New Results

System-level effects of CO2 and RuBisCO concentration on carbon isotope fractionation

Amanda K. Garcia, Mateusz Kedzior, Arnaud Taton, Meng Li, Jodi N. Young, Betül Kaçar
doi: https://doi.org/10.1101/2021.04.20.440233

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), molecular 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 fractionation 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‰, 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.

IMPORTANCE Carbon isotope biosignatures preserved in the geologic record are interpreted to reflect the long-term evolution of microbial carbon fixation and provide the earliest evidence of life on Earth. RuBisCO enzymes, distinctive and early-evolved catalysts that fix atmospheric CO2, have likely been responsible for the bulk of primary productivity through Earth history. Thus, a comprehensive understanding of the molecular, physiological, environmental, and evolutionary factors that influence the isotopic discrimination of cyanobacteria that utilize RuBisCO is essential for the interpretation of ancient isotopic biosignatures. For example, the vastly different atmospheric CO2 levels that characterized the Precambrian may have influenced the expression and regulation of the ancient RuBisCO protein complex. These observations underscore the need to consider how a broader range of environmental conditions and subcellular processes may have shaped isotopic discrimination over geologic time. In this study, we establish a cyanobacterial metabolic-engineering strategy that can test such hypotheses and offer insights into the biogeochemical record of life.

INTRODUCTION

The conserved microbial metabolic pathways that drive global biogeochemistry emerged on Earth billions of years ago, the evolution of which has both shaped and been shaped by large-scale environmental transitions (Falkowski, Fenchel, & Delong, 2008; Knoll, Bergmann, & Strauss, 2016; Lyons, Fike, & Zerkle, 2015). These microbial processes have left distinct signatures that evidence biological activity billions of years in the past. Of these, the oldest and most extensive signature of biological activity on Earth is the deviation in stable carbon isotopic compositions (13C/12C, expressed as δ13C) between preserved inorganic and organic carbon, interpreted to reflect the isotopic discrimination of ancient biological carbon fixation (Des Marais, 2001; Krissansen-Totton, Buick, & Catling, 2015; Lloyd et al., 2020; Schidlowski, 2001). These deviations are primarily shaped by enzymes that preferentially assimilate the lighter 12C isotope from inorganic carbon sources. Carbon biosignatures preserved in the geologic record therefore reflect the long-term evolution of these enzyme-mediated processes and their hosts’ physiologies.

The RuBisCO enzyme (ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase) catalyzes the reduction of inorganic CO2 as the initial step of carbon assimilation into organic biomass via the Calvin-Benson-Bassham (CBB) cycle (Erb & Zarzycki, 2018; Nisbet et al., 2007; Tcherkez, Farquhar, & Andrews, 2006). RuBisCO is one of the most abundant proteins on Earth (Bar-On & Milo, 2019; Ellis, 1979; Raven, 2013), and its presence in photoautotrophic organisms, including early-evolved cyanobacteria, suggests that this enzyme has played a significant role in primary production for much of Earth history (Hamilton, Bryant, & Macalady, 2016; Schirrmeister, Sanchez-Baracaldo, & Wacey, 2016; Schopf, 2011). Thus, the isotopic fractionation behavior of RuBisCO is also thought to have primarily constrained Precambrian carbon isotope signatures preserved in the geologic record (Garcia, Cavanaugh, & Kacar, 2021; Schidlowski, 1988). Though there exist multiple forms of RuBisCO, the known range of Form I RuBisCO isotopic fraction (ε ≈ 20-30‰) (R. D. Guy, Fogel, & Berry, 1993; Scott et al., 2007; von Caemmerer, Tazoe, Evans, & Whitney, 2014) — the form utilized by extant cyanobacteria and responsible for the bulk of modern primary production (Field, 1998) — is largely consistent with the ~25‰ mean deviation between preserved inorganic and organic carbon isotopic compositions across geologic time (Des Marais, 2001; Garcia et al., 2021; Havig, Hamilton, Bachan, & Kump, 2017; Krissansen-Totton et al., 2015; Lloyd et al., 2020; Schidlowski, 2001).

RuBisCO primarily shapes the generation of distinct carbon isotopic biosignatures associated with CBB-utilizing organisms. Nonetheless the carbon isotopic composition of bulk photoautotrophic biomass often deviates from values obtained for purified RuBisCO. This discrepancy might be attributable to several intracellular physiological and metabolic features that additionally shift the isotopic composition of organism biomass. These include the activation of carbon-concentrating mechanisms such as the partitioning of RuBisCO into carboxysomes (Hurley, Wing, Jasper, Hill, & Cameron, 2021; Laws, Popp, Cassar, & Tanimoto, 2002; Price, Badger, Woodger, & Long, 2008; Raven, Cockell, & De La Rocha, 2008), the diffusive transport of CO2 (Hayes, 1993; Rau, Riebesell, & Wolf-Gladrow, 1996), and intermediary carbon-fixing steps (Eungrasamee, Miao, Incharoensakdi, Lindblad, & Jantaro, 2019; H. I. Guy & Evans, 1996; Hayes, 1993; Rothschild & DesMarais, 1989). Further, studies show that photosynthetic carbon isotope discrimination (εp) may vary due to environmental factors and cellular physiological responses, including temperature (Deleens, Treichel, & O’Leary, 1985; Wong & Sackett, 1978), pH (Hinga, Arthur, Pilson, & Whitaker, 1994; Roeske & O’Leary, 1984), growth rate (Bidigare et al., 1997; Laws, Bidigare, & Popp, 1997), and CO2 concentration (Eichner, Thoms, Kranz, & Rost, 2015; Freeman & Hayes, 1992; Hinga et al., 1994; Hurley et al., 2021; Schubert & Jahren, 2012; Wilkes, Lee, McClelland, Rickaby, & Pearson, 2018). These variations indicate a compelling need to comprehensively characterize both internal and external factors that can affect isotope fractionation, though the study of each tend to be siloed in biological and geobiological fields, respectively.

An aspect of photoautotrophic isotopic discrimination that has not been thoroughly investigated is how variable RuBisCO expression can influence host organism growth parameters and the isotopic composition of assimilated biomass. RuBisCO expression has been shown to be CO2-sensitive (Gesch et al., 2003; Onizuka et al., 2002; Sengupta, Sunder, Sohoni, & Wangikar, 2019). This observation is particularly important, considering that atmospheric CO2 concentrations exceeded present day levels by more than an order of magnitude for much of Earth history (between ~0.001 and 0.1 bar CO2 through the Precambrian (Catling & Zahnle, 2020)) and that the RuBisCO catalytic efficiency itself is sensitive to the atmospheric CO2/O2 levels (Erb & Zarzycki, 2018; Kacar, Hanson-Smith, Adam, & Boekelheide, 2017; Poudel et al., 2020; Riebesell, Revill, Holdsworth, & Volkman, 2000; Schubert & Jahren, 2012; Scott et al., 2007; Tcherkez et al., 2006; Wilkes et al., 2018). Thus, interpretation of the carbon isotope record should take into account the coupled influence of CO2 concentration and RuBisCO expression in generating isotopic biosignatures and shaping ancient primary productivity.

To investigate the interplay between RuBisCO expression and CO2 concentrations on cyanobacterial fitness and carbon isotopic discrimination, we generated a genetic system to manipulate the expression levels of RuBisCO in the model organism Synechococcus elongatus PCC 7942 (hereafter S. elongatus). S. elongatus is a naturally competent obligate photoautotroph that utilizes Form I RuBisCO to fix carbon (Bonfil et al., 1998; Gabay, Lieman-Hurwitz, Hassidim, Ronen-Tarazi, & Kaplan, 1998; Maeda, Price, Badger, Enomoto, & Omata, 2000; Omata, Gohta, Takahashi, Harano, & Maeda, 2001; Taton et al., 2020; Tchernov et al., 2001). Specifically, we engineered the S. elongatus genome with an additional copy of the RuBisCO operon in a chromosomal neutral site to overexpress RuBisCO and permit genetic manipulation of the RuBisCO operon for future studies. We confirmed proper assembly of the overexpressed RuBisCO complex and its catalytic activity in the engineered strain. Then, to assess whether physiological and subcellular parameters and varying CO2 concentrations modulate carbon isotopic discrimination in cyanobacteria, we determined growth rate, photosynthetic oxygen evolution rate, and 13C/12C discrimination of the engineered strain in comparison to wild-type S. elongatus. Our results suggest that increased CO2 concentrations result in significantly faster growth in cyanobacteria and increase the magnitude of isotopic discrimination. On the other hand, we show that changing cellular RuBisCO levels do not significantly alter carbon isotope discrimination.

RESULTS

A second copy of the rbc operon results in increased amount of active RuBisCO

The RuBisCO Form I enzyme in S. elongatus is encoded by an operon that includes a CO2-sensitive promoter region (Sengupta et al., 2019) as well as the structural rbcL (large subunit) and rbcS (small subunit) genes (Vijayan, Jain, & O’Shea, 2011). We designed S. elongatus strain Syn02 that harbors the native rbc operon and a second copy inserted in the chromosome neutral site 2 (NS2), a site that permits genetic modification without additional indirect phenotypic impact (Andersson et al., 2000; Clerico, Ditty, & Golden, 2007) (Fig. 1). Syn02 was contructed by transforming wild-type (WT) S. elongatus with plasmid pSyn02 carrying the rbc operon and homologous regions directing recombination at NS2 (Table 1). Additionally, we generated a control strain Syn01 whereby RuBisCO is provided solely by an engineered rbc operon at its NS2 site.

Figure 1.
Figure 1. Experimental strategy.

A – Overall methodology. 1) Genetic engineering: An additional copy of the rbc operon was inserted at the chromosomal NS2 site of S. elongatus PCC 7942 to create the Syn02 strain. 2) RuBisCO characterization was carried out by investigating (a) catalytic activity, (b) assembly into a hexadecameric complex, and (c) expression at both the transcript and protein levels. 3) Physiology: S. elongatus WT and Syn02 strains were evaluated by monitoring microbial growth characteristics, morphology, and rate of photosynthetic oxygen evolution. 4) Carbon isotope fractionation: The impact of RuBisCO overexpression on biomass δ13C and εp under varying CO2 concentrations was assessed. B – Map of the genetic insert in S. elongatus strain Syn02. Plasmid pSyn02 was used to insert the rbc operon and the aph1 gene conferring kanamycin resistance in PCC 7942 WT NS2 to generate the Syn02 strain. Crosslines indicate homologous recombination sites and scale bars show DNA fragment sizes (in bp).

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Table 1.

Strains and plasmids used in this study.

We evaluated whether the additional copy of the rbc operon in strain Syn02 resulted in RuBisCO overexpression by quantifying transcription of the rbcL and purK (located downstream of rbcL in the operon) genes by quantitative reverse-transcription PCR (RT-qPCR). Transcripts were quantified and normalized to those of secA and ppc reference genes (Hood, Higgins, Flamholz, Nichols, & Savage, 2016; Luo et al., 2019; Szekeres, Sicora, Dragoş, & Drugă, 2014). We found that the level of rbcL transcript in Syn02 increased by at least 2-fold relative to WT across all tested CO2 concentrations and growth phases, and as high as ~14-fold in air (p < 0.01; Fig. 2A, Fig. S1A). In addition, we found that rbcL expression increased by >5-fold at elevated CO2 concentrations relative to ambient air (p < 0.001; Fig. 2B, Fig. S1B). Expression of purK in Syn02 increased by >2-fold at 2% and 5% CO2 relative to WT, but decreased by ~0.5-fold in air (Fig. S1C).

Figure 2.
Figure 2. RuBisCO expression, assembly, and activity in S. elongatus strains under varying CO2 concentrations.

A – Expression of Syn02 rbcL relative to WT rbcL. Asterisks indicate t-test results compared to WT rbcL expression at the same growth condition. B – Expression of WT rbcL at 2-5% CO2 relative to ambient air. Asterisks indicate t-test results compared to WT rbcL expression in air. A-B – Expression data was measured by RT-qPCR, normalized to reference genes secA and ppc. Columns represent mean fold expression for three biological replicates. C – Immunodetection of RbcL. (left) Western blot showing RbcL protein detected by anti-RuBisCO antibody and total protein stain from crude cell lysates. (right) RbcL percentage signal intensities were normalized to that for the total soluble protein load. Columns represent mean RbcL intensities for six biological replicates, relative to mean values for WT cultures at the same growth condition. Asterisks indicate t-test results compared to WT RbcL. D – Immunodetection of assembled RuBisCO. Western blot showing the proper assembly of RbcL and RbcS into the L8S8 hexadecameric complex L8S8 (520 kDa), detected by anti-RbcL antibody. Rbc L8S8 percentage signal intensity was normalized to that for the total soluble protein load, and is shown relative to WT. E – Lysate RuBisCO activity. Activity was measured by the RuBP consumption rate, normalized to total soluble protein content of cell lysate. Columns represent the mean activity of three biological replicates. A-C, E – Asterisks indicate t-test results for pairwise comparison indicated by horizontal line. Error bars on all graphs indicate 1 SD. ns – not significant; ** – p <0.01; *** – p <0.001.

To determine rbcL overexpression at the protein-level, we quantified RbcL protein from crude cell lysates by Western blot using rabbit anti-RbcL antibody (see Materials and Methods). In agreement with RuBisCO overexpression indicated by the RT-qPCR results, densitometric analyses revealed a mean ~2 to 4-fold increase of RbcL protein in Syn02 relative to WT across all tested CO2 concentrations (p < 0.01; Fig. 2C). Finally, we confirmed proper assembly of the large (L) and small (S) subunits into the RuBisCO L8S8 complex in Syn02 as well as the Syn01 control strain by native protein electrophoresis and detection by anti-RbcL antibody. We found a ~3-fold increase in assembled RuBisCO protein for Syn02 and ~2-fold increase for Syn01 relative to WT for cultures grown in 2% CO2 (Fig. 2D).

Finally, we tested whether or not RuBisCO overexpression in Syn02 resulted in an increased amount of active RuBisCO by determining the total carboxylase activity of cell lysates. Enzyme activity was evaluated by an in vitro spectrophotometric coupled-enzyme assay that links carboxylase activity to NADH oxidation, reported as the RuBP consumption rate normalized to total soluble protein content (Kubien, Brown, & Kane, 2011). We measured a mean ~0.8-fold (p < 0.01) increase in Syn02 lysate RuBisCO activity relative to WT (Fig. 2E). No significant difference in activity was detected between Syn01 and WT cultures.

RuBisCO overexpression does not strongly influence growth rate or photosynthetic activity

We cultured WT and Syn02 S. elongatus strains in order to determine the influence of Syn02 RuBisCO overexpression on growth rate. Cultures were continuously sparged with ambient air, as well as 2% and 5% CO2, with daily optical density measurements at 750 nm (OD750) as described in Materials and Methods. WT and Syn02 both exhibited ~2.5-fold faster growth rates under 2% and 5% CO2 compared to ambient air (p < 0.001; Fig. 2A; Table 2). Syn02 exhibited a slight ~0.1-fold increase in growth rate in ambient air relative to WT (p < 0.05). No difference in growth rate was observed between the two strains under 2% and 5% CO2. The carrying capacity (maximum cell density measured at OD750 across the total growth period) for cultures varied between different atmospheric conditions, with a maximum carrying capacity of OD750 ≈ 8.4 reached under 2% CO2. No significant difference in carrying capacity was found between WT and Syn02 cultures grown under the same atmospheric conditions. To determine the degree to which sparging impacted growth, we repeated the growth experiments without sparging. Minimal differences were again observed between WT and Syn02 across different CO2 concentrations (Table S1).

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Table 2.

Growth parameters and oxygen evolution rates of S. elongatus strains under varying CO2 concentrationsa.

To further test for differences in photosynthetic activities between the WT and Syn02 strains, we measured their oxygen evolution rates. After brief incubation in the dark, culture samples were exposed to saturated light in an oxygen electrode chamber to detect increased levels of molecular oxygen. Oxygen evolution rates were normalized to chlorophyll a concentrations, following Zavrel et al. (Zavrel, Sinetova, & Červený, 2015). We did not observe a significant difference in mean oxygen evolution rate between WT and Syn02 (342 ± 26 O2·h−1·μg−1 chlorophyll a and 362 ± 26 O2·h−1·μg−1 chlorophyll a, respectively) (Table 2).

CO2 concentration impacts S. elongatus13C/12C fractionation

We tested both the influence of RuBisCO overexpression and CO2 concentration on the magnitude of 13C/12C isotopic discrimination in S. elongatus, which was assessed by measuring the 13C/12C composition of biomass (δ13Cbiomass). The isotopic discrimination associated with photosynthetic CO2 fixation (εp) was then calculated from measured δ13Cbiomass and a reference δ13CCO2 value (8.4‰; (Keeling et al., 2017)), as described in Materials and Methods. We observed that increased CO2 concentration increases the magnitude of S. elongatus 13C/12C discrimination. Mean εp values for S. elongatus strains grown in ambient air were ~10‰ (Fig. 4A). At 2% and 5% CO2, εp values were ~7-9‰ greater than those for cultures grown in air, with mean values ranging between ~17‰ to 20‰ (p < 0.001). Mean εp values were similar for WT and Syn02 strains most tested CO2 conditions, with the exception of 5% CO2, where a significant but slight difference in εp values was detected (18.95‰ and 18.62‰ for WT and Syn02, respectively; p < 0.01). These results demonstrate that RuBisCO overexpression in Syn02 does not strongly impact cyanobacterial 13C/12C discrimination.

DISCUSSION

In this study, we developed an integrative approach to investigate the coupled impact of CO2 levels and RuBisCO expression on cyanobacterial growth, fitness and carbon isotope discrimination. We tested whether the overexpression of RuBisCO influences S. elongatus growth, physiology and carbon isotope fractionation at different CO2 atmosphere concentrations by generating an engineered S. elongatus strain Syn02 that harbors a second copy of the rbc operon at NS2.

Our transcriptional analyses indicate that S. elongatus strain Syn02 overexpresses RuBisCO relative to WT at all tested CO2 concentrations (ambient air, 2%, and 5% CO2) (Fig. 2A, Fig. 2C). In addition, we found that rbcL transcript levels increase for all strains at elevated CO2 levels relative to air (Fig. 2B). This result is in agreement with a previous study reporting a regulatory element in the S. elongatus PCC 7942 rbcL operon that regulates the expression of RbcL with increasing CO2 (Sengupta et al., 2019). We found that the magnitude of increased rbcL transcription between Syn02 and WT was lower at elevated CO2 (Fig. 2A). For example, in air, rbcL transcript levels in Syn02 were ~14-fold greater than in WT, but in 2% CO2, only a ~2-fold increase was observed. These results indicate that, whereas RuBisCO expression is impacted by CO2 concentration, other physiological factors such as the translation efficiency or expression of other ancillary crucial proteins may may be limiting RuBisCO expression and dampen the effects of an additional copy of the rbc operon in Syn02.

Protein-level analyses indicate that increased rbc transcription in Syn02 relative to WT does result in increased cellular RuBisCO concentration. The magnitude of this increase is relatively constant (~2-3 fold) across different CO2 concentrations (Fig. 2C). These results are supported by a ~2-fold increase in assembled RuBisCO L8S8 protein in Syn02 relative to WT (Fig. 3A). However, the ~0.8-fold increase in carboxylase activity of Syn02 cell lysate relative to that of WT was not directly proportional to the increase in protein amount observed by Western blot analyses, suggesting that not all overexpressed RuBisCO was catalytically active (Fig. 3B). Previous studies showed overexpression of RuBisCO in cyanobacteria generally produces an increased pool of active carboxylase (Atsumi, Higashide, & Liao, 2009; Iwaki et al., 2006; Lechno-Yossef et al., 2020; Liang & Lindblad, 2017). However, it is possible that not all overexpressed RuBisCO may be properly assembled, e.g., due to limited chaperonin GroEL/ES, which is known to be involved in RuBisCO folding (Hayer-Hartl, 2017; Liu et al., 2010). Altogether, our findings suggest that additional physiological constraints may be limiting RuBisCO expression beyond transcription. The exact mechanism of the regulation we observed in our experiments await further characterization.

Figure 3.
Figure 3. Growth of S. elongatus strains.

A – Growth profiles. Cultures of each strain were maintained in ambient air or under 2% or 5% CO2. Smoothed profiles were generated from mean optical density (measured at 750 nm, OD750) values for three replicate cultures per growth condition. B – Doubling times. Columns represent the mean doubling time of three replicates. Error bars on all graphs indicate 1 SD, and asterisks indicate t-test results for pairwise comparisons indicated by horizontal lines. ns – not significant; * – p <0.05; *** – p < 0.001.

Various culture conditions including pH, temperature, CO2 concentration, and light intensity may substantially affect the growth characteristics of cyanobacteria, and thus have the potential to influence isotopic discrimination (Kuan, Duff, Posarac, & Bi, 2015; Rillema, MacCready, & Vecchiarelli, 2020; Ungerer, Lin, Chen, & Pakrasi, 2018; Yu et al., 2015). The influence of genetic factors, including the regulation of RuBisCO expression on cyanobacterial fitness, is less well known. Previous studies targeting the correlation between RuBisCO overexpression and fitness in cyanobacteria have yielded mixed results. For instance, faster growth rates and oxygen evolution rates were observed for an engineered Synechocystis PCC 6803 strain that overexpresses RuBisCO (Liang & Lindblad, 2017), as well as in S. elongatus upon co-overexpression of its phosphoribulokinase (Kanno, Carroll, & Atsumi, 2017). However, in Synechococcus sp. PCC 7002, the overexpression of RuBisCO did not alter growth rate (De Porcellinis et al., 2018). The impact of RuBisCO upregulation on cyanobacterial growth appears to be species/strain-specific. Our data shows that RuBisCO overexpression in S. elongatus PCC 7942 strain Syn02 results in a minor increase in growth rate in ambient air relative to WT, and no significant difference was observed at elevated CO2 levels, nor for oxygen evolution rates. Under ambient environmental conditions, RuBisCO has a relatively high baseline level of expression in cyanobacteria (V. Vijayan, I. H. Jain, & E. K. O’Shea, 2011), perhaps explaining why increased expression does not significantly improve fitness. Our results indicate that further study is needed to fully understand the coupled interaction of different environmental conditions and genetic backgrounds in determining cyanobacterial fitness.

Another important aspect to consider is the degree to which genetic regulation and background may impact system-level isotope fractionation in cyanobacteria. Understanding the connection between genetics and biogeochemistry, though often overlooked, can augment interpretation of isotopic biosignatures in deep time. We measured the carbon isotopic composition of cyanobacterial biomass (δ13Cbiomass) and photosynthetic carbon isotopic discrimination (εp) in WT and Syn02 cultured under a range of CO2 concentrations. The increased expression and activity of RuBisCO in Syn02 apparently neither changed the availability of carbon isotopes in the enzyme’s vicinity nor altered the performance of other cellular modules contributing to carbon isotope discrimination. δ13Cbiomass and εp values were comparable for WT and Syn02 grown at all tested CO2 concentrations (Fig. 4).

Figure 4.
Figure 4. 13C/12C discrimination of S. elongatus strains under varying CO2 concentrations.

A – Biomass δ13C. Reported δ13C values are relative to the Vienna PeeDee Belemnite standard (V-PDB). B – 13C/12C discrimination associated with photosynthetic CO2 fixation (εp). εp values are calculated relative to a reference value, δ13CCO2 = −8.4‰ (Keeling et al., 2017) (see Materials and Methods for calculation). A-B – Mean (middle horizontal bars) and 1 SD (vertical error bars) are shown in red. Asterisks indicate t-test results for pairwise comparisons indicated by horizontal lines. ns – not significant; ** – p < 0.01; *** – p < 0.001.

Our results show that at both 2% and 5% CO2, εp values were increased by ~7-9‰ for WT and Syn02 strains relative to air. Theoretically, under high CO2 concentrations, the intrinsic kinetic isotope effect of RuBisCO is expressed and maximizes εp (Bidigare et al., 1997; Hayes, 1993; Schubert & Jahren, 2012; Wilkes et al., 2018). Under these conditions, other cellular modules, such as carbon concentrating mechanisms capable of discriminating carbon isotopes, produce negligible impact on net carbon discrimination at the organismal level (Hurley et al., 2021; Laws et al., 2002). The positive relationship between CO2 concentration and carbon isotope fractionation has been observed empirically for a variety of autotrophs (Freeman & Hayes, 1992; Hinga et al., 1994; Schubert & Jahren, 2012; Wilkes et al., 2018), including for cyanobacteria in particular (Eichner et al., 2015; Hurley et al., 2021). Other studies showed that photoautotrophs that grew under a lower pH, and thus a higher proportion of CO2, also exhibited higher carbon fractionation (Mizutani & Wada, 1982; Roeske & O’Leary, 1984; Wang, Yeager, & Lu, 2016; Yoshioka, 1997). The εp values observed under high inorganic carbon availability fall within the range of 17-20‰ (Fig. 4B), which follows the range known for Form I RuBisCO (R. D. Guy et al., 1993; McNevin, Badger, Kane, & Farquhar, 2006; Scott et al., 2007).

Based on the data generated here, we speculate that, in addition to environmental factors such as CO2 concentrations, RuBisCO variation at the gene and enzymatic level, rather than its overexpression and regulation, may be a more significant determinant of the isotopic composition of produced biosignatures. Less well-characterized RuBisCO forms (i.e., Forms IC, ID, and II) that are found in proteobacteria and marine phytoplankton can exhibit significantly smaller fractionation factors than that observed for forms associated with land plants and cyanobacteria (Boller, Thomas, Cavanaugh, & Scott, 2011; Robinson et al., 2003; Thomas et al., 2019). The full diversity of RuBisCO isotopic fractionation requires further study,. Our results underscore that the integration of synthetic biology, metabolic engineering and geochemistry can offer new insights into the study and interpretation of biogeochemical reservoirs at the global scale. Further work needs to be performed to elucidate how the intracellular factors impact metabolisms responsible for carbon isotope fractionation under ancient transitions in environmental conditions. A deeper understanding of the impact of the RuBisCO regulation and the environmental factors on the resulting organismal behavior is essential to establish the extent to which the RuBisCO isotope fractionation properties can be correlated with evidence for biological activity in the Earth’s deep past.

MATERIALS AND METHODS

Cyanobacterial growth and maintenance

S. elongatus PCC 7942 strains were cultured in BG-11 medium (Rippka, Deruelles, Waterbury, Herdman, & Stanier, 1979) as liquid cultures or on agar plates (1.5% (w/v) agar and 1 mM Na2S2O3·5H2O). For recombinant strains, liquid and solid media were supplemented with appropriate antibiotics: 2 μg·ml−1 Spectinomycin (Sp) plus 2 μg·ml−1 Streptomycin (Sm), 5 μg·ml−1 Kanamycin (Km). The cyanobacterial growth was measured at optical density at 750 nm (OD750).

The strains were archived at −80 °C in 15% (v/v) glycerol. The 2-ml vials were rapidly thawed and inoculated in liquid BG-11 (supplemented with antibiotics as needed). Cultures were shaken at 120 rpm at 30 °C under continuous low illumination of 45 μmol photon·m−2·s−1, and ambient air, until they reached an OD750 between 0.4 and 0.6. These cultures were then used to inoculate fresh cultures that were grown using similar conditions but under moderate illumination of 80 μmol photon·m−2·s−1 and sparged at selected CO2 concentrations (ambient air, 2%, or 5% CO2; cultures were also grown without sparging to assess differences in growth rate; Table S1). When cultures reached an OD750 of ~7 to 7.5, they were sampled for subsequent experiments as described below (cultures were also sampled at an earlier OD750 of 0.5-1.5 for additional RT-qPCR experiments, see below; Fig. S1).

Genetic engineering of cyanobacteria

A recombinant strains of S. elongatus was constructed by natural transformation using standard protocols (Clerico et al., 2007) and the plasmids and methods described below (Table 1). To construct the plasmid pSyn02, pAM4937 was digested with SwaI to release the ccdB toxic gene and produce a plasmid backbone that contains the pBR322 E. coli origin of replication, the base of mobilization site for conjugal transfer, the aph1 gene conferring kanamycin resistance, and sequences for homologous recombination into S. elongatus chromosome at NS2. The rbc operon was amplified from S. elongatus PCC 7942 gDNA with primers F01 and R01 (Table S2) containing 20-nucleotide sequences that overlap with pAM4937 backbone. The resulting DNA fragments were assembled using the GeneArt™ Seamless Cloning and Assembly Kit (Invitrogen, Cat. No. A13288). pSyn02 was used to insert the rbc operon into NS2 of the wild type genome in the strain PCC 7942 through homologous recombination to create the strain of S. elongatus, Syn02, carrying two copies of the rbc operon. Plasmid pSyn-01 was constructed by using two primer pairs F02/R02 and F03/R03 (Table S2), 1) to amplify a fragment of pAM4951 that contains the E. coli origin of replication and the site for conjugal transfer, and 2) to amplify the aadA gene conferring spectinomycin/streptomycin resistance. Native rbc operon flanking sequences were amplified from the S. elongatus PCC 7942 gDNA with the primer pairs F04/R04 and F05/R05 (Table S2) containing 20-nucleotide sequences that overlap with the pAM4951 fragments.

Transformation was carried out after growing the WT strain in liquid culture at 30 °C with shaking (120 rpm) and a light intensity of 80 μmol photon·m−2·s−1 until an OD750 ~0.7. The cells were prepared for transformation according to the protocol by Clerico et al. (2007) and plated on BG-11 agar containing the appropriate antibiotic(s) for recovery. Subsequently, the colonies were picked using sterile pipette tips, patched onto BG-11 agar containing the appropriate antibiotic(s), and further incubated to ensure complete chromosome segregation (i.e., incorporation of the trans-gene into all chromosomes). The patched transformants were screened using colony PCR using the primers F06/R06 and F07/R07 and the genotypes of the engineered strains were confirmed by Sanger sequencing using the primers F08/R08 (Fig. S2, Table S2)

Extraction of total RNA and proteins

Cultures were collected during the exponential growth phase. Cells were pelleted by centrifugation at 4,700 × g for 10 min at room temperature and resuspended in 10 mL of TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). To prepare the crude cell lysate, 8 mL of the cell suspension were pelleted by centrifugation at 4,700 × g for 10 min at room temperature, resuspended in 500 μL of hot (pre-warmed to 95 °C) TE buffer supplemented with 1% (w/v) SDS and incubated at 95 °C for 10 min. Then, the mixture was sonicated at 40% amplitude for 3 × 10 sec with 10 sec intervals, and the cell debris centrifuged at 17,000 × g for 10 min at room temperature. The supernatant was collected and stored at −80 °C. Total RNA was extracted from the remaining 2 mL of the cell suspension using the RNeasy® Protect Bacteria Mini Kit (QIAGEN, Cat. No. 74524) following the manufacturer instructions.

Analysis of rbc operon expression by RT-qPCR

Total RNA was quantified with the NanoDrop spectrophotometer and 1 μg of RNA was treated with the amplification grade deoxyribonuclease I (Invitrogen, Cat. No. 18068-015). 10 μL of DNase I-treated RNA was then used in reverse transcription (RT) performed with the SuperScript™ IV First-Strand Synthesis System (Invitrogen, Cat. No. 18091050). The four pairs of qPCR primers (listed in Table S2) were designed with Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Both reference genes have previously been shown to be stably expressed under diverse conditions in S. elongatus (Luo et al., 2019). The quality of cDNA and primer specificity was assessed by PCR using cDNA templates (RT positive reactions), RT negative controls, and the qPCR primers. The analysis of gene expression levels was performed in the real-time thermal cycler qTOWER3 G (Analytik Jena AG), equipped with the software qPCRsoft, using the cycles: 50 °C/2 min, 95 °C/2 min, 40 × (95 °C/15 sec, 60 °C/1 min). The relative expression of the rbc operon genes (rbcL and purK) was calculated as the average fold change normalized to reference genes using the delta-delta Ct method. The experiment was carried out using three biological replicates and three technical replicates.

Analysis of RbcL protein by Western blot

Total protein concentration in the crude cell lysates was measured using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Cat. No. 23225). The lysates were loaded in the amount of 5 μg of total protein in Laemmli sample buffer onto a 6% (v/v) polyacrylamide stacking gel. Proteins were electrophoresed in a 12% polyacrylamide resolving gel in TGS buffer and blotted in transfer buffer onto a PVDF membrane. Total protein load in each sample was visualized by Revert™ 700 Total Protein Stain (LI-COR Biosciences, Cat. No. 926-11011) and used for the RbcL signal normalization. Detection of RbcL was performed by overnight incubation of the membrane at 4 °C with rabbit anti-RbcL antibody (Agrisera, Cat. No. AS03 037), 1:5000 in TBST with 5% non-fat milk, followed by one-hour incubation at room temperature with IRDye® 800CW goat anti-rabbit IgG secondary antibody (LI-COR Biosciences, Cat. No. 926-32211), 1:20,000 in Intercept® (TBS) blocking buffer (LI-COR Biosciences, Cat. No. 927-60001) with 0.1% (v/v) Tween-20 and 0.01% (w/v) SDS. Both the total protein load and the amount of RbcL in each sample were documented with Odyssey® Fc Imaging System (LI-COR Biosciences, Cat. No. 2800-03) at the near-infrared detection mode. The images were acquired using Image Studio™ software. The densitometric analysis of RbcL signal intensity, normalized to total protein load, was performed with Quantity One® software (Bio-Rad) for six biological replicates. The amount of RbcL produced by each replicate of the Syn02 strain culture was compared to the averaged level of RbcL in the WT PCC 7942 replicate cultures and expressed as the averaged percent of RbcL synthesized by the WT strain.

Assembly of RuBisCO subunits

Assembly of the RuBisCO large and small subunits into a hexadecameric complex in each strain was evaluated by native gel electrophoresis and immunodetection of the RuBisCO complexes. Samples were collected during the exponential growh phase. Cells were pelleted by centrifugation at 4,700 × g for 10 min at room temperature and resuspended in 400 μL of native lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 10% (v/v) glycerol) supplemented with 5 mM DTT, 100 μg/mL lysozyme from chicken egg white, and 1% (v/v) Halt™ Protease Inhibitor Cocktail (Thermo Scientific, Cat. No. 78430). The cell suspensions were incubated at 30 °C for 15 min and subjected to five consecutive freeze-thaw cycles (10 min at −80 °C followed by 5 min at 30 °C), then were sonicated on ice for 3 minutes at 30% amplitude (2-sec on/off intervals), centrifuged at 17,000 × g for 15 min at 4 °C. The concentration of total soluble proteins in the lysates was determined with Pierce™ BCA Protein Assay Kit. The lysates were adjusted to 5 μg of total soluble proteins in native sample buffer and then loaded onto a 4-20% Mini-PROTEAN® TGX™ Precast Protein Gel (Bio-Rad, Cat. No. 4561094). Protein electrophoreses were performed in TG buffer (60 mM Tris, 192 mM glycine) at 100 V for 4 h at 4 °C and blotted in transfer buffer (48 mM Tris, pH 9.2, 39 mM glycine, 0.04% (w/v) SDS) onto a nitrocellulose membrane. After three 10-min washes in wash buffer (48 mM Tris, pH 9.2, 39 mM glycine, 20% (v/v) methanol), total protein load in each sample was visualized by Revert™ 700 Total Protein Stain and used for the normalization of RuBisCO complex quantity. Immunodetection of the RuBisCO complex was performed with the same primary and secondary antibodies that were used to analyze the level of RbcL, as described above.

Catalytic activity of RuBisCO

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). The cyanobacterial strains were cultured, collected, and pelleted as described above. The pellets were resuspended in 1 mL of ice-cold lysis buffer (50 mM EPPS, 1 mM EDTA, 2 mM DTT, pH 8.0) and transferred into 2 mL screw-capped tubes with Lysing Matrix B (MP Biomedical) for lysis by bead beating using FastPrep-24™ 5G bead beater (MP Biomedical) with 4 m/sec for 10 sec, followed by 2-min incubation on ice, repeated six times. The cell lysates were transferred to new Eppendorf tubes to remove the beads and unbroken cells and to pellet the thylakoid membrane by centrifugation at 10,000 × g for 1 min and at 20,000 × g for 30 min at 4 °C, sequentially. The resulting clear supernatants containing cytosolic soluble proteins, including phycobiliproteins and RuBisCO, were used to determine protein concentration by Pierce™ BCA Protein Assay Kit and to measure RuBisCO activity by employing an assay adapted from Kubien et al. (2011). The assay buffer (100 mM HEPES, 25 mM MgCl2, 1 mM EDTA, pH 7.6) was used considering the high Michaelis constant for CO2 (KC) for cyanobacterial RuBisCO. 20 μL of cell lysates were preincubated in the assay mix (with 5 mM NaHCO3) at 25 °C for activation before initiating the reaction by adding synthesized ribulose 1,5-bisphosphate (RuBP) according to Kane et al. (Kane, Wilkin, Portis, & John Andrews, 1998). The absorbance at 340 nm was monitored using the Synergy H1 plate reader (BioTek). RuBisCO activity was reported as RuBP consumption rate normalized to total soluble protein content. The assay was performed for three biological replicates.

Cyanobacterial growth measurements

OD750 values were plotted as a function of time and analyzed in R with the Growthcurver package. Growth curve data was fitted to the standard form of the logistic equation to calculate growth parameters including growth rate, doubling time, and carrying capacity (Sprouffske & Wagner, 2016). Each strain was grown in triplicate for every condition.

Photosynthetic oxygen evolution rate

S. elongatus strain photosynthetic activity was assayed using a Clark-type oxygen electrode chamber to measure the level of molecular oxygen produced in cyanobacterial cultures. Cells were grown in 50 mL of BG-11 at 30 °C, illumination of 80 μmol photon·m−2·s−1, shaking at 120 rpm, in ambient air, and with culture sparging. The samples were collected from triplicate cultures during the exponential growth phase, pelleted by centrifugation at 4,700 × g for 10 min at room temperature, and resuspended in fresh BG-11 to an OD750 of ~1.0. Concentration of chlorophyll a (for normalization) was measured following the protocol by Zavrel et al. (Zavrel et al., 2015). The remaining suspension was incubated in the dark for 20 min with gentle agitation. Samples from each suspension, prepared in three technical replicates, were analyzed in an oxygen electrode chamber under saturated light, using the Oxygraph+ System (Hansatech Instruments) equipped with the OxyTrace+ software. Oxygen evolution rate was monitored for 10 min and expressed as nanomoles of molecular oxygen evolved per hour per microgram of chlorophyll a.

Carbon isotope fractionation in bulk cyanobacterial biomass

The bacteria were pelleted by centrifugation at 4,700 × g for 10 min at room temperature, washed in 10 mL of 10 mM NaCl. The bacteria were resuspended in 1 mL of 10 mM NaCl and transferred to Eppendorf tubes. After centrifugation at 4,700 × g for 10 min at room temperature, the supernatants were completely removed, the pellets were dried in opened tubes in a laboratory oven at 50 °C for 2 days, and the resultant dried biomass samples were transferred into tin capsules.

The carbon isotope composition of bulk biomass (δ13Cbiomass) samples was determined at the UC Davis Stable Isotope Facility. δ13Cbiomass was analyzed using a PDZ Europa ANCA-GSL elemental analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd.). The carbon isotopic composition values were reported relative to the Vienna PeeDee Belemnite standard (V-PDB): Embedded Image

The carbon isotope fractionation associated with photosynthetic CO2 fixation (εp) was calculated relative to a reference δ13CCO2 value (−8.4‰; (Keeling et al., 2017)) according to Freeman and Hayes (1992): Embedded Image

Statistical analyses

Results for experimental analyses were presented as the mean and the sample standard deviation (SD) values of at least three independent experiments. Statistical significance was analyzed swith the two-tailed t-test. The unpaired sample t-test assuming equal variances was used to compare the values obtained for different cyanobacterial strains and the paired sample t-test was used to compare the values for the same strain at different experimental conditions.

ACKNOWLEDGEMENTS

This work was supported by the National Science Foundation Emerging Frontiers Program Award No. 1724090 (BK), NASA Early Career Faculty (ECF) Award No. 80NSSC19K1617 (BK.), NASA Postdoctoral Fellowship (AKG.), Simons Foundation Early Career Award No. 561645 (JNY and ML), and National Institute of General Medical Sciences of the National Institutes of Health award number R01GM118815 (AT, to James W. Golden at the University of California-San Diego). We sincerely thank Bob Blankenship, Zach Adam, Katie McGrath and the members of the Kaçar Lab for their thoughtful comments and suggestions; Sky Dominguez and the University of California-Davis Stable Isotope Facility for the assistance.

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Posted April 20, 2021.
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System-level effects of CO2 and RuBisCO concentration on carbon isotope fractionation
Amanda K. Garcia, Mateusz Kedzior, Arnaud Taton, Meng Li, Jodi N. Young, Betül Kaçar
bioRxiv 2021.04.20.440233; doi: https://doi.org/10.1101/2021.04.20.440233
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