Quantifying Pseudonitzschia multiseries +

Bacteria Coculture Effects

Background
P. multiseries produces domoic acid, a neurotoxin
P. multiseries grows alongside bacteria in the
environment
Domoic acid production by P. multiseries does not
occur in axenic cultures (Kobayashi et. al.)
Suggests a mutualistic relationship between P.
multiseries and marine bacteria
Local P. multiseries
PC4 and PC9 were isolated from Puget Sound
Bacterial strains were isolated from original cultures
and frozen in the -80 freezer for later use.
Coculture: growing P. multiseries with an individual
bacterial strain (the inoculant) from the beginning
and observing effects on growth rate
Two phenotypic effects: synergistic and algicidal
Two Phenotypes: Synergistic and
Algicidal
one Sulfitobacter sp. SA11 increases P. multiseries
growth rate by 30%
Croceibacter atlanticus SA60 (from Puget Sound P.
multiseries culture) is potentially algicidal
Type strain BAA628 (from Sargasso Sea) did not
produce the same effect
Local Bacteria
CFU: colony forming unit; one cell
Bacteria grown in marine broth are measured by
absorbance
OD: optical density
Different bacterial strains may have different CFU
numbers for a given OD
Coculture effect may only be observable at a
particular CFU inoculation
Growing on a Curve
1. Dilute by 100,000x
2. Perform serial dilution
3. Plate 50uL of each serial dilution
4. Counted colonies = CFUs
Method

= #
0.05
y = 5E+09x - 9E+07
R = 0.996
-1.00E+08
0.00E+00
1.00E+08
2.00E+08
3.00E+08
4.00E+08
5.00E+08
6.00E+08
7.00E+08
8.00E+08
0 0.05 0.1 0.15 0.2
C
F
U
/
m
L
OD
SA60 CFU/mL vs OD at 1E-6
Dilution Factor
y = 4E+09x + 4E+08
R = 0.9974
0.00E+00
2.00E+08
4.00E+08
6.00E+08
8.00E+08
1.00E+09
1.20E+09
1.40E+09
0 0.05 0.1 0.15 0.2 0.25
C
F
U
/
m
L
OD
BAA628 CFU/mL vs OD at 1E-6
Dilution Factor
Goals
1. Determine at which inoculation concentration
Croceibacter atlanticus produces a noticeable
algicidal effect
2. Establish if other bacteria related to Sulfitobacter
have an effect on Pseudonitzschia growth rate.
A) Croceibacter atlanticus concentration dependence
Growth Curves for P. multiseries + SA60
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
0 10 20 30 40 50 60 70 80 90 100
L
n
(
R
F
U
)
Hours after Transfer
Natural Log of RFU vs Hours after Transfer
PC4ax
PC4ax+SA60x1.0
PC4ax+SA60x0.1
PC4ax+SA60x0.01
Growth Curves for P. multiseries + SA60
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
AX OD 1.0 OD 0.1 OD 0.01
A
v
e
r
a
g
e
d

G
r
o
w
t
h

R
a
t
e
Averaged Growth Rate/Day vs Added OD
Growth Curves for P. multiseries + BAA628
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
0 10 20 30 40 50 60 70 80 90 100
L
n
(
R
F
U
)
Hours after Transfer
Natural Log of RFU vs Hours after Transfer
PC4ax
PC4ax+BAA628x1.0
PC4ax+BAA628x0.1
PC4ax+BAA628x0.01
Growth Curves for P. multiseries + BAA628
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
AX x1.0 x0.1 x0.01
A
v
e
r
a
g
e
d

G
r
o
w
t
h

R
a
t
e
/
D
a
y
Averaged Growth Rate/Day vs Added OD
B) Synergistic bacteria coculturing
Marine Bacteria Phylogeny
GS35 = Ruegeria
mobilis F1926
GS36 =
Phaeobacter
gallaeciensis
BS107
SA44 & SA52 =
Sulfitobacter sp.
Marine Bacteria Phylogeny by 16S ribosomal
RNA
Growth Curves for P. multiseries + synergistic
bacteria
-1
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
0 20 40 60 80 100 120 140 160 180
L
n
(
F
R
U
)
Hours after Transfer
Natural Log of RFU vs Hours after Transfer
PC9ax
PC9ax+GS36
PC9ax+GS35
PC9ax+SA44
PC9ax+SA52
Marine Bacteria Phylogeny
GS35 = Ruegeria
mobilis F1926
GS36 =
Phaeobacter
gallaeciensis
BS107
SA44 & SA52 =
Sulfitobacter sp.
Marine Bacteria Phylogeny by 16S ribosomal
RNA
Further experiments
Perform Croceibacter atlanticus coculture with
different initial inoculant concentration
Check for reproducibility of SA44 coculture effects
(repeat experiments)
Sequence SA11, SA52
Same genus = easier time trying to figure out which
genes are responsible for the P. multiseries mutualism
More cocultures within and outside of the
Sulfitobacter genus