1. Preliminary
experiments
with
P.
aeruginosa
show
that
there
are
very
few
pvDD
colonies
and
very
many
PA01
colonies
when
each
strain
is
grown
on
their
own.
The
number
of
colony-­‐forming
units
(CFUs)
is
calculated
from
the
colony
count.
Further
analysis
of
the
compeDDon
culture
by
comparing
the
pvDD
colonies
(cheater)
and
PA01
(cooperator)
for
each
experiment
reveals
that
the
number
of
pvDD
colonies
is
higher
in
the
compeDDon
cultures
as
opposed
to
the
pvDD
only
culture,
the
values
of
each
were
averaged.
InteresDngly,
the
50/50
culture
has
a
higher
percentage
of
pvDD
than
the
99/1.
Cheating within Pseudomonas aeruginosa
and its Relationship to Frequency-Dependence
Erica Ryu, Fredrik Inglis, David Queller, Joan Strassmann
Washington University in St. Louis
Introduction
Methods
Current and Future Experiments
Conclusions
Experiments
with
P.
aeruginosa
1. PA01
–
cooperator
strain;
pvDD
–
cheater
strain
(gene
necessary
for
siderophore
producDon
has
been
knocked
out)
2. Eight
trials
each
of
PA01
and
pvDD
strains
were
grown
separately
(control),
while
eight
trials
of
PA01
and
pvDD
were
mixed
and
therefore
grown
together
(experiment)
• The
bacteria
were
grown
in
iron-­‐deficient
CAA
media
and
incubated
for
24
hours
at
35ºC.
3. ASer
growth,
cultures
were
diluted
to
10-­‐6
and
spread
on
iron-­‐rich
plates
colonies
counted
under
a
UV
light
• Two
experiments
–
50/50
raDo,
and
99/1
raDo
(PA01/pvDD)
References and Acknowledgements
Results
CooperaDon
and
compeDDon
are
social
behaviors
that
are
essenDal
in
evoluDonary
biology
because
they
drive
certain
species
to
thrive
while
others
are
pushed
towards
exDncDon.
P.
aeruginosa
is
a
bacterium
that
exhibits
both
compeDDon
and
cooperaDon.
• In
iron-­‐deficient
environments,
the
bacteria
produce
siderophores,
which
are
parDcles
that
scavange
iron.
They
take
up
siderophores
bound
to
iron,
because
iron
is
a
necessary
component
for
survival.
• These
siderophore-­‐producing
bacteria
can
be
thought
of
as
cooperators
because
they
rarely
reabsorb
their
own
siderophores.
• Some
bacteria,
the
cheaters,
do
not
produce
siderophores,
yet
sDll
absorb
siderophores
carrying
iron.
(Griffin
2004).
Siderophore
Without
iron
Cheaters
should
outcompete
the
cooperators
because
they
have
more
energy
to
grow.
This
also
means
that
the
cheaters
should
grow
be[er
in
a
lower
concentraDon
compared
to
the
cooperators,
because
more
siderophores
will
be
present
in
the
media.
Thus,
it
is
possible
that
frequency-­‐dependent
selecDon
will
operate.
Ques0on:
How
does
frequency-­‐dependence
affect
compe00on
between
the
cheater
and
cooperator
strains
of
Pseudomonas
aeruginosa?
Predic0on:
Cooperators
grow
be@er
alone,
cheaters
will
outcompete
cooperators,
and
cheaters
will
grow
be@er
when
at
a
lower
concentra0on.
Griffin,
A.S.,
West,
S.A.,
Buckling,
A.
(2004).
CooperaDon
and
compeDDon
in
pathogenic
bacteria.
Nature,
430,
1024-­‐1027.
Thanks
to
the
Queller-­‐Strassmann
lab
at
Washington
University,
especially
Fred
Inglis
for
personally
mentoring
me.
I
would
also
like
to
thank
Tammy
Haselkorn
and
Joan
Strassmann
for
their
advice.
I
would
like
to
thank
Odion
Asikhia
for
being
a
wonderful
lab
partner.
Lastly,
I
would
like
to
thank
my
parents
for
helping
me
with
everything.
This
material
is
based
upon
work
supported
by
the
NaDonal
Science
FoundaDon
under
grant
numbers
IOS
1256416
and
DEB
1146375
and
the
John
Templeton
FoundaDon.
Future
P.
fluorescens
experiments
• PF3
and
PF2
grown
in
the
sori
of
D.
discoideum
with
other
amoeba
spores
• CompeDDon
cultures
grown
on
iron-­‐rich
agar
plates.
• Colonies
counted
in
order
to
determine
pvDD/PA01
and
therefore
the
effect
of
D.
discoideum
on
P.
fluorescens
compeDDon.
P.
fluorescens
tagging
• Tagging
of
P.
fluorescens
with
Mcherry
and
GFP
has
been
a[empted
but
is
not
yet
successful.
• Various
plasmid
extracDon
kits
have
been
used,
and
both
electroporaDon
and
conjugaDon
have
been
a[empted.
• A
high
quanDty
of
Mcherry
plasmid
has
been
obtained
(~400ng/µl).
• Because
pvDD
cannot
grow
in
a
iron-­‐deficient
without
PA01,
pvDD
is
the
cheater
and
PA01
is
the
cooperator.
• pvDD
grows
be[er
in
compeDDon,
but
not
as
much
as
expected.
• pvDD
grows
be[er
in
the
50/50
culture,
contradicDng
frequency
dependence
expectaDons.
• pvDD
does
not
outcompete
PA01,
contradicDng
compeDDon
expectaDons.
• Given
that
previous
studies
show
that
cheater
and
cooperator
strains
of
P.
aeruginosa
cannot
coexist,
these
results
likely
derive
from
contaminaDon
and
clumping
issues.
Cooperator
Cheater
Siderophore
with
iron
The
cheater-­‐cooperator
relaDonship
is
also
apparent
in
Pseudomonas
fluorescens,
a
bacterium
consumed
by
the
social
amoeba
Dictyostelium
discoideum.
The
strains
are
surprisingly
able
to
coexist,
countering
P.
aeruginosa
findings
from
previous
studies
(keeping
in
mind
that
my
results
are
probably
due
to
contaminaDon
issues).
It
is
possible
that
growth
with
D.
discoideum
is
affecDng
the
cheater
and
cooperator
populaDons,
which
is
why
P.
fluorescens
strains
are
able
to
coexist.