Transcript + MLSA on Vibrio Hopkins Microbiology course 2013 Monica Abrudan, David Fronk, Marius Linkevicius, Lea Madsen, Laura-Carlota Paz & Brian Piasecki.

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MLSA on Vibrio
Hopkins Microbiology course 2013
Monica Abrudan, David Fronk, Marius Linkevicius, Lea Madsen,
Laura-Carlota Paz & Brian Piasecki
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Sampling sites
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Sampling 2013

Sampling of Vibrio from seawater and anemones at 2
different sites and plating on TCBS agar plates.
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2x restreaks and isolation of colonies for genetic analysis
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Anemones differ between samples
+ Hopkins and Point Lobos Sampling
Hopkins (2013.06.23)
Hopkins (2013.06.23)
Point Lobos (2013.06.23)
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MLSA steps on the road
DNA Extraction
Gene Amplification
Sequencing
Evaluation
Looking at the
sequences +
Analysis
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The genes
recA
- Repair and maintenance
of DNA
- Central role in
homologous
recombination
- 38 kDa
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The genes
gyrB
- Topoisomerase Type II
- ATP dependent
- enzyme that relieves strain
while double-stranded DNA
is being unwound by helicase
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The genes
mdh
- Malate dehydrogenase
- Oxidation of malate to
oxaloacetate (NDA+dependent)
- Homodimeric soluble
30-35 kDa protein
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The genes
OmpK
- Only non
Housekeeping gene
- Outer membrane
protein of ~26 kDA
- Transmembrane iontransporter
- Putative receptor for
KVP40 phage
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Null Hypotheses
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Ecological niches:
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No structural differences in populations within:
1. sites (PL vs. H; CA vs. NZ)
2. anemone vs. water
3. years
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Genes
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No differences between ompK and the housekeeping genes
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Overview
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Housekeeping genes
 Phylogeny
 Dot tree
 Tree trees (recA, mdh, gyrB)
 Tree of 2013
 Tree for 2007-2013
 Diversity
 Evenness and richness
 Rarefraction
 Frequency of alleles
 Persistent alleles
 Structure data (populations)
 PCA
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ompK
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Phylogeny
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dN/dS
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Tree
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Gene network
+ Concatenation of recA, mdh, and
gyrB
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Diversity
Richness and evenness based on sequence types
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Diversity
Richness and evenness based on sequence types
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Comparison over the years (water)
Frequency
Point Lobos
1.5
1.0
0.5
0.0
1
5
8 18 20 34 62 67 2
8 20 25 29 36 62 64 1
gyrB
8 22 25 27 42 65 82 89 91 99
mdh
racA
Locus
2007
2008
2009
2010
2011
2012
2013
Frequency
Hopkins
1.200
1.000
0.800
0.600
0.400
0.200
0.000
1
4
9 13 19 27 45 53 58 1 11 14 25 40 50 56 1
gyrB
mdh
Locus
8 14 20 33 38 41 59 74 85
racA
+Comparison of NZ coasts (anemone)
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West coast New Zealand
0.200
0.180
Frequency
0.160
0.140
0.120
0.100
0.080
Bethels
0.060
O'Neils
0.040
0.020
212
187
173
165
150
134
123
86
50
46
21
12
165
143
135
123
109
88
75
50
46
20
11
174
166
140
127
116
100
68
44
40
20
10
3
0.000
Locus
East coast New Zealand
0.250
0.150
Narrow Neck
0.100
Westwell
0.050
Locus
209
194
178
144
132
113
104
91
50
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3
156
144
129
111
105
95
79
40
32
8
172
160
145
132
106
90
81
67
44
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9
0.000
3
Frequency
0.200
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+ recA 2007-2013 colored by site
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Diversity measures
New Zealand
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Point Lobos and Hopkins Samples, 2007-2012
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Population structure (CA vs. NZ)
K=4
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The sequences are divided into 2 populations by sampling sites.
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Structure tool has arranged housekeeping gene sequences into 4 clusters
(represented by different colors).
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The blue and green clusters dominate in California population.
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The red and yellow clusters are mainly found in New Zealand population.
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Different colors could represent different sampling sites.
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Indications of recombination were observed (the lines with different colors).
+ Population structure change in
California (2007-2013)
K=5
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The sequences are divided into 7 predetermined populations by year.
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The program has assigned the housekeeping gene sequences into 5 clusters
(represented by different colors).
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The blue cluster dominates 2007-2010 and it is back in 2013.
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The red cluster dominates 2011-2012.
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Effects from strong El Niño in the beginning of 2010?
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+ PCA plot of sites
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gyrB
CA
mdh
recA
NZ
NZ
NZ W
CA
CA
NZ E (water)
NZ E
PL
Ho
p
NE
NW
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Summary
Null hypotheses touched
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No structural differences in populations within:
1.
Sites
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Results
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Clear population structure differences between CA and New Zealand.
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Clustering in PCA of CA vs. NZ and NZ east vs. NZ west in the mdh gene.
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Apparent population structure differs between Point Lobos and Hopkins.
 Evenness (diversity) differs between Point Lobos and Hopkins.
2.
3.
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Anemone vs. water
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Results
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A difference in diversity pattern is seen between anemone and water.
Years
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Results
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Population structure and diversity (richness and evenness) differ between 2007-2009 and 20102013
No differences between individual housekeeping genes.
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Results
recA, mdh seem more conserved with regard to the persistent “core alleles” compared to gyrB.
+ ompK is under purifying selection
1.8
1.6
Mean dN/dS = 0.127
76/95 sites (p< 0.1)
dN/dS
1.4
1.2
1
0.8
0.6
0.4
0
Codon
4
8
12
16
20
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36
40
44
48
52
56
60
64
68
72
76
80
84
88
92
0.2
dN/dS < 1.0 – selection maintains similar codon sequences
dN/dS = 1.0 – neutral evolution
dN/dS > 1.0 – selection favors codon changes
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ompK phylogram depicts many
related and divergent groups
Tree generated with MCMC in MrBayes
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Protein similarity
networks reveal
dominant and “invader”
types
• Sequential clustering
done using 99% and 97%
identities respectively
•At least 3 types
maintained over space
and distance
• Years of 2008 and 2011
show influx of new types
2007
2008
2009
2010
2011
2012
2013
Hopkins
Point Lobos
New Zealand
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2007
2008
2009
2010
2011
2012
2013
Hopkins
Point Lobos
New Zealand
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http://ggweather.com/enso/oni.htm
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+ Conclusions
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Different analysis methods converged to the same results
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We can see difference between sites and years
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However we also see maintenance of housekeeping genes over sites
and over years
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El niño in winter 2009
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Structured (anemone) vs. unstructured (water)
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ompK appear more conserved than initially thought
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However this might be influenced by the exclusion of indels
We see a similar pattern between ompK and the housekeeping
genes
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Future work
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Sample new sites and/or sample more
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More metadata on environmental factors
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Look at the variable regions of ompK
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Thanks All’y’all

Christina Machak
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Koshlan Mayer-Blackwell
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Paul Rainey
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Alfred Spormann
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Chrisopher Francis
+ WATER-Samples
NEW
ZEALAND
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CALIFORNIA
Allele Frequency at
recA for o (n=8)
Allele Frequency
at recA for Pop1
(n=51)
Allele Frequency at
recA for w (n=7)
Allele Frequency at
recA for Pop2
(n=42)
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K=3
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K=4
K=5
New Zealand W, Water
New Zealand W, Anemone
New Zealand N, Anemone
New Zealand O, Water
New Zealand O, Anemone
New Zealand B, Anemone
Point Lobos Water
Point Lobos Anemone
Hopkins Water
Hopkins Anemone
New Zealand
East
New Zealand
West
Californi
a
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Conclusions