Genetic Variation of
Renibacterium salmoninarum
genes in infected salmonids
Jeffrey Burnett
HHMI Summer Investigator
Dr. Dan Rockey Laboratory
Biomedical Sciences
Renibacterium salmoninarum
• Causes bacterial kidney disease (BKD)
Introduction
• Wild and farmed salmonid species
Hypothesis
Methods
Results
Discussion
Conclusion
13 Oct 2007
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Jeffrey Burnett - HHMI
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Relevance
• Why is this a problem?
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
13 Oct 2007
• We eat salmonids
• We depend on salmonids to keep an
ecosystemic balance in our local
rivers and streams
• R. salmoninarum devastates whole
populations; endangered fish stocks
Jeffrey Burnett - HHMI
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Relevance
• All salmonids are susceptible to BKD
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
13 Oct 2007
coho salmon (Oncorhynchus kisutch)
brook trout (Salvelinus fontinalis)
brown trout (Salmo trutta)
chinook salmon (Oncorhynchus
tshawytscha)
• rainbow trout (Oncorhynchus mykiss)
•
•
•
•
Jeffrey Burnett - HHMI
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Renibacterium salmoninarum
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
http://oregonstate.edu/dept/salmon/projects/images/4BKD.jpg
13 Oct 2007
Jeffrey Burnett - HHMI
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Relevance
Introduction
Hypothesis
Methods
• Prevalence
• Found in majority of countries
• Economic impact felt worldwide
Results
Discussion
Conclusion
13 Oct 2007
Jeffrey Burnett - HHMI
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Relevance
• Economics close to home
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
13 Oct 2007
• Local: Oregon Hatcheries
• 2004 - $143,000
• Largest Global Impact:
• Chile,S.A. and Europe
Jeffrey Burnett - HHMI
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Big Picture
Introduction
Hypothesis
Methods
Results
Discussion
• Drug / Vaccine to eliminate
bacteria
• Difficult to treat
• Current treatments ineffective
Conclusion
13 Oct 2007
Jeffrey Burnett - HHMI
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Goal of My Project
• Genome analysis
Introduction
Hypothesis
Methods
Results
Discussion
• American Tissue Culture Collection
(ATCC) 33209
• Accurate representation
• ERGO by Integrated Genomics
Conclusion
13 Oct 2007
Jeffrey Burnett - HHMI
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Specific Goal
Renibacterium salmoninarum vs. Arthrobacter sp.
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
gene affected by frameshift
transport and binding proteins
central intermediary metabolism
Fatty acid and phospholipid metabolism
energy metabolism
regulatory functions
Biosynthesis of cofactors, prosthetic groups, and carriers
cellular processes
DNA metabolism
protein fate
amino acid biosynthesis
unknown/hypothetical/unclassified/not called
signal transduction
cell envelope
Purines, pyrimidines, nucleosides, and nucleotides
protein synthesis
Mobile and extrachromosomal element functions
transcription
total
13 Oct 2007
Jeffrey Burnett - HHMI
# frameshifted total of type percentage
158
346
45.66
53
170
31.18
31
100
31.00
91
304
29.93
59
240
24.58
26
109
23.85
21
90
23.33
23
108
21.30
29
152
19.08
21
111
18.92
215
1182
18.19
1
6
16.67
31
215
14.42
6
61
9.84
9
128
7.03
10
151
6.62
2
34
5.88
786
3507
9
Hypothesis
Introduction
Hypothesis
Methods
Results
Discussion
• Due to extended laboratory
culture, the genome of strain
ATCC33209 has extensive
mutations not representative of
what is found in nature
Conclusion
13 Oct 2007
Jeffrey Burnett - HHMI
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Genetic analysis
•
Fibronectin Binding Protein
•
Dipeptide Permease Protein
Introduction
Hypothesis
Methods
Results
•
Citrate Synthase Protein
•
Tetracycline Resistance Protein P
Discussion
Conclusion
13 Oct 2007
Jeffrey Burnett - HHMI
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Genomic DNA
Introduction
Hypothesis
Methods
Results
Discussion
•
•
•
•
wt - 2 fish kidneys (A,B)
Mt239
ATCC33209
684
Conclusion
13 Oct 2007
Jeffrey Burnett - HHMI
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Primer Design
Introduction
• Flank apparent frameshifts identified
by ERGO
Hypothesis
Methods
Results
Discussion
Conclusion
•
13 Oct 2007
Tetracycline Resistance Protein P
Jeffrey Burnett - HHMI
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Experiment
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
• PCR products inserted into expression
vectors
• Plasmids transformed into
Escherichia coli
• Plasmids purified from bacteria
• Sent for sequencing
13 Oct 2007
Jeffrey Burnett - HHMI
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Results
Introduction
• Center for Genome Research and
Biocomputing (CGRB) - OSU
Hypothesis
Methods
Results
• 20 sequences in both directions
Discussion
Conclusion
13 Oct 2007
Jeffrey Burnett - HHMI
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Results
Introduction
Hypothesis
ATCC
mt239
684
A
B
Fibronectin Binding Protein
Y
Y
N
N
Y
dppD/F
Y
Y
N
Y
N
Citrate Synthase
Y
Y
Y
Y
Y
Tetracycline Resistance P
Y
Y
Y
N
Y
Methods
Results
Discussion
Conclusion
13 Oct 2007
Y = yes, the sequence is identical to the
ATCC sequence
N = no, the sequence received is
different from the ATCC sequence
Jeffrey Burnett - HHMI
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Results
Introduction
Hypothesis
Methods
Results
Discussion
• Verification of first round of
results
• Reconstruct plasmids from
different samples of DNA strains
Conclusion
13 Oct 2007
Jeffrey Burnett - HHMI
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Results
ATCC
mt239
684
A
B
Fibronectin Binding Protein
Y
Y
N
N
Y
Introduction
dppD/F
Y
Y
N
Y
N
Hypothesis
Citrate Synthase
Y
Y
Y
Y
Y
Tetracycline Resistance P
Y
Y
Y
N
Y
Methods
Results
Discussion
•All of the samples marked “N” ran in
duplicate, returned the same results
Conclusion
13 Oct 2007
Jeffrey Burnett - HHMI
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Discussion
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
13 Oct 2007
• Findings are contrary to what we
had originally hypothesized
• Genes are actually more mutated
in the other strain isolate DNA that
we tested
Jeffrey Burnett - HHMI
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Conclusion
Introduction
Hypothesis
Methods
Results
Discussion
Conclusion
13 Oct 2007
• My research suggests that the
ATCC sequence is representative
of what is found in nature
• The bacteria is acquiring more
mutations in its genome than the
original ATCC strain
Jeffrey Burnett - HHMI
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Acknowledgements
• Howard Hughes Medical Institute
• Dr. Kevin Ahern
• Dr. Dan Rockey Laboratory
• Sara Weeks
• Gina Capri
• Integrated Genomics
13 Oct 2007
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