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Origins of the modern reductive tricarboxylic acid cycle in the evolution of life

Authors:

Abstract

Fundamental to astrobiology is origin of autotrophy with reductive TCA cycle (rTCA) being often proposed as a primordial carbon fixation pathway. In this study we examined evolution of citrate cleavage reaction in rTCA done either by citryl-CoA synthase (CCS) and citryl-CoA ligase (CCL) with the release of citryl-CoA intermediate or by ATP citrate lyase (ACL), a result of fusion between the two. We analyzed sequences from 60 mostly novel prokaryotic taxa identified to possess either CCS/CCL or ACL and examined various metabolic characteristics of these organisms. We found that most of CCS/CCL-possessing taxa were thermophilic and aerobic compared to ACL and fraction of taxa possessing a complete version of oTCA was the same in both groups. Our phylogenetic analysis of CCS/CCL, ACL and their homologs succinyl-CoA synthetase (SCS) and citrate synthase (CS) shows that SCS from distant bacterial taxa capable of citrate cleavage are monophyletic. We also found that Cyanobacterial SCS originated from a horizontal gene transfer from Archaea. Overall, our results support a hypothesis that ancient origins of cyclical rTCA is unlikely and highlight the role of syntrophy in the evolution of life with the example of Nanohaloarchaea-Haloarchaea symbiosis that as we show, could be capable of rTCA.
Origins of the modern reductive tricarboxylic acid cycle in the evolution of life
Tymofii Sokolskyi1,2, Shiladitya DasSarma2,3
1Botany department, University of Wisconsin-Madison, Madison, WI, USA; sokolskyi@wisc.edu, 2Blue Marble Space
Institute of Science, Seattle, WA, USA, 3 Institute of Marine and Environmental Technology, University of Maryland
School of Medicine, Baltimore, MD, USA
Introduction: Better understanding of the evolution of
life on Earth is essential to astrobiology. This includes the
transition from protocells to the last universal common
ancestor, with establishment of chemiosmotic coupling and
metabolic pathways as essential life processes. We have
proposed the Purple Earth hypothesis, which posits that
retinal-based phototrophy preceded photosynthesis and
provided an early mechanism for generating proton motive
gradients for cellular bioenergetics (DasSarma &
Schwieterman, 2021). From this perspective, we sought to
evaluate ifthe reductive tricarboxylic acid cycle (rTCA;
Figure 1, A) may be a primordial carbon fixation pathway
(Becerra et al., 2014).
Results: We examined the evolution of the rarest of
rTCA reactions citrate cleavage via citryl-CoA synthase
(CCS) and citryl-CoA ligase (CCL) with the release
of citryl-CoA intermediate or by ATP citrate lyase (ACL), a
result of fusion of the two enzymes (Figure 1, B) (Aoshima,
2007). We identified sequences of citrate cleavage enzymes
in the proteomes of multiple archaeal and bacterial groups
for which it was previously unknown and found that most of
CCS/CCL-possessing taxa were thermophilic and oxygen-
tolerant compared to ACL and fraction of taxa possessing a
complete version of oxidative TCA (oTCA) was the same in
both groups. We also found that some Haloarchaea possess
all but one rTCA enzymes, missing citrate cleavage, which
is the most complete rTCA pathway in any Archaea.
Our phylogenetic analysis of polypeptide sequences of
CCS/CCL, ACL and CCS homolog succinyl-CoA
synthetase (SCS) and CCL homolog citrate synthase (CS)
shows that SCS from distant bacterial taxa capable of citrate
cleavage form a monophyletic clade, while their CS is
mostly lacking. Additionally, we provide phylogenetic
evidence that Cyanobacteria acquired their SCS horizontally
from some groups of anaerobic thermophilic Archaea.
Overall, we suggest that these results support a hypothesis
that complete cyclical rTCA originated relatively late in the
evolution of life, at least in its modern form in Bacteria
(Becerra et al., 2014). 
Discussion: Multiple studies suggested ancient origins
of rTCA, implying it might even be a primordial carbon
fixation pathway in early organisms due to the possibility of
non-enzymatic catalysis (Zubarev et al., 2015). There also is
growing body of evidence that the Last Universal Common
Ancestor (LUCA) was capable of autotrophic carbon fixation
(Sutherland, 2017), implying a possibility that rTCA could
have been involved in LUCA’s metabolism. However, our
phylogenetic analyses demonstrate that enzymes catalyzing
citrate cleavage, one of the key reactions of rTCA, were
obtained by all modern taxa known to use this pathway
through horizontal gene transfers. Additionally, we identify
several new taxa for which operation of a complete rTCA
cycle is possible (Syntrophobacterales, Calditrichales, Ca.
Acidulodesulfobacterales, Desulfofundulus, Beggiatoa) and
several taxa for which it is possible through syntrophic
associations, particularly in Asgard Archaea and
Haloarchaea-Nanohaloarchaea symbioses (La Cono et al.,
2020).
References:
Aoshima, M. (2007) Appl Microbiol Biotech 75, 249.
Becerra, A. et al. (2014) Int Microbiol, 17, 91.
DasSarma, S. and Schwieterman, E.W. (2018) Int J
Astrobiol, 20, 241.
La Cono, V. et al. (2020) Proc Natl Acad Sci, 117, 20223.
Sutherland, J. (2017) Nature Rev Chem, 1(2), 1-7.
Zubarev, D. et al. (2015) Scientific reports, 5(1), 1-7.
Figure 1. A – scheme of rTCA reactions. B evolutionary
relationships between citrate cleavage enzymes based on
Aoshima, 2007 and confirmed by our study.
A
B
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