenorhabditis species encountering in the wild?
• Future:
– How do differences in the source of nutrition influence the demography of populations?

• Koffler Scientific Reserve at Jokers Hill (KSR)
• King Township, Ontario
• 348 hectares:
– Fields
– Wetlands
– Grasslands
– Forest

Path
Site 3
Site 2
Pond
Site 1
Site 4
Site 5

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, KH
2
PO
4
, and K
2
HPO
4
)
Barrière & Felix (2006)

• Molecular methods:
 Soil DNA extraction
~ 1,500 bp 
 PCR amplification of 28S
(LSU) ribosomal RNA gene

Isolation of Nematode-Associated Bacteria
1
Picked colonies of bacteria in close association with nematodes onto
LB plates
1-2 grams of soil plated onto non-seeded NGM agar plates
2
Picked individual worms onto Luria-
Bertani (LB) plates
Picked individual colonies of bacteria
Dilution streaking to obtain pure cultures

Identification of Bacterial Isolates
1.
Genomic DNA extraction

Identification of Bacterial Isolates
1.
Genomic DNA extraction
2.
PCR amplification of 16S rDNA
500 bp 
500 bp 

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
4,000 bp 
500 bp 
4.
Transform into E. coli
5.
Purification of plasmid DNA
6.
Restriction digest with EcoRI
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

Site
1
2
3
Isolate
B
C
D
E
A
C
D
A
B
D
E
A
B
C
Species
Pseudomonas putida
Comomonas testosteroni
Pseudomonas entomophila
Mesorhizobium sp.
Ochrobactrum sp.
Acinetobacter haemolyticus
Enterobacter sp.
Acinetobacter sp.
Serratia proteamaculans
Serratia proteamaculans
Acinetobacter iwoffii
Serratia proteamaculans
Stenotrophomonas maltophilia
Stenotrophomonas maltophilia
Sequence
Similarity*
99.0%
100%
99.0%
97.0%
89.7%
99.0%
99.0%
98.0%
100%
99.0%
98.0%
99.0%
100%
100%
Gram (+) or (-)
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
Neg.
*
Nucleotide sequence similarity to known prokaryotic 16S rDNA sequences in the NCBI Database

Site 1
P. putida
C. testosteroni
P. entomophilia
Mesorhiuzobium sp.
Ocrobactrum sp.
N/A
A.haemolyticus
Enterobacter sp.
Acinetobacter sp.
Site 2
N/A
N/A
S. proteamaculans
A. iwoffii
S. maltophilia
Site 3

1
Surface sterilization of worms via 25% glycerol wash protocol
2
Supernatants spread onto LB plates to check for residual bacteria
3,000 rpm x 8 mins x 8 washes
3
Freeze crack with liquid N
2
4
PCR amplification of 16S rDNA
5
Cloning and sequencing

• 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?

• 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?

Source: Rachel Bigley

Source: Rachel Bigley

• 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???