Figure 1. Twenty genes form the basis of a bacterial CCM.

(A) The bacterial CCM consists of at least two essential components - energy-coupled inorganic carbon uptake and carboxysome structures that encapsulate rubisco with a carbonic anhydrase (CA) enzyme (Desmarais et al., 2019; Kaplan et al., 1980; Price and Badger, 1989a, Price and Badger, 1989b; Rae et al., 2013; Shively et al., 1973). Transport generates a large cytosolic HCO₃^- pool, which is rapidly converted to high carboxysomal CO₂ concentration by the carboxysomal CA (Mangan et al., 2016; McGrath and Long, 2014). (B) Elevated CO₂ increases the rubisco carboxylation rate (green) and suppresses oxygenation by competitive inhibition (grey). [O₂] was set to 270 μM for rate calculations. A more detailed version of this calculation is described in Figure 1—figure supplement 1. (C) H. neapolitanus CCM genes are mostly contained in a 20 gene cluster (Desmarais et al., 2019) expressing rubisco and its associated chaperones (green), carboxysome structural proteins (purple), and an inorganic carbon transporter (orange). Supplementary file 1 gives fuller description of the functions of these 20 genes along with a per-gene bibliography. Figure 1—figure supplement 2 demonstrates that the operon beginning with acRAF indeed encodes a functional inorganic carbon transporter.

Figure 1—figure supplement 1. Elevated CO₂ overcomes limitations associated with rubisco catalysis.

(A) Kinetic data assembled for ≈300 rubiscos from diverse organisms show that there is limited variation (less than one order of magnitude) in CO₂ specificity (S_C/O) and maximum carboxylation rate (k_cat,C) among the Form I rubiscos found in all photoautotrophs and all bacteria harboring carboxysome CCMs (Flamholz et al., 2019). Moreover, S_C/O and k_cat,C appear to trade-off with each other. Although this relationship is not strict, rubiscos with high k_cat,C values also typically have lower S_C/O(Savir et al., 2010; Tcherkez et al., 2006). As carboxylation and oxygenation reactions occur at the same active site, elevated CO₂ will both increase the carboxylation rate (until it reaches k_cat,C) and also inhibit oxygenation by exclusion of oxygen from the active site. As it relies only on the well-founded assumption that catalysis with CO₂ and O₂ substrates are mutually exclusive, this mechanism should function for any rubisco. Panels B-D depict this effect for three distinct rubiscos, which are highlighted with black borders in (A). Panels give carboxylation (light green), oxygenation (red) and net carboxylation (dark green) rates as a function of the aqueous CO₂ concentration at ambient O₂ levels (270 uM at 25 ℃). All curves were calculated using standard kinetic equations for rubisco. Net carboxylation was calculated as the carboxylation rate less ½ the oxygenation rate, which presumes a plant-type photorespiratory pathway that loses one CO₂ for every two oxygenation reactions. (B) Bacterial Form II rubiscos are typically found in organisms living in low O₂ environments and, accordingly, display low CO₂ specificities (S_C/O ≈ 10) and relatively high maximum carboxylation rates (k_cat,C ≈10–20 s⁻¹, Davidi et al., 2020). As such, Form II rubiscos do not perform well in ambient CO₂ and O₂ concentrations. (C) C₃ plants like spinach do not have CCMs. Furthermore, the CO₂ concentration inside the leaf is typically measured to be lower than ambient due to a balance of stomatal conductance and CO₂ fixation by rubisco itself (Caemmerer and Evans, 1991). Accordingly, C₃ plant rubiscos display high CO₂ specificities (S_C/O ≈ 100), modest k_cat,C ≈ 3 s⁻¹, and perform well at ambient and sub-ambient CO₂ levels, displaying relatively little oxygenation and, consequently, net carboxylation rates that are similar to the total carboxylation rate. (D) Rubiscos found in bacteria with a carboxysome CCM typically have relatively low CO₂ specificities (S_C/O ≈ 50) and fast maximum carboxylation rates relative to other Form I rubiscos (k_cat,C ≈ 10 s⁻¹). In general, rubiscos from organisms bearing CCMs (whether bacteria, algae, or plants) tend to have lower CO₂ specificities and higher k_cat,C than enzymes from related organisms without CCMs (Iñiguez et al., 2020; Savir et al., 2010). The carboxysomal rubisco from S. elongatus PCC 7942 performs worse than a typical C₃ plant rubisco in ambient air, but much better in the elevated CO₂ environment we presume is maintained by the carboxysome CCM. The aqueous CO₂ and O₂ concentrations were calculated assuming Henry’s law equilibrium at 25 ℃. Notably, changes in temperature will affect CO₂ and O₂ solubility (Milo and Phillips, 2015; Sander, 2015) and rubisco kinetics, most notably decreasing CO₂-specificity at elevated temperatures (Boyd et al., 2019; Sage et al., 2012).

Figure 1—figure supplement 2. The 20 gene CCM cluster includes a functional DAB-type inorganic carbon transporter.

In previous work, we showed that the H. neapolitanus genome contains two homologous complexes DAB1 and DAB2 that are required for growth in ambient air (Desmarais et al., 2019). We further demonstrated that DAB2 is a two-gene operon whose protein products form a membrane associated complex that is capable of energetically active inorganic carbon uptake, but did not investigate DAB1 in detail. Here, we use DAB1 because it is encoded in the same genomic locus as the carboxysome, in a putative operon that also contains other potentially CCM-relevant genes like rubisco chaperones. See Figure 1C and Supplementary file 1 for further detail on the contents of this operon. As before, we rely on a reporter strain, CAfree, to test inorganic carbon transporters. This strain lacks all endogenous carbonic anhydrases and fails to grow in ambient air as a result (Desmarais et al., 2019). Growth of CAfree is complemented by elevating the CO₂ level in the growth chamber, expressing carbonic anhydrases, or by supplying intracellular HCO₃^- via an inorganic carbon transporter like DAB2. Panel (A) reproduces our previous result - that expression of DAB2 from the pFA backbone complements growth of CAfree in ambient air, such that CAfree:pFA-DAB2 (orange) grows similarly to the wild-type strain (WT:vec, light grey). A negative control (CAfree:vec, dark grey) fails to grow, as expected. (B) DAB1 genes, which are marked in orange in Figure 1C, also rescue growth of CAfree in ambient air. pFA-DAB1 expresses only the DAB1 genes and omits the remaining eight genes in the operon. (C) The pCCM plasmid encodes all 11 genes found in the same putative operon as DAB1. CAfree:pCCM also well grows in ambient air. Cells were grown in LB media with 100 nM aTc induction throughout, with ‘vec’ denoting a vector control of pFA-sfGFP.