skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Functional reconstitution of a bacterial CO2 concentrating mechanism in Escherichia coli
Many photosynthetic organisms employ a CO 2 concentrating mechanism (CCM) to increase the rate of CO 2 fixation via the Calvin cycle. CCMs catalyze ≈50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an Escherichia coli strain engineered to depend on rubisco carboxylation for growth. Expression of 20 CCM genes enabled E. coli to grow by fixing CO 2 from ambient air into biomass, with growth in ambient air depending on the components of the CCM. Bacterial CCMs are therefore genetically compact and readily transplanted, rationalizing their presence in diverse bacteria. Reconstitution enabled genetic experiments refining our understanding of the CCM, thereby laying the groundwork for deeper study and engineering of the cell biology supporting CO 2 assimilation in diverse organisms.  more » « less
Award ID(s):
1818377
PAR ID:
10299719
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ;
Date Published:
Journal Name:
eLife
Volume:
9
ISSN:
2050-084X
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; (, Ecology)
    Abstract Carbon‐concentrating mechanisms (CCMs) are a widespread phenomenon in photosynthetic organisms. In vascular plants, the evolution of CCMs ([C44‐carbon compound] and crassulacean acid metabolism [CAM]) is associated with significant shifts, most often to hot, dry and bright, or aquatic environments. If and how CCMs drive distributions of other terrestrial photosynthetic organisms, remains little studied. Lichens are ecologically important obligate symbioses between fungi and photosynthetic organisms. The primary photosynthetic partner in these symbioses can include CCM‐presenting cyanobacteria (as carboxysomes), CCM‐presenting green algae (as pyrenoids) or green algae lacking any CCM. We use an extensive dataset of lichen communities from eastern North America, spanning a wide climatic range, to test the importance of CCMs as predictors of lichen ecology and distribution. We show that the presence or absence of CCMs leads to opposite responses to temperature and precipitation in green algal lichens, and different responses in cyanobacterial lichens. These responses contrast with our understanding of lichen physiology, whereby CCMs mitigate carbon limitation by water saturation at the cost of efficient use of vapor hydration. This study demonstrates that CCM status is a key functional trait in obligate lichen symbioses, equivalent in importance to its role in vascular plants, and central for studying present and future climate responses. 
    more » « less
  2. ; ; ; ; ; ; ; ; ; ; et al (, Applied and Environmental Microbiology)
    Biddle, Jennifer F (Ed.)
    ABSTRACT Autotrophic bacteria are able to fix CO2in a great diversity of habitats, even though this dissolved gas is relatively scarce at neutral pH and above. As many of these bacteria rely on CO2fixation by ribulose 1,5-bisphospate carboxylase/oxygenase (RubisCO) for biomass generation, they must compensate for the catalytical constraints of this enzyme with CO2-concentrating mechanisms (CCMs). CCMs consist of CO2and HCO3−transporters and carboxysomes. Carboxysomes encapsulate RubisCO and carbonic anhydrase (CA) within a protein shell and are essential for the operation of a CCM in autotrophicBacteriathat use the Calvin-Benson-Basham cycle. Members of the genusThiomicrospiralack genes homologous to those encoding previously described CA, and prior to this work, the mechanism of function for their carboxysomes was unclear. In this paper, we provide evidence that a member of the recently discovered iota family of carbonic anhydrase enzymes (ιCA) plays a role in CO2fixation by carboxysomes from members ofThiomicrospiraand potentially otherBacteria. Carboxysome enrichments fromThiomicrospira pelophilaandThiomicrospira aerophilawere found to have CA activity and contain ιCA, which is encoded in their carboxysome loci. When the gene encoding ιCA was interrupted inT. pelophila, cells could no longer grow under low-CO2conditions, and CA activity was no longer detectable in their carboxysomes. WhenT. pelophilaιCA was expressed in a strain ofEscherichia colilacking native CA activity, this strain recovered an ability to grow under low CO2conditions, and CA activity was present in crude cell extracts prepared from this strain. IMPORTANCEHere, we provide evidence that iota carbonic anhydrase (ιCA) plays a role in CO2fixation by some organisms with CO2-concentrating mechanisms; this is the first time that ιCA has been detected in carboxysomes. While ιCA genes have been previously described in other members of bacteria, this is the first description of a physiological role for this type of carbonic anhydrase in this domain. Given its distribution in alkaliphilic autotrophic bacteria, ιCA may provide an advantage to organisms growing at high pH values and could be helpful for engineering autotrophic organisms to synthesize compounds of industrial interest under alkaline conditions. 
    more » « less
  3. ; (, Annual review of plant biology)
    Although cyanobacteria and algae represent a small fraction of the biomass of all primary producers, their photosynthetic activity accounts for roughly half of the daily CO2 fixation that occurs on Earth. These microorganisms are able to accomplish this feat by enhancing the activity of the CO2-fixing enzyme Rubisco using biophysical CO2 concentrating mechanisms (CCMs). Biophysical CCMs operate by concentrating bicarbonate and converting it into CO2 in a compartment that houses Rubisco (in contrast with other CCMs that concentrate CO2 via an organic intermediate, such as malate in the case of C4 CCMs). This activity provides Rubisco with a high concentration of its substrate, thereby increasing its reaction rate. The genetic engineering of a biophysical CCM into land plants is being pursued as a strategy to increase crop yields. This review focuses on the progress toward understanding the molecular components of cyanobacterial and algal CCMs, as well as recent advances toward engineering these components into land plants. 
    more » « less
  4. ; ; ; ; ; ; ; ; ; ; et al (, Journal of Bacteriology)
    null (Ed.)
    In nature, concentrations of dissolved inorganic carbon (DIC; = CO 2 + HCO 3 - + CO 3 2- ) can be low, and autotrophic organisms adapt with a variety of mechanisms to elevate intracellular DIC concentrations to enhance CO 2 fixation. Such mechanisms have been well-studied in Cyanobacteria , but much remains to be learned about their activity in other phyla. Novel multi-subunit membrane-spanning complexes capable of elevating intracellular DIC were recently described in three species of bacteria. Homologs of these complexes are distributed among 17 phyla in Bacteria and Archaea, and are predicted to consist of one, two, or three subunits. To determine whether DIC accumulation is a shared feature of these diverse complexes, seven of them, representative of organisms from four phyla, from a variety of habitats, and with three different subunit configurations were chosen for study. A high-CO 2 requiring, carbonic anhydrase-deficient ( yadF - cynT - ) strain of E. coli Lemo21(DE3), which could be rescued via elevated intracellular DIC concentrations, was created for heterologous expression and characterization of the complexes. Expression of all seven complexes rescued the ability of E. coli Lemo21(DE3) yadF - cynT - to grow under low CO 2 conditions, and six of the seven generated measurably elevated intracellular DIC concentrations when their expression was induced. For complexes consisting of two or three subunits, all subunits were necessary for DIC accumulation. Isotopic disequilibrium experiments clarified that CO 2 was the substrate for these complexes. In addition, the presence of an ionophore prevented the accumulation of intracellular DIC, suggesting that these complexes may couple proton potential to DIC accumulation. IMPORTANCE To facilitate the synthesis of biomass from CO 2 , autotrophic organisms use a variety of mechanisms to increase intracellular DIC concentrations. A novel type of multi-subunit complex has recently been described, which has been shown to generate measurably elevated intracellular DIC concentrations in three species of bacteria, begging the question of whether these complexes share this capability across the 17 phyla of Bacteria and Archaea where they are found. This study shows that DIC accumulation is a trait shared by complexes with varied subunit structures, from organisms with diverse physiologies and taxonomies, suggesting that this trait is universal among them. Successful expression in E. coli suggests the possibility of their expression in engineered organisms synthesizing compounds of industrial importance from CO 2 . 
    more » « less
  5. ; (, Photosynthesis Research)
    Abstract Marine diatoms are key primary producers across diverse habitats in the global ocean. Diatoms rely on a biophysical carbon concentrating mechanism (CCM) to supply high concentrations of CO2around their carboxylating enzyme, RuBisCO. The necessity and energetic cost of the CCM are likely to be highly sensitive to temperature, as temperature impacts CO2concentration, diffusivity, and the kinetics of CCM components. Here, we used membrane inlet mass spectrometry (MIMS) and modeling to capture temperature regulation of the CCM in the diatomPhaeodactylum tricornutum (Pt). We found that enhanced carbon fixation rates byPtat elevated temperatures were accompanied by increased CCM activity capable of maintaining RuBisCO close to CO2saturation but that the mechanism varied. At 10 and 18 °C, diffusion of CO2into the cell, driven byPt’s ‘chloroplast pump’ was the major inorganic carbon source. However, at 18 °C, upregulation of the chloroplast pump enhanced (while retaining the proportion of) both diffusive CO2and active HCO3−uptake into the cytosol, and significantly increased chloroplast HCO3−concentrations. In contrast, at 25 °C, compared to 18 °C, the chloroplast pump had only a slight increase in activity. While diffusive uptake of CO2into the cell remained constant, active HCO3−uptake across the cell membrane increased resulting inPtdepending equally on both CO2and HCO3−as inorganic carbon sources. Despite changes in the CCM, the overall rate of active carbon transport remained double that of carbon fixation across all temperatures tested. The implication of the energetic cost of thePtCCM in response to increasing temperatures was discussed. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email