CO2-dependent carbon isotope fractionation in Archaea, Part I: Modeling the 3HP/4HB pathway
Introduction
Three new pathways of carbon fixation have been discovered in recent decades: the 3HP cycle of Chloroflexi (Strauss and Fuchs, 1993), the 3HP/4HB cycle of Archaea (Berg et al., 2007, Könneke et al., 2014), and the dicarboxylic/4-HB (Di/4HB) cycle of Archaea (Huber et al., 2008). These three, plus the globally dominant Calvin-Benson-Bassham pathway (CBB; Calvin and Benson, 1948), the reverse tricarboxylic acid (rTCA) cycle (Evans et al., 1966), and the Wood-Ljungdahl, or Acetyl-CoA, pathway (e.g., Wood et al., 1986) represent current knowledge of microbial autotrophy.
The rTCA, 3HP, 3HP/4HP, and Di/4HB pathways share the common property of being catalytic cycles in which one or more of the carbon-fixing enzymes is specific for as a substrate. The biomass of organisms containing these pathways is enriched in 13C (has higher values of δ13C) relative to biomass produced via the CBB and Acetyl-CoA pathways (Sirevag et al., 1977, Holo and Sirevag, 1986, van der Meer et al., 2001a, van der Meer et al., 2001b, House et al., 2003, van der Meer et al., 2003, Könneke et al., 2012, Jennings et al., 2014). Such organism-level biosynthetic isotope effects, or ε values, reflect the difference between total dissolved inorganic carbon (DIC) and the resulting biomass, and are reported as ε ≈ Δδ = δ13CDIC – δ13Cbiomass (Hayes, 1993, Hayes, 2001). The δ13C characteristics of this family of autotrophic pathways reflect both the enrichment of 13C in when in equilibrium with CO2 (Mook et al., 1974), plus the small (2–3‰) kinetic isotope effect (KIE) associated with enzymes specific for (O’Leary et al., 1981, McNevin et al., 2006). Here we specify εAr to mean the ε value for biomass of Archaea using the 3HP/4HB pathway.
The ca. 20‰ value of εAr for marine 3HP/4HB Thaumarchaeota, specifically the model organism N. maritimus (Könneke et al., 2005, Könneke et al., 2012), is markedly different from the ca. 3‰ value for the 3HP/4HB Sulfolobales (van der Meer et al., 2001a, House et al., 2003, Jennings et al., 2014). Genomic (Walker et al., 2010) and enzymatic (Könneke et al., 2014) evidence verify the 3HP/4HB metabolism in N. maritimus – i.e., it contains a single carbon-fixing enzyme known to be solely dependent on , not CO2 – yet isotopically, it is distinct.
The marked difference in εAr values between thermoacidophilic Sulfolobales and the ammonia-oxidizing marine Thaumarchaeota is well recognized (e.g., Könneke et al., 2012, Könneke et al., 2014) but has not yet been quantitatively explained. Explanation of the 20‰ εAr value in Thaumarchaeota generally invokes both direct enzymatic fractionation during carbon fixation (Könneke et al., 2012) and fractional contributions to biomass from 13C-depleted organic carbon, either by true mixotrophy or in communities that contain heterotrophs in addition to autotrophs (e.g., Herndl et al., 2005, Ingalls et al., 2006, Pearson et al., 2016). However, recent work indicates that N. maritimus and other ammonia-oxidizing marine Thaumarchaeota do not assimilate organic carbon into biomass and lack genes to acquire inorganic carbon from organic substrates like urea (Walker et al., 2010, Santoro et al., 2015, Kim et al., 2016). Therefore the ca. 20‰ value of εAr for N. maritimus in pure culture as determined by Könneke et al. (2012) must solely reflect autotrophy. By analogy, similar values of εAr in the marine environment (Schouten et al., 2013, Pearson et al., 2016, Hurley et al., 2019) suggest dominantly autotrophic processes as well. If so, prior reports of mixo- or heterotrophy may be attributed to artifacts of sampling or analysis.
Here we seek to reconcile these issues by modeling εAr for both N. maritimus and the well-studied member of the thermoacidophilic Sulfolobales, M. sedula. The results suggest that the extent of intracellular inorganic carbon disequilibrium may be the critical factor determining εAr values expressed by the 3HP/4HB cycle. Because the intracellular carbon budget depends on the supply of CO2, εAr should be sensitive to changes in carbon availability and growth rate. Our accompanying Part II paper supports this idea, showing that εAr as recorded in thaumarchaeal membrane lipids isolated from marine suspended particulate organic matter (POM) correlates significantly with the in-situ CO2 concentration (Hurley et al., 2019).
Section snippets
Isotope flux-balance model
We constructed a carbon isotope and flux-balance model for bulk cellular carbon resulting from fixation in the 3HP/4HB cycle (Fig. 1). The model follows established principles (e.g., Hayes, 2001, Tang et al., 2017) for interrogating stable isotope distributions in open systems and assumes steady state. Fluxes (φ) and kinetic isotope effects (KIEs, ε) are combined in a system of dependent linear equations of the form:where Xi is the fraction of carbon in
Simplified model, M. sedula
The predicted flux balances for M. sedula and N. maritimus are notably different in their magnitude of intracellular conversion between CO2 and (Fig. 2a). When normalized to carbon fixation rate, the hydration flux (φ4) for M. sedula is nearly 200-fold faster than for N. maritimus, resulting in dehydration backflow (B) ratios > 0.99 vs. ≤ 0.47, respectively (Table 1).
Rapid exchange of DIC species within M. sedula has isotopic consequences, namely, that Hi and Ci are predicted to approach
Physiological and evolutionary role of the 3HP/4HB pathway
Growth under conditions of extreme resource limitation is thought to be a common feature not only of the Archaea, but of Earth’s microbial biosphere in general (Valentine, 2007, Lever et al., 2015). While typically discussed in the context of energy limitation, here we argue that in the case of marine Thaumarchaeota, carbon limitation also likely plays an important role in their physiology, due to the apparent absence of a functional CA. The ammonia-oxidizing, marine Thaumarchaeota have a low
Conclusions
The flux-balance model developed here provides a simple explanation for why the carbon isotope signatures for 3HP/4HB pathway Sulfolobales and Thaumarchaeota are so different, despite both taxonomic groups of Archaea utilizing the same enzymatic pathway of autotrophic carbon fixation. The key observation is that the total biosynthetic isotope effect (εAr) in Thaumarchaeota is larger than the probable magnitude of the isotope effect for the carbon-fixing enzyme (εfix). This requires that the
Acknowledgements
We thank Martin Könneke, Jed Fuhrman, David Johnston, Emma Bertran, Itay Halevy, Rich Pancost, and Anne Dekas for valuable discussions. We are grateful to Joe Werne for editorial assistance and to three anonymous reviewers for their valuable comments. Funding from NSF-1129343, NSF-1702262, and the Gordon and Betty Moore Foundation (to A.P.); from National Science Foundation Graduate Research Fellowship 1144152 (to E.B.W.); and from the Agouron Institute (to S.J.H.) supported this work.
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