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PNAS
2022 Vol. 119 No. 49 e2210539119
https://doi.org/10.1073/pnas.2210539119
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RESEARCH ARTICLE
|
Significance
The emergence of biological
novelty is often coupled to the
evolution of Earth’s chemical
environment. Here, we studied
how the evolution of a bacterial
CO
2
-concentrating mechanism
(CCM)—a complex,
multicomponent system that
enables modern CO
2
-fixing
bacteria to grow robustly in
environments with low CO
2
depends on environmental CO
2
levels. Using a “synthetic
biological” approach to assay the
growth of the present-day
bacteria engineered to resemble
ancient ones, we show that it is
possible to explain the
emergence of bacterial CCMs if
atmospheric CO
2
was once much
higher than today, consistent
with geochemical proxies. Taken
together, our results delineated
an unexpected “CO
2
-catalyzed”
pathway for the evolution of
bacterial CCMs, whose multiple
emergence has been challenging
to understand.
Author contributions: A.I.F., E.D., W.W.F., and D.F.S.
designed research; A.I.F., E.D., J.P., J.J.D., and L.M.O.
performed research; S.W.S. contributed new reagents/
analytic tools; A.I.F., E.D., J.P., J.J.D., L.M.O., W.W.F., and
D.F.S. analyzed data; and A.I.F. and E.D. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Copyright © 2022 the Author(s). Published by PNAS.
This open access article is distributed under
Creative
Commons Attribution License 4.0 (CC BY)
.
1
A.I.F. and E.J.D. contributed equally to this work.
2
To whom correspondence may be addressed. Email:
aflamhol@caltech.edu or savage@berkeley.edu.
This article contains supporting information online at
https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.
2210539119/-/DCSupplemental
.
Published December 1, 2022.
OPEN ACCESS
MICROBIOLOGY
ENVIRONMENTAL SCIENCES
Trajectories for the evolution of bacterial CO
2
-concentrating
mechanisms
Avi I. Flamholz
a
,b,c,1,2
, Eli Dugan
1
,a
, Justin Panich
d
, John J. Desmarais
a
, Luke M. Oltrogge
a
, Woodward W. Fischer
c
,e
, Steven W. Singer
d
, and
David F. Savage
a
,f
,g
,2
Edited by François Morel, Princeton University, Princeton, NJ; received June 24, 2022; accepted October 24, 2022
Cyanobacteria rely on CO
2
-concentrating mechanisms (CCMs) to grow in today’s
atmosphere (0.04% CO
2
). These complex physiological adaptations require ≈15 genes
to produce two types of protein complexes: inorganic carbon (Ci) transporters and 100+
nm carboxysome compartments that encapsulate rubisco with a carbonic anhydrase
(CA) enzyme. Mutations disrupting any of these genes prohibit growth in ambient air.
If any plausible ancestral form—i.e., lacking a single gene—cannot grow, how did the
CCM evolve? Here, we test the hypothesis that evolution of the bacterial CCM was
“catalyzed” by historically high CO
2
levels that decreased over geologic time. Using an
E. coli
reconstitution of a bacterial CCM, we constructed strains lacking one or more
CCM components and evaluated their growth across CO
2
concentrations. We expected
these experiments to demonstrate the importance of the carboxysome. Instead, we
found that partial CCMs expressing CA or Ci uptake genes grew better than controls
in intermediate CO
2
levels (≈1%) and observed similar phenotypes in two autotrophic
bacteria,
Halothiobacillus neapolitanus
and
Cupriavidus necator
. To understand how
CA and Ci uptake improve growth, we model autotrophy as colimited by CO
2
and
HCO
3
, as both are required to produce biomass. Our experiments and model deline-
ated a viable trajectory for CCM evolution where decreasing atmospheric CO
2
induces
an HCO
3
deficiency that is alleviated by acquisition of CA or Ci uptake, thereby
enabling the emergence of a modern CCM. This work underscores the importance
of considering physiology and environmental context when studying the evolution of
biological complexity.
carbon fixation | evolution | photosynthesis | Earth history | synthetic biology
Nearly all carbon enters the biosphere through CO
2
fixation in the Calvin–Benson–Bassham
(CBB) cycle. Rubisco, the carboxylating enzyme of that pathway, is often considered inef-
ficient due to relatively slow carboxylation kinetics (1–3) and nonspecific oxygenation of
its five-carbon substrate ribulose 1,5-bisphosphate, or RuBP (4, 5). However, rubisco arose
more than 2.5 billion years ago, when the Earth’s atmosphere contained virtually no O
2
and, many argue, far more CO
2
than today (6, 7). Over geologic timescales, photosynthetic
O
2
production (6), organic carbon burial (8), and CO
2
-consuming silicate weathering
reactions (9) caused a gradual increase in atmospheric levels of O
2
(≈20% of 1 bar atmos-
phere today) and depletion of atmospheric CO
2
to the present-day levels of a few hundred
parts per million (≈280 ppm preindustrial, ≈420 ppm or ≈0.04% today). Historical CO
2
levels are challenging to estimate, but are thought to have been substantially higher than
today, perhaps as high as 0.1–1 bar early in Earth history (7). It is likely, therefore, that
contemporary autotrophs grow on much lower levels of CO
2
than their ancestors did.
Many CO
2
-fixing organisms evolved CO
2
-concentrating mechanisms (CCMs), which
help meet the challenge of fixing carbon in a low CO
2
atmosphere. CCMs concentrate CO
2
near rubisco and are found in several varieties in all Cyanobacteria, some Proteobacteria, as
well as many eukaryotic algae and diverse plants (10). Because CO
2
and O
2
addition occur
at the same active site in rubisco (4), elevated CO
2
has the dual effects of accelerating car-
boxylation and suppressing oxygenation of RuBP by competitive inhibition (10). As shown
in Fig. 1
A
, bacterial CCMs are encoded by ≈15 genes comprising three primary features:
Zi) an energy-coupled inorganic carbon (Ci) transporter at the cell membrane and ii) a
cytosolic 100+ nm protein compartment called the carboxysome that iii) coencapsulates
rubisco with a carbonic anhydrase (CA) enzyme (2, 11). Energized Ci transport produces
a high HCO
3
concentration in the cytosol (≈30 mM, Fig. 1
A
), which is converted into a
high carboxysomal CO
2
concentration by CA activity, localized exclusively to the carbox-
ysome (12, 13). While HCO
3
-
undergoes spontaneous dehydration to CO
2
, the uncatalyzed
reaction is slow enough (≈10s equilibration times) that HCO
3
-
can enter the carboxysome
diffusively, where it undergoes rapid CA-catalyzed dehydration (10). This description applies
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to both varieties of bacterial CCM: the
α
-carboxysome variety
found in Proteobacteria and many Cyanobacteria, as well as the
β
form found exclusively in Cyanobacteria (11, 14, 15).
CCM genes are straightforward to identify experimentally as
mutations disrupting essential CCM components prohibit
growth in ambient air (Fig. 1
B
) and mutants are typically grown
in 1% CO
2
or more (16–20). At first glance, therefore, the CCM
appears to be “irreducibly complex” as all plausible recent ances
-
tors—e.g., strains lacking individual CCM genes—are not viable
in the present-day atmosphere. Irreducible complexity is incom
-
patible with evolution by natural selection, so we and others
supposed that bacterial CCMs evolved over a protracted interval
of Earth history when atmospheric CO
2
concentrations were
much greater than that today (10, 21–23). We therefore hypoth
-
esized that ancestral forms of the bacterial CCM (i.e., those
lacking some genes and complexes required today) would have
improved organismal growth in the elevated CO
2
environments
that prevailed when they arose.
To test the hypothesis, we constructed the present-day analogs
of plausible CCM ancestors (henceforth “analogs of ancestral
CCMs”) and tested their growth across a range of CO
2
partial
pressures. Note that we have not endeavored to generate precise
reconstructions of ancient taxa—indeed, we lack sufficient infor-
mation about the organisms and their environments to do so.
Rather, we aimed to identify the key components that appear in
all CCMs (10) and ask whether or not they improve growth on
their own in some defined CO
2
concentration. Our goal was to
identify a stepwise pathway of gene acquisition supporting the
evolutionary emergence of a bacterial CCM by improving growth
in ever-decreasing CO
2
concentrations (Fig. 1
C
). We focused on
trajectories involving sequential acquisition of genetic components
because CAs (24), Ci transporters (20), and homologs of carbox-
ysome shell genes (14, 25) are widespread among bacteria and
could therefore be acquired horizontally.
One approach to constructing contemporary analogs of CCM
ancestors is to remove CCM genes from a native host. If CCM
components were acquired sequentially, some single-gene knock-
outs would be analogous to recent ancestors, e.g., those lacking a
complete carboxysome shell (26). We tested this approach by
assaying a whole-genome knockout library of a
ɣ
-proteobacterial
chemoautotroph,
H. neapolitanus,
in five CO
2
partial pressures
(20, 27). As shown in Fig. 2 and elaborated below, we found that
many CCM genes contribute substantially to growth even at CO
2
concentrations tenfold greater than the present-day atmosphere
(0.5% CO
2
, ≈12.5 times the present atmospheric levels in 2020,
PAL), supporting the view that CCM components play an impor
-
tant physiological role even in relatively high environmental CO
2
concentrations.
B
A
C
Fig. 1.
Mechanism and potential routes for the evolution of the bacterial CO
2
-concentrating mechanism. (
A
) Today, the bacterial CCM functions through the
concerted action of three primary features - (i) an inorganic carbon (Ci) transporter at the cell membrane, and (ii) a properly-formed carboxysome structure (iii)
co-encapsulating rubisco with carbonic anhydrase (CA). Ci uptake leads to a high intracellular HCO
3
concentration, well above equilibrium with the external
environment. Elevated HCO
3
is converted to a high carboxysomal CO
2
concentration by CA activity located only there, which promotes carboxylation by
rubisco. (
B
) Mutants lacking genes coding for essential CCM components grow in elevated CO
2
but fail to grow in ambient air, as shown here for mutations
to the
α
-carboxysome in the proteobacterial chemoautotroph
H. neapolitanus
. Strains lacking the carboxysomal CA (
Δ
csosCA
) or an unstructured protein
required for carboxysome formation (
Δ
csos2
) failed to grow in ambient air, but grew robustly in 5% CO
2
(>10
8
colony-forming units/ml,
SI Appendix
, Fig. S1
). See
SI Appendix
, Table S4
for description of mutant strains. (
C
) We consider the CCM to be composed of three functionalities beyond rubisco itself: a CA enzyme
(magenta), a Ci transporter (dark brown), and carboxysome encapsulation of rubisco with CA (light brown). If CO
2
levels were sufficiently high, primordial CO
2
-
fixing bacteria would not have needed a CCM. We sought to discriminate experimentally between the six sequential trajectories (dashed arrows) in which CCM
components could have been acquired.
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2022 Vol. 119 No. 49 e2210539119
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Removing single CCM genes from a native host can only produce
analogs of recent ancestors, however. We recently constructed a func
-
tional
α
-carboxysome CCM in an engineered
E. coli
strain called
CCMB1 (2). This strain depends on rubisco carboxylation for
growth and expression of a full complement of CCM genes from
the chemoautotroph
H. neapolitanus
enabled growth in ambient air.
Here, we used CCMB1 to construct analogs of ancestral CCMs,
including several lacking one or more essential components of mod
-
ern CCMs. We assayed the growth of these putative ancestors across
a range of CO
2
pressures to determine whether any ancestral forms
contribute to organismal fitness—i.e., improve growth relative to a
control strain expressing only rubisco—across a range of CO
2
pres-
sures. We had expected these experiments to highlight the central
role of the carboxysome compartment (23), but instead found that
CAs and/or Ci uptake systems were likely early drivers of CCM
evolution. In the following sections, we describe these experiments,
discuss how they highlight the central role of bicarbonate (HCO
3
)
in all metabolisms, and comment on how these results can inform
our entwined understandings of bacterial physiology, CCM evolu-
tion, and the CO
2
content of Earth’s ancient atmosphere.
Results and Discussion
H. neapolitanus
CCM Genes Contribute to Fitness Even in Elevated
CO
2
.
Using barcoded genome-wide transposon mutagenesis, we
previously demonstrated that a 20-gene cluster in
H. neapolitanus
contains all the genes necessary for a functional CCM (2, 20). Our
original screen measured the effect of gene disruption across the entire
genome via batch competition assays, comparing the abundance of
disruptive mutants in high CO
2
(5%, ≈125 PAL) and ambient air
(≈0.04%) via high-throughput sequencing (20, 28). If the relative
abundance of mutants in a particular gene decreased reproducibly
in ambient air, but not in 5% CO
2
, we concluded that the gene is
linked to autotrophic growth in ambient air and, therefore, likely
participates in the CCM. Our mutant “library” includes an average
≈35 distinct mutants per gene, so each “mutant fitness assay” contains
multiple internal biological replicates.
To mimic the changes in atmospheric CO
2
that likely occurred
over Earth history, we assayed the same library in three additional
CO
2
pressures to cover five CO
2
levels: ambient (≈0.04% CO
2
),
low (0.5%, 12.5 PAL), moderate (1.5%, 37.5 PAL), high (5%,
125 PAL), and very high (10%, 250 PAL). Replicate experiments
were strongly correlated (R > 0.85,
SI Appendix
, Fig. S2
), implying
a high degree of reproducibility. We, therefore, proceeded to ask
whether
H. neapolitanus
CCM genes contribute to growth in a
range of CO
2
concentrations.
Fig. 2 plots the effect of disrupting CCM genes across five CO
2
pressures, with genes grouped by their documented roles in the
CCM. Each point in Fig. 2
A
represents the average fitness of 5–50
individual mutants. Surprisingly, we found that many CCM genes
also contributed substantially to growth in 0.5% and 1.5% CO
2
,
as indicated by large growth defects in disruptive mutants (nega-
tive values in Fig. 2
A
), resulting in a negative average impact of
CCM mutants when the CO
2
pressure was 1.5% or less (Fig. 2
B
).
Carboxysome genes, for example, were critical for growth in 0.5%
CO
2
, while certain Ci transport genes contributed substantially
to growth in 0.5% and 1.5% CO
2
.
In high CO
2
, however, the
H. neapolitanus
CCM appears to be
entirely dispensable (5–10%, Fig. 2
B
). As such, the data presented
in Fig. 2 indicated that individual CCM components such as the
AB
Fig. 2.
H. neapolitanus
CCM genes contribute to growth even in super-ambient CO
2
concentrations.
H. neapolitanus
is a chemoautotroph that natively utilizes
a CCM in low CO
2
environments. We profiled the contributions of CCM genes to autotrophic growth across a range of CO
2
levels by assaying a barcoded
transposon mutant library (20) in sequencing-based batch culture competition assays (
Methods
). Mutational effects were estimated as the log
2
ratio of strain
counts between the end point sample (e.g., 0.5% CO
2
) and the 5% CO
2
preculture for each barcoded mutant (20, 28). As the library contained an average of ≈40
mutants per CCM gene, each point in (
A
) gives the average effect of multiple distinct mutants to a single CCM gene in a given CO
2
condition. A value of log
2
(n/n
0
)
= −2 therefore indicates that gene disruption was, on average, associated with a fourfold decrease in mutant abundance as measured by Illumina sequencing of
mutant strain barcodes (20, 28). Negative values indicate that the gene, e.g., the carboxysomal CA, contributes positively to the growth of wild-type
H. neapolitanus
in the given condition. Observed log
2
(n/n
0
) values indicated that many
H. neapolitanus
CCM genes contribute to growth in super-ambient CO
2
concentrations,
including genes coding for Ci uptake, carboxysome shell proteins and the carboxysomal CA. Biological replicates are indicated by shading, and the gray bar
gives the interquartile range of fitness effects for all ≈1700 mutants across all CO
2
levels (−0.15-0.065). Replicates were highly concordant (
SI Appendix
, Fig. S2
).
“Ci transport mutants” include 4 DAB genes in two operons (20), “carboxysome” includes 6 nonenzymatic carboxysome genes, “rubisco mutants” denote the two
subunits of the carboxysomal rubisco, and “CA mutants” denote the carboxysomal CA gene
csosCA
.
H. neapolitanus
also expresses a secondary rubisco, which
explains why disruption of the carboxysomal rubisco is not lethal in high CO
2
(
SI Appendix
, Fig. S3
). Panel (
B
) gives the average mutational effect of CCM genes
as a function of the CO
2
concentration. Negative average values in 0.5-1.5% CO
2
highlight the positive contribution of CCM genes to growth in these conditions.
See
SI Appendix
, Figs. S2 and S3
for analysis of reproducibility and
SI Appendix
, Tables S1–S3
for detailed description of genes.