Supplementary Information for
Trajectories for the evolution of bacterial CO
2
-concentrating
mechanisms
Avi I. Flamholz
1,2,3,
*
,†
, Eli Dugan
1,
*, Justin Panich
4
,
John J. Desmarais
1
, Luke M. Oltrogge
1
, Woodward W.
Fischer
3,5
, Steven W. Singer
4
, David F. Savage
1,6,†
1
Department of Molecular and Cell Biology, University
of California, Berkeley,
California
94720,
United
States
2
Division of Biology and Biological Engineering,
California Institute of Technology, Pasadena, CA 91125
3
Resnick Sustainability Institute, California Institute
of Technology, Pasadena, CA 91125, USA
4
Biological Systems and Engineering Division, Lawrence
Berkeley National Laboratory, Berkeley, CA
94720, USA
5
Division of Geological & Planetary Sciences, California
Institute of Technology, Pasadena, CA 91125
6
Howard Hughes Medical Institute, University of California,
Berkeley, California 94720
* Equal contribution
†
Corresponding Authors: Avi I. Flamholz, David F.
Savage
Emails:
aflamhol@caltech.edu
,
savage@berkeley.edu
This PDF file includes:
Supplementary text
Figures S1 to S14
SI References
Other supplementary materials for this manuscript include the following:
Supplementary Tables S1-S5
Supplementary Methods
3
Plasmid Construction
3
Electroporation of CCMB1
E. coli
3
Manipulation of the C. necator genome
3
Plasmid transformation of C. necator
4
Modeling the co-limitation of autotrophic growth
5
Carbonic anhydrase cannot reasonable act as a CO2 pump alone
5
A model of autotrophy including the HCO3--dependence of growth
6
Choosing realistic ranges for parameter values
8
On the requirement for bicarbonate for biosynthesis
9
Supplementary Figures
11
Supplementary References
25
Supplementary Methods
Plasmid Construction
Genes
of
interest
were
amplified
by
PCR
and
cloned
into
their
respective
vectors
using
Gibson
Assembly.
Plasmids
were
then
transformed
into
chemically
competent
NEB
Turbo
E.
coli
cells
in
most
cases.
Single
colonies
were
inoculated
into
5-8
mL
LB
media
with
appropriate
antibiotics
and
mini-prepped
once
turbid
(Qiagen
QIAPrep
spin
kit).
For
the
construction
of
p1Ac,
which
constitutively
expresses
prk
,
we
used
CCMB1
as
the
cloning
strain
(1)
.
In
addition,
to
ensure
that
prk
expression
was
not
deleterious,
as
it
is
for
wild-type
E.
coli
(2)
,
CCMB1:p1Ac
transformants
were
cultured
in
a
prk
-dependent
manner
M9
glycerol
media.
Plasmid
sequences
were
verified
by
Sanger
sequencing
at
the
UC
Berkeley
DNA
sequencing
facility.
Details
of
plasmids
used
in
this
study
are
documented
in
Table
S5
and
plasmids
have
been deposited to Addgene at
https://www.addgene.org/David_Savage/
.
Electroporation of CCMB1
E. coli
Electrocompetent
CCMB1
stocks
were
prepared
by
standard
methods
from
cultures
grown
in
LB
media
under
10%
CO
2
.
Plasmids
were
transformed
via
electroporation
with
the
following
protocol.
A
50
μ
l
aliquot
of
electrocompetent
CCMB1
was
placed
on
ice
until
thawed.
100
ng
of
mini-prepped
plasmid
(100
ng
each
if
a
double
transformation)
was
then
added,
gently
mixed,
and
left
to
incubate
for
10
minutes.
The
transformation
aliquot
was
subsequently
transferred
to
a
chilled
1
mm
cuvette
(Biorad
Gene
Pulser)
and
pulsed
in
a
Gene
Pulser
Xcell
Microbial
System
electroporator
(1800
V,
200
Ω
,
25μF).
500
μ
l
SOC
was
added
to
the
cuvette
and
the
resulting
culture
was
pipetted
into
a
14
ml
round-bottom
falcon
tube
and
placed
in
10%
CO
2
to
incubate
for
1
hour.
Elevated
CO
2
was
found
to
be
critical
to
ensure
that
CCMB1
recovery
is
independent
of
the
transformed
plasmid(s).
After
incubation,
200
μ
l
of
the
culture
was
plated
on
an
LB
agar
plate
with
appropriate
antibiotics.
When
preparing
S17
E.
coli
donor
cells
for
conjugation
with
C.
necator
,
plasmids
were
transformed
by
the
same
method,
except
the
recovery was done in ambient CO
2
for 30 minutes.
Spotting assays of CO
2
dependent viability
Figure
1B
and
Figure
S8
report
spotting-based
titer
plating
assays
of
H.
neapolitanus
and
CCMB1
E.
coli
strains
respectively.
For
these
assays,
precultures
of
the
respective
strains
were
grown
in
liquid
media
in
high
CO
2
.
Stationary
phase
precultures
were
diluted
to
a
defined
optical
density
(600
nm,
Genesys
20
spectrophotometer,
Thermo
Scientific)
after
which
they
were
tenfold
serial
diluted
in
media
a
96-well
plate.
3
uL
of
tenfold
serial
dilutions
were
then
spotted
on
agar
plates
with
appropriate
antibiotics.
Plates
were
dried
in
a
laminar
flow
hood
before
and
after
spotting.
After
the
spots
dried,
plates
were
incubated
in
the
reported
CO
2
conditions
in
a
CO
2
controlled
incubator
(S41i,
New
Brunswick).
After
growth
for
a
defined
period
of
time,
colonies
were
counted
in
the
highest
dilution
to
show
>
1
colony
and
colony
forming
units
(CFU/OD/mL)
were
back-calculated
from
the
dilution
factor,
spotted
volume
and
optical
density.
Precultures
for
the
H.
neapolitanus
spotting
assay
reported
in
Figure
1B
were
grown
in
DSMZ-68
media
in
5%
CO
2
on
a
platform
shaker
(New
Brunswick
Scientific
Innova
2000,
200
RPM)
in
a
Percival
Intellus
Incubator
and
then
washed
and
diluted
to
OD
0.1
in
DSMZ-68
lacking
pH
indicator
and
thiosulfate
before
spotting.
Tenfold
dilutions
were
plated
on
DSMZ-68
agar
plates
supplemented
with
spectinomycin
(10
μ
g/mL)
for
the
mutants
but
not
the
wild-type,
which
lacks
a
resistance
marker.
Precultures
for
the
E.
coli
experiment
reported
in
Figure
S8
were
grown
in
10%
CO
2
in
a
CO
2
controlled
incubator
(S41i,
New
Brunswick)
in
M9
glycerol
media
supplemented
with
30
μ
g/ml
kanamycin,
12.5
μ
g/ml
chloramphenicol.
E.
coli
strains
that
cannot
grow
in
minimal
medium
(e.g.
CCMB1
strains
lacking
rubisco)
were
precultured
in
LB
media
in
10%
CO
2
.
Cultures
were
then
washed
and
diluted
in
M9
media
with
no
carbon
source
(i.e.
without
glycerol)
and
spotted
onto
M9
glycerol
plates
supplemented
with
12.5
μ
g/ml
chloramphenicol.
Both
E.
coli
and
H.
neapolinatus
plates
were
incubated
for
4
days
before
counting.
The
relevant
strains
and plasmids are documented in Tables S4 and S5 respectively.
Manipulation of the
C. necator
genome
The
knockout
mutant
C.
necator
Δ
A0006
Δ
can
Δ
caa
was
produced
by
iterative
rounds
homologous
recombination
(to
generate
a
desired
mutation)
followed
by
sacB
counterselection
to
cure
the
kanamycin
resistance
marker
integrated
at
the
target
locus
(3)
.
Homologous
recombination
was
achieved
by
conjugation
with
E.
coli
S17
carrying
a
mobilizable
vector
encoding
500
bp
homology
arms
flanking
a
cassette
encoding
kanamycin
resistance
and
sacB
counter
selection.
For
each
individual
knockout,
a
pKD19-mobSacB
plasmid
was
generated
with
500
bp
homology
arms
directly
flanking
the
target
gene.
This
plasmid
was
transformed
into
C.
necator
by
conjugation
with
E.
coli
S17
and
plated
onto
LB
agar
supplemented
with
200
μ
g/ml
kanamycin
to
select
for
integrants
and
10
μ
g/ml
gentamicin
to
select
against residual
E. coli
.
Single
integrant
colonies
were
inoculated
into
LB
with
10
μ
g/ml
gentamicin
and
20
μ
g/ml
kanamycin
and
incubated
in
30
°C
until
turbid.
Genomic
integration
was
verified
by
colony
PCR
using
a
primer
set
where
one
primer
annealed
to
the
genome
and
the
other
primer
annealed
to
the
plasmid
backbone.
Verified
colonies
were
inoculated
into
salt-free
LB
(10
g/L
tryptone,
5
g/L
yeast
extract)
supplemented
with
10
μ
g/ml
gentamicin
and
100
mg/ml
sucrose
and
incubated
at
30
°C
for
48-72
hours
to
select
against
sacB
activity.
Strains
were
then
streaked
on
two
different
LB
plates:
one
without
NaCl,
but
containing
10
μ
g/ml
gentamicin
and
50
mg/ml
sucrose
and
a
second
plate
with
NaCl,
10
μ
g/ml
gentamicin
and
200
μ
g/ml
kanamycin.
Colonies
that
grew
on
sucrose
but
not
on
kanamycin
were
genotyped
by
colony
PCR
using
a
pair
of
primers
that
annealed
upstream
and
downstream
of
the
target
gene.
PCRs
were
run
on
an
agarose
gel
to
ensure
prospective
knockouts
were
not
wild-type
revertants.
The
final
strain,
C.
necator
Δ
A0006
Δ
can
Δ
caa
was
further
verified
by
phenotype:
it
fails
to
grow
heterotrophically
in
ambient
air,
but is able to grow under elevated CO
2
(4, 5)
.
Plasmid transformation of
C. necator
To
enable
routine
electroporation
of
plasmids
into
C.
necator
H16,
we
first
knocked
out
the
hdsR
homolog
A0006
as
removal
of
this
restriction
enzyme
increases
electroporation
efficiency
(3,
6)
.
Electrocompetent
stocks
of
C.
necator
Δ
A0006-
derived
strains
(including
the
various
knockouts)
were
made
according
to
a
protocol
from
(3)
with
the
following
modifications.
A
colony
of
the
strain
was
inoculated
into
LB
with
10
μ
g/ml
gentamicin.
Once
turbid,
the
pre-culture
was
added
to
100
mL
fresh
media
and
let
grow
until
it
reached
an
OD600
between
0.6-0.8.
Δ
A0006
was
grown
in
ambient
CO
2
and
Δ
A0006
Δ
can
Δ
can
was
grown
in
10%
CO
2
.
Cells
were
then
chilled,
shaking
in
an
ice
slurry
until
they
reached
4
°C.
The
culture
was
split
into
two
50
ml
Falcon
tubes
and
centrifuged
at
4000
g
for
10
minutes
at
4
°C.
The
supernatant
was
decanted
and
pellets
were
washed
twice
with
50
ml
ice
cold
sterile
water
and
once
with
50
ml
10%
glycerol.
The
pellets
were
then
resuspended
in
0.75
ml
10%
glycerol,
pooled,
and 100 ul aliquots were flash-frozen in liquid nitrogen for storage at -80 °C.
For
plasmid
transformation,
a
100
μ
l
aliquot
of
C.
necator
was
thawed
on
ice.
Upon
thawing,
500
ng
of
plasmid
was
added,
gently
mixed,
and
left
to
incubate
on
ice
for
5
minutes.
The
aliquot
was
then
transferred
to
a
1
mm
electroporation
cuvette
(Biorad
Gene
Pulser)
and
pulsed
in
a
Gene
Pulser
Xcell
Microbial
System
electroporator
(2300
V,
200
Ω
,
25μF).
The
sample
was
then
immediately
resuspended
in
1
ml
of
LB
supplemented
with
10
mg/ml
fructose,
transferred
into
a
14
ml
round-bottom
falcon
tube,
and
recovered
in
a
30
°C
for
2
hours
(H16
Δ
A0006
in
ambient
CO
2
,
H16
Δ
A0006
Δ
can
Δ
caa
in
10%
CO
2
).
200
μ
l
was
then
plated
on
LB
agar
plates
with
10
μ
g/ml
gentamicin,
200
μ
g/ml
kanamycin,
and
10
mg/ml
fructose
and
placed
in
a
30
°C
incubator
at
ambient
CO
2
or
10%
CO
2
(depending
on
the
strain)
for
48
hours.
Modeling, data and analysis
The dual-limitation model was elaborated in Mathematica 12 (Wolfram) and steady-state solutions were
translated to Python for further analysis and plotting. All data analysis was performed using Python 3.8
and Jupyter notebooks. Data and code required to generate all figures is available at
https://github.com/flamholz/ccm_evolution
.
Modeling the co-limitation of autotrophic growth
Carbonic anhydrase cannot reasonably act as a CO
2
pump alone
Our
model
considers
an
autotroph
with
no
CCM
that
uses
rubisco
to
fix
CO
2
in
an
environment
with
fixed
extracellular
CO
2
and
HCO
3
-
concentrations,
C
out
and
H
out
.
We
further
assume
that
these
extracellular
species
are
in
equilibrium
with
respect
to
the
pH,
i.e.
that
H
out
/C
out
=
K
EQ
(pH),
and
that
the
intracellular
pH
is
the
same
as
the
extracellular
pH
so
that
the
pH-dependent
equilibrium
constant
K
EQ
(pH)
is
equal
on
both
sides
of
the
cell
membrane.
This
assumption
of
equal
pH
equilibrium
is
not
required
but
simplifies
the
model
(7)
.
We
now
write
differential
equations
describing
the
time
evolution
of
the
intracellular
CO
2
and
HCO
3
-
concentrations,
C
in
and
H
in
,
at
first
ignoring
the
HCO
3
-
dependence
of
growth
to
illustrate
that
it must be included.
Since
CO
2
and
HCO
3
-
have
diffusion
constants
of
≈
10
3
μ
m
2
/s
in
water
,
corresponding
to
diffusion
timescales
of
R
2
/6D
≈
10
-4
s
over
the
≈
1
micron
lengths
of
bacterial
cells,
we
assume
that
their
concentrations
are
spatially
homogeneous
inside
and
outside
the
cell
(8)
.
While
cytoplasm
is
more
viscous
than
water
(9,
10)
,
these
effects
depend
on
the
size
of
the
diffusant.
Diffusion
constants
measured
for
smaller
molecules
(<
1
kDa)
are
about
fourfold
smaller
in
cytoplasm
than
in
water
(9)
,
which
does
not
affect
our
calculation
of
millisecond
diffusion
timescales
over
bacterial
cell
lengths.
We
also
assume
all
enzyme-catalyzed
reactions
have
first-order
kinetics,
i.e.
substrate
concentrations
are
substantially lower than Michaelis constants ([S]
≪
K
M
). These assumptions give the following equations:
푑
퐶
푖
푛
푑
푡
=
α
(
퐶
표
푢
푡
−
퐶
푖
푛
)
−
γ
퐶
푖
푛
−
(
δ
퐶
푖
푛
−
φ
퐻
푖
푛
)
푑
퐻
푖
푛
푑
푡
=
β
(
퐻
표
푢
푡
−
퐻
푖
푛
)
+
(
δ
퐶
푖
푛
−
φ
퐻
푖
푛
)
Here
we
treat
both
CO
2
and
HCO
3
-
as
entering
the
cell
passively
with
“effective
permeabilities”
ɑ
and
β
.
These
effective
permeabilities
account
for
the
surface
area
to
volume
ratio
of
bacterial
cells,
which,
for
rod
shaped
cells
around
the
size
of
E.
coli
,
is
(BNIDs
101792
and
114924
)
as
we
discuss
푆
퐴
/
푉
≈
4
μ
푚
−
1
below.
is
a
linearized
expression
for
rate
of
irreversible
CO
2
fixation
by
rubisco,
where
γ
퐶
푖
푛
assuming
a
Michaelis-Menten
formalism
and
C
in
≪
K
M
.
In
contrast
to
rubisco,
the
γ
=
푘
푐
푎
푡
[
푟
푢
푏
푖
푠
푐
표
]
/
퐾
푀
CA
reaction
is
reversible.
As
such,
the
balance
of
the
rates
of
CO
2
hydration
and
(
δ
퐶
푖
푛
−
φ
퐻
푖
푛
)
(
δ
퐶
푖
푛
)
HCO
3
-
dehydration
,
assuming
each
of
these
reactions
are
in
their
linear
regimes
as
well.
While
the
(
φ
퐻
푖
푛
)
assumption
of
linearity
is
not
required,
it
is
also
not
counterfactual:
typical
K
M
values
measured
for
bacterial
rubiscos
(11)
and
carbonic
anhydrases
(12)
are
comparable
to
equilibrium
concentrations
of
CO
2
and HCO
3
-
in water in equilibrium with ambient
air at 25 C (Figure S11).
We set both derivatives to 0 and solve for the steady-state values of C
in
and H
in
.
퐶
푖
푛
=
퐶
표
푢
푡
(
퐾
퐸
푄
β
φ
+
α
(
β
+
φ
)
)
β
(
α
+
γ
+
δ
)
+
φ
(
α
+
γ
)
퐻
푖
푛
=
퐶
표
푢
푡
(
α
δ
+
퐾
퐸
푄
β
(
α
+
γ
+
δ
)
)
β
(
α
+
γ
+
δ
)
+
φ
(
α
+
γ
)
If
we
further
assume
that
CA
activity
is
negligible,
i.e.
that
,
then
we
recover
the
solution
from
δ
,
φ
≈
0
our
simplified
main-text
calculation
where
is
independent
of
H
in
.
As
a
reminder,
we
used
this
퐶
푖
푛
=
퐶
표
푢
푡
α
α
+
γ
equation
to
calculate
that
in
the
absence
of
CA
activity,
even
when
rubisco
comprises
20%
퐶
푖
푛
>
0
.
9
퐶
표
푢
푡
of total protein.
The
above
calculation
implies
that
CA
expression
could
increase
C
in
by
at
most
10%
because
CAs
are
not
coupled
to
any
energy
source
and,
therefore,
cannot
increase
C
in
above
C
out
.
This
calculation
depends,
of
course,
on
the
rubisco
kinetics
and
expression
(
)
and
membrane
permeability
to
CO
2
(
).
Rubisco
γ
α
kinetics
have
been
studied
in
great
depth
and
are
well-constrained
(11,
13)
.
Similarly,
many
generations
of
physical
chemists
have
studied
the
permeability
of
lipid
membranes
to
small
molecules
and
developed
theory
to
estimate
membrane
permeabilities
(14–16)
.
Nonetheless,
membrane
permeabilities
can
depend
on
the
lipid
composition
of
the
membrane
and
the
complement
of
protein
channels
embedded
therein
(17)
.
Assuming
that
rubisco
fixation
is
the
sole
growth-limiting
reaction,
we
can
estimate
the
exponential
growth
rate
from
C
in
by
calculating
the
rubisco
fixation
rate
훾
C
in
≈
9x10
3
μ
M/s.
Here
we
took
C
out
≈
10
μ
M,
which
is
roughly
Henry’s
law
equilibrium
with
present-day
atmosphere
at
25
°C
(Fig.
S16),
훼
=
10
4
s
-1
and
훾
=
10
3
s
-1
.
We
expound
on
this
choice
of
values
in
the
main
text
and
below.
Assuming
a
cell
volume
of
≈
1
fL
(BNIDs
104843
,
100004
),
9x10
3
μ
M/s
equals
a
fixation
rate
of
roughly
5x10
6
CO
2
/s
or
≈
10
10
CO
2
/hr.
An
E.
coli
cell
of
this
volume
contains
≈
10
10
carbon
atoms
(BNID
103010
)
and
Cyanobacteria
do
not
differ
substantially
from
E.
coli
in
carbon
content
(compare
BNIDs
105530
and
111459
).
Therefore,
assuming
no
loss
of
fixed
carbon,
such
a
Cyanobacterium
would
double
once
an
hour.
Autotrophic
respiration,
which
equals
the
difference
between
gross
and
net
fixation,
is
typically
less
than
50%
of
gross
both
in
pure
cyanobacterial
cultures
(18)
and
natural
ecosystems
(19)
implying
a
doubling
time
of
at most 2 hours.
Given
the
model
articulated
above,
a
10%
increase
in
C
in
(e.g.
due
to
CA
expression)
can
increase
the
rubisco
carboxylation
rate
by
at
most
10%.
As
rubisco
is
required
for
producing
all
biomass
carbon
in
autotrophy,
a
10%
increase
in
the
rate
of
rubisco
carboxylation
can
increase
the
exponential
growth
rate
by
at
most
10%.
However,
in
Figures
S5-6
the
“rubisco
alone”
strain
did
not
meaningfully
grow
in
0.5%
CO
2
while
the
strains
expressing
a
CA
or
Ci
transporter
grew
robustly.
These
qualitative
effects
indicated
that
we
should
look
for
a
mechanism
that
can
improve
growth
by
more
than
≈
10%.
As
described
in
the
main
text
and
the
following
section,
the
cellular
demand
for
HCO
3
-
,
which
is
required
for
several
anabolic
carboxylation reactions
(20–23)
, is one such mechanism.
Notably,
CO
2
and
HCO
3
-
do
interconvert
spontaneously.
The
spontaneous
reaction
is
associated
with