Widespread use of proton-pumping rhodopsin in Antarctic phytoplankton.

Photosynthetic carbon (C) fixation by phytoplankton in the Southern Ocean (SO) plays a critical role in regulating air-sea exchange of carbon dioxide and thus global climate. In the SO, photosynthesis (PS) is often constrained by low iron, low temperatures, and low but highly variable light intensities. Recently, proton-pumping rhodopsins (PPRs) were identified in marine phytoplankton, providing an alternate iron-free, light-driven source of cellular energy. These proteins pump protons across cellular membranes through light absorption by the chromophore retinal, and the resulting pH energy gradient can then be used for active membrane transport or for synthesis of adenosine triphosphate. Here, we show that PPR is pervasive in Antarctic phytoplankton, especially in iron-limited regions. In a model SO diatom, we found that it was localized to the vacuolar membrane, making the vacuole a putative alternative phototrophic organelle for light-driven production of cellular energy. Unlike photosynthetic C fixation, which decreases substantially at colder temperatures, the proton transport activity of PPR was unaffected by decreasing temperature. Cellular PPR levels in cultured SO diatoms increased with decreasing iron concentrations and energy production from PPR photochemistry could substantially augment that of PS, especially under high light intensities, where PS is often photoinhibited. PPR gene expression and high retinal concentrations in phytoplankton in SO waters support its widespread use in polar environments. PPRs are an important adaptation of SO phytoplankton to growth and survival in their cold, iron-limited, and variable light environment.

13C NMR metabolomics: J-resolved STOCSY meets INADEQUATE.

Robust annotation of metabolites is a challenging task in metabolomics. Among available applications, 13C NMR experiment INADEQUATE determines direct 13C-13C connectivity unambiguously, offering indispensable information on molecular structure. Despite its great utility, it is not always practical to collect INADEQUATE data on every sample in a large metabolomics study because of its relatively long experiment time. Here, we propose an alternative approach that maintains the quality of information but saves experiment time. In this approach, individual samples in a study are first screened by 13C homonuclear J-resolved experiment (JRES). Next, JRES data are processed by statistical total correlation spectroscopy (STOCSY) to extract peaks that behave similarly among samples. Finally, INADEQUATE is collected on one internal pooled sample to select STOCSY peaks that originate from the same compound. We tested this concept using the 13C-labeled endometabolome of a model marine diatom strain incubated under various settings, intending to cover a range of metabolites produced under different external conditions. This scheme was able to extract known diatom metabolites proline, 2,3-dihydroxypropane-1-sulfonate (DHPS), ß-1,3-glucan, choline, and glutamate. This pipeline also detected unknown compounds with structural information, which is valuable in metabolomics where a priori knowledge of metabolites is not always available. The ability of this scheme was seen even in sugar regions, which are usually challenging in 1H NMR due to severe peak overlap. JRES and INADEQUATE were highly complementary; INADEQUATE provided directly-bonded 13C networks, whereas JRES linked INADEQUATE networks within the same compound but broken by nitrogen or sulfur atoms, highlighting the advantage of this integrated approach.

The BBSome restricts entry of tagged carbonic anhydrase 6 into the cis-flagellum of Chlamydomonas reinhardtii.

The two flagella of Chlamydomonas reinhardtii are of the same size and structure but display functional differences, which are critical for flagellar steering movements. However, biochemical differences between the two flagella have not been identified. Here, we show that fluorescence protein-tagged carbonic anhydrase 6 (CAH6-mNG) preferentially localizes to the trans-flagellum, which is organized by the older of the two flagella-bearing basal bodies. The uneven distribution of CAH6-mNG is established early during flagellar assembly and restored after photobleaching, suggesting that it is based on preferred entry or retention of CAH6-mNG in the trans-flagellum. Since CAH6-mNG moves mostly by diffusion, a role of intraflagellar transport (IFT) in establishing its asymmetric distribution is unlikely. Interestingly, CAH6-mNG is present in both flagella of the non-phototactic bardet-biedl syndrome 1 (bbs1) mutant revealing that the BBSome is involved in establishing CAH6-mNG flagellar asymmetry. Using dikaryon rescue experiments, we show that the de novo assembly of CAH6-mNG in flagella is considerably faster than the removal of ectopic CAH6-mNG from bbs flagella. Thus, different rates of flagellar entry of CAH6-mNG rather than its export from flagella is the likely basis for its asymmetric distribution. The data identify a novel role for the C. reinhardtii BBSome in preventing the entry of CAH6-mNG specifically into the cis-flagellum.

Molecular composition and biodegradation of loggerhead sponge Spheciospongia vesparium exhalent dissolved organic matter.

Sponges are critical components of marine reefs due to their high filtering capacity, wide abundance, and alteration of biogeochemical cycling. Here, we characterized dissolved organic matter (DOM) composition in the sponge holobiont exhalent seawater of a loggerhead sponge (Spheciospongia vesparium) and in ambient seawater in Florida Bay (USA), as well as the microbial responses to each DOM pool through dark incubations. The sponge holobiont removed 6% of the seawater dissolved organic carbon (DOC), utilizing compounds that were low in carbon and oxygen, yet high in nitrogen content relative to the ambient seawater. The microbial community accessed 7% of DOC from the ambient seawater during a 5-day incubation but only 1% of DOC from the sponge exhalent seawater, suggesting a decrease in lability possibly due to holobiont removal of nitrogen-rich compounds. If this holds true for other sponges, it may have important implications for DOM lability and cycling in coastal environments.

Influence of Temperature and CO2 On Plasma-membrane Permeability to CO2 and HCO3- in the Marine Haptophytes Emiliania huxleyi and Calcidiscus leptoporus (Prymnesiophyceae).

Membrane permeabilities to CO2 and HCO3- constrain the function of CO2 concentrating mechanisms that algae use to supply inorganic carbon for photosynthesis. In diatoms and green algae, plasma membranes are moderately to highly permeable to CO2 but effectively impermeable to HCO3- . Here, CO2 and HCO3- membrane permeabilities were measured using an 18 O-exchange technique on two species of haptophyte algae, Emiliania huxleyi and Calcidiscus leptoporus, which showed that the plasma membranes of these species are also highly permeable to CO2 (0.006-0.02 cm · s-1 ) but minimally permeable to HCO3- . Increased temperature and CO2 generally increased CO2 membrane permeabilities in both species, possibly due to changes in lipid composition or CO2 channel proteins. Changes in CO2 membrane permeabilities showed no association with the density of calcium carbonate coccoliths surrounding the cell, which could potentially impede passage of compounds. Haptophyte plasma-membrane permeabilities to CO2 were somewhat lower than those of diatoms but generally higher than membrane permeabilities of green algae. One caveat of these measurements is that the model used to interpret 18 O-exchange data assumes that carbonic anhydrase, which catalyzes 18 O-exchange, is homogeneously distributed in the cell. The implications of this assumption were tested using a two-compartment model with an inhomogeneous distribution of carbonic anhydrase to simulate 18 O-exchange data and then inferring plasma-membrane CO2 permeabilities from the simulated data. This analysis showed that the inferred plasma-membrane CO2 permeabilities are minimal estimates but should be quite accurate under most conditions.

Automated classification of three-dimensional reconstructions of coral reefs using convolutional neural networks.

Coral reefs are biologically diverse and structurally complex ecosystems, which have been severally affected by human actions. Consequently, there is a need for rapid ecological assessment of coral reefs, but current approaches require time consuming manual analysis, either during a dive survey or on images collected during a survey. Reef structural complexity is essential for ecological function but is challenging to measure and often relegated to simple metrics such as rugosity. Recent advances in computer vision and machine learning offer the potential to alleviate some of these limitations. We developed an approach to automatically classify 3D reconstructions of reef sections and assessed the accuracy of this approach. 3D reconstructions of reef sections were generated using commercial Structure-from-Motion software with images extracted from video surveys. To generate a 3D classified map, locations on the 3D reconstruction were mapped back into the original images to extract multiple views of the location. Several approaches were tested to merge information from multiple views of a point into a single classification, all of which used convolutional neural networks to classify or extract features from the images, but differ in the strategy employed for merging information. Approaches to merging information entailed voting, probability averaging, and a learned neural-network layer. All approaches performed similarly achieving overall classification accuracies of ~96% and >90% accuracy on most classes. With this high classification accuracy, these approaches are suitable for many ecological applications.

Reduced nitrogenase efficiency dominates response of the globally important nitrogen fixer Trichodesmium to ocean acidification.

The response of the prominent marine dinitrogen (N2)-fixing cyanobacteria Trichodesmium to ocean acidification (OA) is critical to understanding future oceanic biogeochemical cycles. Recent studies have reported conflicting findings on the effect of OA on growth and N2 fixation of Trichodesmium. Here, we quantitatively analyzed experimental data on how Trichodesmium reallocated intracellular iron and energy among key cellular processes in response to OA, and integrated the findings to construct an optimality-based cellular model. The model results indicate that Trichodesmium growth rate decreases under OA primarily due to reduced nitrogenase efficiency. The downregulation of the carbon dioxide (CO2)-concentrating mechanism under OA has little impact on Trichodesmium, and the energy demand of anti-stress responses to OA has a moderate negative effect. We predict that if anthropogenic CO2 emissions continue to rise, OA could reduce global N2 fixation potential of Trichodesmium by 27% in this century, with the largest decrease in iron-limiting regions.

Pervasive iron limitation at subsurface chlorophyll maxima of the California Current.

Subsurface chlorophyll maximum layers (SCMLs) are nearly ubiquitous in stratified water columns and exist at horizontal scales ranging from the submesoscale to the extent of oligotrophic gyres. These layers of heightened chlorophyll and/or phytoplankton concentrations are generally thought to be a consequence of a balance between light energy from above and a limiting nutrient flux from below, typically nitrate (NO3). Here we present multiple lines of evidence demonstrating that iron (Fe) limits or with light colimits phytoplankton communities in SCMLs along a primary productivity gradient from coastal to oligotrophic offshore waters in the southern California Current ecosystem. SCML phytoplankton responded markedly to added Fe or Fe/light in experimental incubations and transcripts of diatom and picoeukaryote Fe stress genes were strikingly abundant in SCML metatranscriptomes. Using a biogeochemical proxy with data from a 40-y time series, we find that diatoms growing in California Current SCMLs are persistently Fe deficient during the spring and summer growing season. We also find that the spatial extent of Fe deficiency within California Current SCMLs has significantly increased over the last 25 y in line with a regional climate index. Finally, we show that diatom Fe deficiency may be common in the subsurface of major upwelling zones worldwide. Our results have important implications for our understanding of the biogeochemical consequences of marine SCML formation and maintenance.

Plasma Membrane-Type Aquaporins from Marine Diatoms Function as CO2/NH3 Channels and Provide Photoprotection.

Aquaporins (AQPs) are ubiquitous water channels that facilitate the transport of many small molecules and may play multiple vital roles in aquatic environments. In particular, mechanisms to maintain transmembrane fluxes of important small molecules have yet to be studied in marine photoautotrophic organisms. Here, we report the occurrence of multiple AQPs with differential cellular localizations in marine diatoms, an important group of oceanic primary producers. The AQPs play a role in mediating the permeability of membranes to CO2 and NH3 In silico surveys revealed the presence of five AQP orthologs in the pennate diatom Phaeodactylum tricornutum and two in the centric diatom Thalassiosira pseudonana GFP fusions of putative AQPs displayed clear localization to the plasma membrane (PtAGP1 and PtAQP2), the chloroplast endoplasmic reticulum (CER; PtAGP1 and PtAQP3), and the tonoplast (PtAQP5) in P. tricornutum In T. pseudonana, GFP-AQP fusion proteins were found on the vacuole membrane (TpAQP1) and CER (TpAQP2). Transcript levels of both PtAQP1 and PtAQP2 were highly induced by ammonia, while only PtAQP2 was induced by high (1%[v/v]) CO2 Constitutive overexpression of GFP-tagged PtAQP1 and PtAQP2 significantly increased CO2 and NH3 permeability in P. tricornutum, strongly indicating that these AQPs function in regulating CO2/NH3 permeability in the plasma membrane and/or CER. Cells carrying GFP-tagged PtAQP1 and PtAQP2 had higher nonphotochemical quenching under high light relative to that of wild-type cells, suggesting that these AQPs are involved in photoprotection. These AQPs may facilitate the efflux of NH3, preventing the uncoupling effect of high intracellular ammonia concentrations.

Dynamic changes in carbonate chemistry in the microenvironment around single marine phytoplankton cells.

Photosynthesis by marine diatoms plays a major role in the global carbon cycle, although the precise mechanisms of dissolved inorganic carbon (DIC) uptake remain unclear. A lack of direct measurements of carbonate chemistry at the cell surface has led to uncertainty over the underlying membrane transport processes and the role of external carbonic anhydrase (eCA). Here we identify rapid and substantial photosynthesis-driven increases in pH and [CO32-] primarily due to the activity of eCA at the cell surface of the large diatom Odontella sinensis using direct simultaneous microelectrode measurements of pH and CO32- along with modelling of cell surface inorganic carbonate chemistry. Our results show that eCA acts to maintain cell surface CO2 concentrations, making a major contribution to DIC supply in O. sinensis. Carbonate chemistry at the cell surface is therefore highly dynamic and strongly dependent on cell size, morphology and the carbonate chemistry of the bulk seawater.

Mechanisms of carbon dioxide acquisition and CO2 sensing in marine diatoms: a gateway to carbon metabolism.

Diatoms are one of the most successful marine eukaryotic algal groups, responsible for up to 20% of the annual global CO2 fixation. The evolution of a CO2-concentrating mechanism (CCM) allowed diatoms to overcome a number of serious constraints on photosynthesis in the marine environment, particularly low [CO2]aq in seawater relative to concentrations required by the CO2 fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), which is partly due to the slow diffusion rate of CO2 in water and a limited CO2 formation rate from [Formula: see text] in seawater. Diatoms use two alternative strategies to take up dissolved inorganic carbon (DIC) from the environment: one primarily relies on the direct uptake of [Formula: see text] through plasma-membrane type solute carrier (SLC) 4 family [Formula: see text] transporters and the other is more reliant on passive diffusion of CO2 formed by an external carbonic anhydrase (CA). Bicarbonate taken up into the cytoplasm is most likely then actively transported into the chloroplast stroma by SLC4-type transporters on the chloroplast membrane system. Bicarbonate in the stroma is converted into CO2 only in close proximity to RubisCO preventing unnecessary CO2 leakage. CAs play significant roles in mobilizing DIC as it is progressively moved towards the site of fixation. However, the evolutionary types and subcellular locations of CAs are not conserved between different diatoms, strongly suggesting that this DIC mobilization strategy likely evolved multiple times with different origins. By contrast, the recent discovery of the thylakoid luminal θ-CA indicates that the strategy to supply CO2 to RubisCO in the pyrenoid may be very similar to that of green algae, and strongly suggests convergent coevolution in CCM function of the thylakoid lumen not only among diatoms but among eukaryotic algae in general. In this review, both experimental and corresponding theoretical models of the diatom CCMs are discussed.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

The potential for co-evolution of CO2-concentrating mechanisms and Rubisco in diatoms.

Diatoms are a diverse group of unicellular algae that contribute significantly to global photosynthetic carbon fixation and export in the modern ocean, and are an important source of microfossils for paleoclimate reconstructions. Because of their importance in the environment, diatoms have been a focus of study on the physiology and ecophysiology of carbon fixation, in particular their CO2-concentrating mechanisms (CCMs) and Rubisco characteristics. While carbon fixation in diatoms is not as well understood as in certain model aquatic photoautotrophs, a greater number of species have been examined in diatoms. Recent work has highlighted a large diversity in the function, physiology, and kinetics of both the CCM and Rubisco between different diatom species. This diversity was unexpected since it has generally been assumed that CCMs and Rubiscos were similar within major algal lineages as the result of selective events deep in evolutionary history, and suggests a more recent co-evolution between the CCM and Rubisco within diatoms. This review explores our current understanding of the diatom CCM and highlights the diversity of both the CCM and Rubisco kinetics. We will suggest possible environmental, physiological, and evolutionary drivers for the co-evolution of the CCM and Rubisco in diatoms.

The diversity of CO2-concentrating mechanisms in marine diatoms as inferred from their genetic content.

Marine diatoms are one of the most ecologically significant primary producers in the ocean. Most diatoms use a CO2-concentrating mechanism (CCM) to overcome the scarcity of CO2 in the ocean and limitations of the carbon-fixing enzyme Rubisco. However, the CCMs in model diatoms differ substantially in their genetic make-up and structural organization. To assess the extent of CCM diversity in marine diatoms more generally, we analyzed genome and transcriptome data from 31 diatom strains to identify putative CCM genes, examine the overall CCM architecture, and study CCM development in the context of the evolutionary history of these diatoms. Key CCM genes [carbonic anhydrases (CAs) and solute carrier 4 (SLC4) bicarbonate transporters] identified in the diatoms were placed into groups of likely orthologs by sequence similarity (OrthoMCL) and phylogenetic methods. These analyses indicated that diatoms seem to share similar HCO3- transporters, but possess a variety of CAs that have either undergone extensive diversification within the diatom lineage or have been acquired through horizontal gene transfer. Hierarchical clustering of the diatom species based on their CCM gene content suggests that CCM development is largely congruent with evolution of diatom species, despite some notable differences in CCM genes even among closely related species.

Microelectrode characterization of coral daytime interior pH and carbonate chemistry.

Reliably predicting how coral calcification may respond to ocean acidification and warming depends on our understanding of coral calcification mechanisms. However, the concentration and speciation of dissolved inorganic carbon (DIC) inside corals remain unclear, as only pH has been measured while a necessary second parameter to constrain carbonate chemistry has been missing. Here we report the first carbonate ion concentration ([CO3(2-)]) measurements together with pH inside corals during the light period. We observe sharp increases in [CO3(2-)] and pH from the gastric cavity to the calcifying fluid, confirming the existence of a proton (H(+)) pumping mechanism. We also show that corals can achieve a high aragonite saturation state (Ωarag) in the calcifying fluid by elevating pH while at the same time keeping [DIC] low. Such a mechanism may require less H(+)-pumping and energy for upregulating pH compared with the high [DIC] scenario and thus may allow corals to be more resistant to climate change related stressors.

The physiology and genetics of CO2 concentrating mechanisms in model diatoms.

Diatoms, a diverse and ecologically-important group of unicellular algae, use a CO2 concentrating mechanism to enhance the performance of RubisCO and overcome the limited availability of CO2 in their habitats. The recent development of genetic manipulation techniques for the model diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana and the sequencing of their genomes have enabled the rapid identification of genes involved in their CO2 concentrating mechanisms (CCMs). These include numerous carbonic anhydrases (CAs), which are localized to distinct subcellular compartments in the two diatom species, and putative bicarbonate transporters, one of which has been functionally characterized. New physiological data on the P. tricornutum CCM are consistent with this molecular data and suggest that the major driver of the CCM is a 'chloroplast-pump' that actively transports bicarbonate into the chloroplast. In T. pseudonana, the localization of a CA in the chloroplast stroma presents a paradox as this would be expected to impede function of a biophysical CCM, though the recent proposal of a modified C4 CCM offers a potential explanation.

Marine diatom proteorhodopsins and their potential role in coping with low iron availability.

Proteorhodopsins (PR) are retinal-binding membrane proteins that function as light-driven proton pumps to generate energy for metabolism and growth. Recently PR-like genes have been identified in some marine eukaryotic protists, including diatoms, dinoflagellates, haptophytes and cryptophytes. These rhodopsins are homologous to green-light-absorbing, ATP-generating PRs present within bacteria. Here we show that in the oceanic diatom Pseudo-nitzschia granii, PR-like gene and protein expressions increase appreciably under iron limitation. In a survey of available transcriptomes, PR-like genes in diatoms are generally found in isolates from marine habitats where seasonal to chronic growth limitation by the micronutrient iron is prevalent, yet similar biogeographical patterns are not apparent in other phytoplankton taxa. We propose that rhodopsin-based phototrophy could account for a proportion of energy synthesis in marine eukaryotic photoautotrophs, especially when photosynthesis is compromised by low iron availability. This alternative ATP-generating pathway could have significant effects on plankton community structure and global ocean carbon cycling.

Internal carbonic anhydrase activity in the tissue of scleractinian corals is sufficient to support proposed roles in photosynthesis and calcification.

Reef-building corals import inorganic carbon (Ci) to build their calcium carbonate skeletons and to support photosynthesis by the symbiotic algae that reside in their tissue. The internal pathways that deliver Ci for both photosynthesis and calcification are known to involve the enzyme carbonic anhydrase (CA), which interconverts CO2 and HCO3 (-). We have developed a method for absolute quantification of internal CA (iCA) activity in coral tissue based on the rate of (18)O-removal from labeled Ci. The method was applied to three Caribbean corals (Orbicella faveolata, Porites astreoides and Siderastrea radians) and showed that these species have similar iCA activities per unit surface area, but that S. radians has ∼10-fold higher iCA activity per unit tissue volume. A model of coral Ci processing shows that the measured iCA activity is sufficient to support the proposed roles for iCA in Ci transport for photosynthesis and calcification. This is the case even when iCA activity is homogeneously distributed throughout the coral, but the model indicates that it would be advantageous to concentrate iCA in the spaces where calcification (the calcifying fluid) and photosynthesis (the oral endoderm) take place. We argue that because the rates of photosynthesis and calcification per unit surface area are similar among the corals studied here, the areal iCA activity used to deliver Ci for these reactions should also be similar. The elevated iCA activity per unit volume of S. radians compared with that of the other species is probably due to the thinner effective tissue thickness in this species.

Low temperature reduces the energetic requirement for the CO2 concentrating mechanism in diatoms.

The goal of this study is to investigate the CO2 concentrating mechanism (CCM) of the dominant phytoplankton species during the growing season at Palmer station in the Western Antarctic Peninsula. Key CCM parameters (cellular half-saturation constants for CO2 fixation, carbonic anhydrase activity, CO2 /HCO3 (-) uptake, δ(13) Corg ) in natural phytoplankton assemblages were determined. Those results, together with additional measurements on CO2 membrane permeability from Fragilariopsis cylindrus laboratory cultures, were used to develop a numerical model of the CCM of cold water diatoms. The field data demonstrate that the dominant species throughout the season possess an effective CCM, which achieves near saturation of CO2 for fixation. The model provides a means to examine the role of eCA activity and HCO3 (-) /CO2 uptake in the functioning of the CCM. According to the model, the increase in δ(13) Corg during the bloom results chiefly from decreasing ambient CO2 concentration (which reduces the gross diffusive flux across the membrane) rather than a shift in inorganic carbon uptake from CO2 to HCO3 (-) . The CCM of diatoms in the Western Antarctic Peninsula functions with a relatively small expenditure of energy, resulting chiefly from the low half-saturation constant for Rubisco at cold temperatures.

Size scaling of extracellular carbonic anhydrase activity in centric marine diatoms.

Many microalgae have a surface-associated extracellular carbonic anhydrase (eCA) that converts HCO3 (-) to CO2 for uptake and subsequent photosynthetic fixation. We investigated eCA activity and assessed its importance for photosynthetic CO2 supply in six centric diatom species spanning nearly the full range of cell sizes for centric diatoms (equivalent spherical radius 3-67 µm). Since larger cells are more susceptible to diffusion limitation, we hypothesized that eCA activity would increase with cell size as would its importance for CO2 supply. eCA activity did increase with cell size, increasing with cell radius by a size-scaling exponent of 2.6 ± 0.3. The rapid increase in eCA activity with cell radius keeps the absolute CO2 concentration difference between bulk seawater and the cell surface very low (<~0.2 µM) allowing high rates of CO2 uptake even for large diatoms. Although inhibiting eCA did reduce photosynthesis in the diatoms, there was no overall relationship between the extent of inhibition of photosynthesis and cell size. The only indication that eCA may be more important for larger diatoms was that photosynthesis in the smallest diatoms (<4 µm radius) was only affected by eCA inhibition when CO2 concentrations were very low, while photosynthesis in some larger diatoms was affected even at typical seawater CO2 concentrations. eCA is ubiquitous in centric marine diatoms, in contrast to other taxa where its presence is irregularly distributed among different species, and plays an important role in supplying CO2 for photosynthesis across the size spectrum.

The minimal CO2-concentrating mechanism of Prochlorococcus spp. MED4 is effective and efficient.

As an oligotrophic specialist, Prochlorococcus spp. has streamlined its genome and metabolism including the CO2-concentrating mechanism (CCM), which serves to elevate the CO2 concentration around Rubisco. The genomes of Prochlorococcus spp. indicate that they have a simple CCM composed of one or two HCO3(-) pumps and a carboxysome, but its functionality has not been examined. Here, we show that the CCM of Prochlorococcus spp. is effective and efficient, transporting only two molecules of HCO3(-) per molecule of CO2 fixed. A mechanistic, numerical model with a structure based on the CCM components present in the genome is able to match data on photosynthesis, CO2 efflux, and the intracellular inorganic carbon pool. The model requires the carboxysome shell to be a major barrier to CO2 efflux and shows that excess Rubisco capacity is critical to attaining a high-affinity CCM without CO2 recovery mechanisms or high-affinity HCO3(-) transporters. No differences in CCM physiology or gene expression were observed when Prochlorococcus spp. was fully acclimated to high-CO2 (1,000 µL L(-1)) or low-CO2 (150 µL L(-1)) conditions. Prochlorococcus spp. CCM components in the Global Ocean Survey metagenomes were very similar to those in the genomes of cultivated strains, indicating that the CCM in environmental populations is similar to that of cultured representatives.