Isotopic discrimination and kinetic parameters of RubisCO from the marine bloom‐forming diatom,Skeletonema costatum

The cosmopolitan, bloom‐forming diatom, Skeletonema costatum, is a prominent primary producer in coastal oceans, fixing CO₂ with ribulose 1,5‐bisphosphate carboxylase/oxygenase (RubisCO) that is phylogenetically distinct from terrestrial plant RubisCO. RubisCOs are subdivided into groups based on sequence similarity of their large subunits (IA–ID, II, and III). ID is present in several major oceanic primary producers, including diatoms such as S. costatum, coccolithophores, and some dinoflagellates, and differs substantially in amino acid sequence from the well‐studied IB enzymes present in most cyanobacteria and in green algae and plants. Despite this sequence divergence, and differences in isotopic discrimination apparent in other RubisCO enzymes, stable carbon isotope compositions of diatoms and other marine phytoplankton are generally interpreted assuming enzymatic isotopic discrimination similar to spinach RubisCO (IB). To interpret phytoplankton δ¹³C values, S. costatum RubisCO was characterized via sequence analysis, and measurement of its K_CO2 and V_max, and degree of isotopic discrimination. The sequence of this enzyme placed it among other diatom ID RubisCOs. Michaelis‐Menten parameters were similar to other ID enzymes (K_CO2 = 48.9 ± 2.8 μm ; V_max = 165.1 ± 6.3 nmol min^?1 mg^?1). However, isotopic discrimination (ε = [¹²k/¹³k ? 1] × 1000) was low (18.5‰; 17.0–19.9, 95% CI) when compared to IA and IB RubisCOs (22–29‰), though not as low as ID from coccolithophore, Emiliania huxleyi (11.1‰). Variability in ε‐values among RubisCOs from primary producers is likely reflected in δ¹³C values of oceanic biomass. Currently, δ¹³C variability is ascribed to physical or chemical factors (e.g. illumination, nutrient availability) and physiological responses to these factors (e.g. carbon‐concentrating mechanisms). Estimating the importance of these factors from δ¹³C measurements requires an accurate ε‐value, and a mass‐balance model using the ε‐value for S. costatum RubisCO is presented. Clearly, appropriate ε‐values must be included in interpreting δ¹³C values of environmental samples.     

Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity

Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO₂/O₂ specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions.     

Aerobic chemolithoautotrophic growth and RubisCO function in Rhodobacter capsulatus and a spontaneous gain of function mutant of Rhodobacter sphaeroides

Photosynthetic prokaryotes that assimilate CO₂ under anoxic conditions may also grow chemolithoautotrophically with O₂ as the electron acceptor. Among the nonsulfur purple bacteria, two species (Rhodobacter capsulatus and Rhodopseudomonas acidophilus), exhibit aerobic chemolithoautotrophic growth with hydrogen as the electron donor. Although wild-type strains of Rhodobacter sphaeroides grow poorly, if at all, with hydrogen plus oxygen in the dark, we report here the isolation of a spontaneous mutant (strain HR-CAC) of Rba. sphaeroides strain HR that is fully capable of this mode of growth. Rba. sphaeroides and Rba. capsulatus fix CO₂ via the reductive pentose phosphate pathway and synthesize two forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). RubisCO levels in the aerobic-chemolithoautotrophic-positive strain of Rba. sphaeroides were similar to those in wild-type strains of Rba. sphaeroides and Rba. capsulatus during photoheterotrophic and photolithoautotrophic growth. Moreover, RubisCO levels of Rba. sphaeroides strain HR-CAC approximated levels obtained in Rba. capsulatus when the organisms were grown as aerobic chemolithoautotrophs. Either form I or form II RubisCO was able to support aerobic chemolithoautotrophic growth of Rba. capsulatus strain SB 1003 and Rba. sphaeroides strain HR-CAC at a variety of CO₂ concentrations, although form II RubisCO began to lose the capacity to support aerobic CO₂ fixation at high O₂ to CO₂ ratios. The latter property and other facets of the physiology of this system suggest that Rba. sphaeroides and Rba. capsulatus strains may be effectively employed for the biological selection of RubisCO molecules of altered substrate specificity. Received: 8 August 1997 / Accepted: 26 December 1997     

Genomic resolution of a cold subsurface aquifer community provides metabolic insights for novel microbes adapted to high CO2 concentrations

As in many deep underground environments, the microbial communities in subsurface high‐CO₂ ecosystems remain relatively unexplored. Recent investigations based on single‐gene assays revealed a remarkable variety of organisms from little studied phyla in Crystal Geyser (Utah, USA), a site where deeply sourced CO₂‐saturated fluids are erupted at the surface. To provide genomic resolution of the metabolisms of these organisms, we used a novel metagenomic approach to recover 227 high‐quality genomes from 150 microbial species affiliated with 46 different phylum‐level lineages. Bacteria from two novel phylum‐level lineages have the capacity for CO₂ fixation. Analyses of carbon fixation pathways in all studied organisms revealed that the Wood‐Ljungdahl pathway and the Calvin‐Benson‐Bassham Cycle occurred with the highest frequency, whereas the reverse TCA cycle was little used. We infer that this, and selection for form II RuBisCOs, are adaptions to high CO₂‐concentrations. However, many autotrophs can also grow mixotrophically, a strategy that confers metabolic versatility. The assignment of 156 hydrogenases to 90 different organisms suggests that H₂ is an important inter‐species energy currency even under gaseous CO₂‐saturation. Overall, metabolic analyses at the organism level provided insight into the biochemical cycles that support subsurface life under the extreme condition of CO₂ saturation.     

A Non-radioisotopic Anion-exchange Chromatographic Method to Measure the CO2/O2 Specificity Factor for Ribulose Bisphosphate Carboxylase/Oxygenase

A non-radioisotopic anion-exchange ion chromatographic method for measuring the carboxylation/ oxygenation specificity (τ) of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is presented. The assay measures the amounts of fixation products at varying [CO₂]/[O₂] ratios to measure the relative rates of CO₂ and O₂ fixation reactions. The amount of 3-phosphoglycerate (3-PGA) and phosphoglycolate (PG) in the reaction mixture were measured with a conductivity detector and the specific factor was calculated using the following equations: ν_c = ([3-PGA] – [PG])/2 and ν_o = [PG]. By this method, specificity factors for RubisCOs were measured without using radioactive reagents.     

Isotope discrimination by form IC RubisCO from Ralstonia eutropha and Rhodobacter sphaeroides,metabolically versatile members of ‘Proteobacteria’ from aquatic and soil habitats

RubisCO, the CO₂ fixing enzyme of the Calvin–Benson–Bassham (CBB) cycle, is responsible for the majority of carbon fixation on Earth. RubisCO fixes ¹²CO₂ faster than ¹³CO₂ resulting in ¹³C-depleted biomass, enabling the use of δ¹³C values to trace CBB activity in contemporary and ancient environments. Enzymatic fractionation is expressed as an ε value, and is routinely used in modelling, for example, the global carbon cycle and climate change, and for interpreting trophic interactions. Although values for spinach RubisCO (ε = ~29‰) have routinely been used in such efforts, there are five different forms of RubisCO utilized by diverse photolithoautotrophs and chemolithoautotrophs and ε values, now known for four forms (IA, B, D and II), vary substantially with ε = 11‰ to 27‰. Given the importance of ε values in δ¹³C evaluation, we measured enzymatic fractionation of the fifth form, form IC RubisCO, which is found widely in aquatic and terrestrial environments. Values were determined for two model organisms, the ‘Proteobacteria’ Ralstonia eutropha (ε = 19.0‰) and Rhodobacter sphaeroides (ε = 22.4‰). It is apparent from these measurements that all RubisCO forms measured to date discriminate less than commonly assumed based on spinach, and that enzyme ε values must be considered when interpreting and modelling variability of δ¹³C values in nature.     

Expression of the cbbLcbbS and cbbM genes and distinct organization of the cbb Calvin cycle structural genes of Rhodobacter capsulatus

Rhodobacter capsulatus fixes CO₂ via the Calvin reductive pentose phosphate pathway and, like some other nonsulfur purple bacteria, is known to synthesize two distinct structural forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Cosmid clones that hybridized to form I (cbbLcbbS) and form II (cbbM) RubisCO gene probes were isolated from a genomic library of R. capsulatus strain SB1003. Southern blotting and hybridization analysis with gene-specific probes derived from Rhodobacter sphaeroides revealed that R. capsulatus cbbM is clustered with genes encoding other enzymes of the Calvin cycle, including fructose 1,6/sedoheptulose 1,7-bisphosphatase (cbbF), phosphoribulokinase (cbbP), transketolase (cbbT), glyceraldehyde-3-phosphate dehydrogenase (cbbG), and fructose 1,6-bisphosphate aldolase (cbbA), as well as a gene (cbbR) encoding a divergently transcribed LysR-type regulatory protein. Surprisingly, a cosmid clone containing the R. capsulatus form I RubisCO genes (cbbL and cbbS) failed to hybridize to the other cbb structural gene probes, unlike the situation with the closely related organism R. sphaeroides. The form I and form II RubisCO genes were cloned into pUC-derived vectors and were expressed in Escherichia coli to yield active recombinant enzyme in each case. Complementation of a RubisCO-deletion strain of R. sphaeroides to photosynthetic growth by R. capsulatus cbbLcbbS or cbbM was achieved using the broad host-range vector, pRK415, and R. sphaeroides expression vector pRPS-1. Received: 6 June 1995 / Accepted: 29 September 1995     

Influence of form IA RubisCO and environmental dissolved inorganic carbon on the delta13C of the clam-chemoautotroph symbiosis Solemya velum

Many nutritive symbioses between chemoautotrophic bacteria and invertebrates, such as Solemya velum, have delta(13)C values of approximately -30 to -35%, considerably more depleted than phytoplankton. Most of the chemoautotrophic symbionts fix carbon with a form IA ribulose 1,5-bisphosphate carboxylase (RubisCO). We hypothesized that this form of RubisCO discriminates against (13)CO(2) to a greater extent than other forms. Solemya velum symbiont RubisCO was cloned and expressed in Escherichia coli, purified and characterized. Enzyme from this recombinant system fixed carbon most rapidly at pH 7.5 and 20-25 degrees C. Surprisingly, this RubisCO had an epsilon-value (proportional to the degree to which the enzyme discriminates against (13)CO(2)) of 24.4 per thousand, similar to form IB RubisCOs, and higher than form II RubisCOs. Samples of interstitial water from S. velum's habitat were collected to determine whether the dissolved inorganic carbon (DIC) could contribute to the negative delta(13)C values. Solemya velum habitat DIC was present at high concentrations (up to approximately 5 mM) and isotopically depleted, with delta(13)C values as low as approximately -6%. Thus environmental DIC, coupled with a high degree of isotopic fractionation by symbiont RubisCO likely contribute to the isotopically depleted delta(13)C values of S. velum biomass, highlighting the necessity of considering factors at all levels (from environmental to enzymatic) in interpreting stable isotope ratios.     

CO2 assimilation strategies in stratified lakes: Diversity and distribution patterns of chemolithoautotrophs

While mechanisms of different carbon dioxide (CO₂) assimilation pathways in chemolithoautotrohic prokaryotes are well understood for many isolates under laboratory conditions, the ecological significance of diverse CO₂ fixation strategies in the environment is mostly unexplored. Six stratified freshwater lakes were chosen to study the distribution and diversity of the Calvin‐Benson‐Bassham (CBB) cycle, the reductive tricarboxylic acid (rTCA) cycle, and the recently discovered archaeal 3‐hydroxypropionate/4‐hydroxybutyrate (HP/HB) pathway. Eleven primer sets were used to amplify and sequence genes coding for selected key enzymes in the three pathways. Whereas the CBB pathway with different forms of RubisCO (IA, IC and II) was ubiquitous and related to diverse bacterial taxa, encompassing a wide range of potential physiologies, the rTCA cycle in Epsilonproteobacteria and Chloribi was exclusively detected in anoxic water layers. Nitrifiying Nitrosospira and Thaumarchaeota, using the rTCA and HP/HB cycle respectively, are important residents in the aphotic and (micro‐)oxic zone of deep lakes. Both taxa were of minor importance in surface waters and in smaller lakes characterized by an anoxic hypolimnion. Overall, this study provides a first insight on how different CO₂ fixation strategies and chemical gradients in lakes are associated to the distribution of chemoautotrophic prokaryotes with different functional traits.     

Distribution and diversity of autotrophic bacteria in groundwater systems based on the analysis of RubisCO genotypes

A molecular approach, based on the detection of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large subunit genes, was applied to investigate the distribution and diversity of autotrophic bacteria in groundwater systems. DNA extracts from 48 sampling stations, including a variety of pristine and polluted, shallow and deep-subsurface groundwater samples obtained from Germany and Austria, served as a template for the PCR amplification of form I (cbbL) and form II (cbbM) large subunit RubisCO genes. The majority of the samples (>80%) contained two different forms of RubisCO. In 17 samples, all three forms of RubisCO were identified. PCR products from four selected groundwater habitats containing all three forms of RubisCO were used to construct clone libraries. Based on restriction fragment length polymorphism (RFLP) analysis, 109 RubisCO-clone-inserts were subjected to sequencing and phylogenetic analysis. With the exception of a form IA RubisCO sequence cluster obtained from deep subsurface samples, which was identical to the RubisCO genes described for Ralstonia metallidurans CH34, most sequences were distantly related to a variety of RubisCO species in chemolithoautotrophic Proteobacteria. Several sequences occurred in isolated lineages. These findings suggest that autotrophic bacteria with the capability to assimilate CO₂ via the Calvin Cycle pathway are widespread inhabitants of groundwater systems.     

Carboxysomal carbonic anhydrases: Structure and role in microbial CO2 fixation

Cyanobacteria and some chemoautotrophic bacteria are able to grow in environments with limiting CO₂ concentrations by employing a CO₂-concentrating mechanism (CCM) that allows them to accumulate inorganic carbon in their cytoplasm to concentrations several orders of magnitude higher than that on the outside. The final step of this process takes place in polyhedral protein microcompartments known as carboxysomes, which contain the majority of the CO₂-fixing enzyme, RubisCO. The efficiency of CO₂ fixation by the sequestered RubisCO is enhanced by co-localization with a specialized carbonic anhydrase that catalyzes dehydration of the cytoplasmic bicarbonate and ensures saturation of RubisCO with its substrate, CO₂. There are two genetically distinct carboxysome types that differ in their protein composition and in the carbonic anhydrase(s) they employ. Here we review the existing information concerning the genomics, structure and enzymology of these uniquely adapted carbonic anhydrases, which are of fundamental importance in the global carbon cycle.     

Photoautotrophic Cell Suspension Cultures from Mesembryanthemum crystallinum and their Response to Salt Stress

For the first time, photoautotrophic cell suspension cultures of Mesembryanthemum crystallinum have been established. The cells are growing in a sugar-free culture medium in the presence of 2 % (v/v) CO₂ as the sole carbon source. A 16 h light photoperiod is applied. Increase in fresh and dry weight during a 21 days growth cycle was more than 3-fold. Treatment of the cells with 200 mM NaCl from day 10 to day 21 of subculture stimulated cell culture growth, enhanced CO₂ fixation and elicited an increase in the extractable activities of enzymes related to CO₂ fixation (RubisCO; PEP carboxylase) and malic acid metabolism (NAD / NADP dependent malic enzyme and malic acid dehydrogenase). The cells performed osmotic adjustment to high salinity by uptake of K⁺, Na⁺, Cl^? and formation of proline as well as by a reduction in cell size. Although sugar and starch content of the cells changed during light/dark transition, a CAM-related diurnal fluctuation of malic acid was not observed.     

Differential effects of ocean acidification on carbon acquisition in two bloom‐forming dinoflagellate species

Dinoflagellates represent a cosmopolitan group of phytoplankton with the ability to form harmful algal blooms. Featuring a Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RubisCO) with very low CO₂ affinities, photosynthesis of this group may be particularly prone to carbon limitation and thus benefit from rising atmospheric CO₂ partial pressure (pCO₂) under ocean acidification (OA). Here, we investigated the consequences of OA on two bloom‐forming dinoflagellate species, the calcareous Scrippsiella trochoidea and the toxic Alexandrium tamarense. Using dilute batch incubations, we assessed growth characteristics over a range of pCO₂ (i.e. 180–1200 µatm). To understand the underlying physiology, several aspects of inorganic carbon acquisition were investigated by membrane‐inlet mass spectrometry. Our results show that both species kept growth rates constant over the tested pCO₂ range, but we observed a number of species‐specific responses. For instance, biomass production and cell size decreased in S. trochoidea, while A. tamarense was not responsive to OA in these measures. In terms of oxygen fluxes, rates of photosynthesis and respiration remained unaltered in S. trochoidea whereas respiration increased in A. tamarense under OA. Both species featured efficient carbon concentrating mechanisms (CCMs) with a CO₂‐dependent contribution of HCO₃^? uptake. In S. trochoidea, the CCM was further facilitated by exceptionally high and CO₂‐independent carbonic anhydrase activity. Comparing both species, a general trade‐off between maximum rates of photosynthesis and respective affinities is indicated. In conclusion, our results demonstrate effective CCMs in both species, yet very different strategies to adjust their carbon acquisition. This regulation in CCMs enables both species to maintain growth over a wide range of ecologically relevant pCO₂.     

Predicting adaptive evolution under elevated atmospheric CO2 in the perennial grass Bromus erectus

Increasing concentrations of CO₂ in the atmosphere are likely to affect the ecological dynamics of plant populations and communities worldwide, yet little is known about potential evolutionary consequences of high CO₂. We employed a quantitative genetic framework to examine how the expression of genetic variation and covariation in fitness‐related traits, and thus, the evolutionary potential of a species, is influenced by CO₂. In two field experiments, genotypes of the dominant grassland perennial Bromus erectus were grown for several years in plots maintained at present‐day or at elevated CO₂ levels. Under noncompetitive conditions (experiment 1), elevated CO₂ had little impact on plant survival, growth, and reproduction. Under competitive conditions in plots with diverse plant communities (experiment 2), performance of B. erectus was reduced by elevated CO₂. This suggests that the effect of CO₂ was largely indirect, intensifying competitive interactions. Elevated CO₂ had significant effects on the expression of genetic variation in both the competitive and noncompetitive environment, but the effects were in opposite direction. Heritability of plant size was generally higher at elevated than at ambient CO₂ in the noncompetitive environment, but lower in the competitive environment. Selection analysis revealed a positive genotypic selection differential for plant size at ambient CO₂, indicating selection favoring genotypes with high growth rate. At elevated CO₂, the corresponding selection differential was nonsignificant and slightly negative. This suggests that elevated CO₂ is unlikely to stimulate the evolution of high biomass productivity in this species.     

Function, structure, and evolution of the RubisCO-like proteins and their RubisCO homologs.

About 30 years have now passed since it was discovered that microbes synthesize RubisCO molecules that differ from the typical plant paradigm. RubisCOs of forms I, II, and III catalyze CO(2) fixation reactions, albeit for potentially different physiological purposes, while the RubisCO-like protein (RLP) (form IV RubisCO) has evolved, thus far at least, to catalyze reactions that are important for sulfur metabolism. RubisCO is the major global CO(2) fixation catalyst, and RLP is a somewhat related protein, exemplified by the fact that some of the latter proteins, along with RubisCO, catalyze similar enolization reactions as a part of their respective catalytic mechanisms. RLP in some organisms catalyzes a key reaction of a methionine salvage pathway, while in green sulfur bacteria, RLP plays a role in oxidative thiosulfate metabolism. In many organisms, the function of RLP is unknown. Indeed, there now appear to be at least six different clades of RLP molecules found in nature. Consideration of the many RubisCO (forms I, II, and III) and RLP (form IV) sequences in the database has subsequently led to a coherent picture of how these proteins may have evolved, with a form III RubisCO arising from the Methanomicrobia as the most likely ultimate source of all RubisCO and RLP lineages. In addition, structure-function analyses of RLP and RubisCO have provided information as to how the active sites of these proteins have evolved for their specific functions.     

SHORT‐ VERSUS LONG‐TERM RESPONSES TO CHANGING CO2 IN A COASTAL DINOFLAGELLATE BLOOM: IMPLICATIONS FOR INTERSPECIFIC COMPETITIVE INTERACTIONS AND COMMUNITY STRUCTURE

Increasing pCO₂ (partial pressure of CO₂) in an “acidified” ocean will affect phytoplankton community structure, but manipulation experiments with assemblages briefly acclimated to simulated future conditions may not accurately predict the long‐term evolutionary shifts that could affect inter‐specific competitive success. We assessed community structure changes in a natural mixed dinoflagellate bloom incubated at three pCO₂ levels (230, 433, and 765 ppm) in a short‐term experiment (2 weeks). The four dominant species were then isolated from each treatment into clonal cultures, and maintained at all three pCO₂ levels for approximately 1 year. Periodically (4, 8, and 12 months), these pCO₂‐conditioned clones were recombined into artificial communities, and allowed to compete at their conditioning pCO₂ level or at higher and lower levels. The dominant species in these artificial communities of CO₂‐conditioned clones differed from those in the original short‐term experiment, but individual species relative abundance trends across pCO₂ treatments were often similar. Specific growth rates showed no strong evidence for fitness increases attributable to conditioning pCO₂ level. Although pCO₂ significantly structured our experimental communities, conditioning time and biotic interactions like mixotrophy also had major roles in determining competitive outcomes. New methods of carrying out extended mixed species experiments are needed to accurately predict future long‐term phytoplankton community responses to changing pCO₂.     

Non-canonical localization of RubisCO under high-light conditions in the toxic cyanobacterium Microcystis aeruginosa PCC7806

The frequent production of the hepatotoxin microcystin (MC) and its impact on the lifestyle of bloom-forming cyanobacteria are poorly understood. Here, we report that MC interferes with the assembly and the subcellular localization of RubisCO, in Microcystis aeruginosa PCC7806. Immunofluorescence, electron microscopic and cellular fractionation studies revealed a pronounced heterogeneity in the subcellular localization of RubisCO. At high cell density, RubisCO particles are largely separate from carboxysomes in M. aeruginosa and relocate to the cytoplasmic membrane under high-light conditions. We hypothesize that the binding of MC to RubisCO promotes its membrane association and enables an extreme versatility of the enzyme. Steady-state levels of the RubisCO CO₂ fixation product 3-phosphoglycerate are significantly higher in the MC-producing wild type. We also detected noticeable amounts of the RubisCO oxygenase reaction product secreted into the medium that may support the mutual interaction of M. aeruginosa with its heterotrophic microbial community.