Effects of CO₂and RuBisCO concentration on cyanobacterial growth and carbon isotope fractionation

Abstract

Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO2 concentrations) and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a range of genetic and environmental factors that may have impacted ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO and characterized the resultant physiological and isotope fractionation effects. We specifically investigated how both increased atmospheric CO2 concentrations and RuBisCO regulation influence cell growth, oxygen evolution rate, and carbon isotope discrimination in cyanobacteria. We found that >2% CO2 increases the growth rate of wild-type and mutant strains, and that the pool of active RuBisCO enzyme increases with increased expression. At elevated CO2, carbon isotope discrimination (ϵp) is increased by ~8 per mille, whereas RuBisCO overexpression does not significantly affect isotopic discrimination at all tested CO2 concentrations. Our results show that understanding the environmental factors that impact RuBisCO regulation, physiology, and evolution is crucial for reconciling microbially driven carbon isotope fractionation with the geologic record of organic and inorganic carbon isotope signatures.

Resurrected Rubisco suggests uniform carbon isotope signatures over geologic time

“…2B). The nucleotide sequence for the reconstructed AncIB RbcL protein was codon-optimized for S. elongatus and cloned within a copy of the rbc operon into pSyn02 (Garcia et al, 2021b) for insertion into the S. elongatus chromosomal neutral site 2 (NS2). The native rbc operon was subsequently deleted to create the AncIB strain.…”

“…Proteins were electrophoresed in a 12% polyacrylamide resolving gel and blotted onto a nitrocellulose membrane. Detection of RbcL and total protein load was performed as previously described (50). The densitometric analysis of RbcL signal intensity, normalized to total protein load, was performed with Quantity One® software (Bio-Rad) for three to six biological replicates.…”

“…The activity of RuBisCO in cyanobacterial lysates was measured using a spectrophotometric coupled-enzyme assay that links this activity with the rate of NADH oxidation (Kubien et al, 2011). Cell lysis and the activity assay were carried out as previously described (Garcia et al, 2021b) with either 2.5 mM or 5 mM NaHCO 3 . After 20 min at 25 °C for activation of Rubisco, the reaction was initialized with the addition of ribulose 1,5-bisphosphate (RuBP) (0.5 mM) and the absorbance at 340 nm was recorded using a Synergy H1 plate reader (BioTek).…”

“…Proteins were electrophoresed in a 12% polyacrylamide resolving gel and blotted onto a nitrocellulose membrane. Detection of RbcL and total protein load was performed as previously described (Garcia et al, 2021b; Kędzior and Kacar, 2021). Detection of RbcL was performed with rabbit anti-RbcL antibody (Agrisera, Cat No.…”

“…Cyanobacteria genetic engineering. Recombinant strains of S. elongatus were constructed by natural transformation using standard protocols (Clerico et al, 2007) with minor modifications (Garcia et al, 2021b). The plasmids and strains used in this study are listed in Table S1.…”