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.The marine picocyanobacteria genus Prochlorococcus along with its sister group the marine genus Synechococcus dominate primary production in oligotrophic marine environments (Partensky et al., 1999). Prochlorococcus spp. is an oligotrophic specialist with several key adaptations allowing it to outcompete other phytoplankton in the stable, low-nutrient regions where it thrives. These adaptations include small cell size (less than 1 μm), allowing it to effectively capture nutrients and light, and genome streamlining, which minimizes nutrient requirements (Partensky and Garczarek, 2010). At approximately 1,900 genes, the genomes of high-light-adapted Prochlorococcus spp. are the smallest known among photoautotrophs, suggesting that this is about the minimum number of genes needed to make a cell from inorganic constituents and light (Rocap et al., 2003). Genome reduction has been accomplished by both the loss of entire pathways and complexes, such as the phycobilisomes and many regulatory capabilities, and the paring down of systems to their minimal components, as is the case for the circadian clock and the photosynthetic complexes (Rocap et al., 2003; Kettler et al., 2007; Partensky and Garczarek, 2010).As part of this genome streamlining, the CO₂-concentrating mechanism (CCM), which enhances the efficiency of photosynthesis by elevating the concentration of CO₂ around Rubisco, has been reduced to what appears to be the minimal number of components necessary for a functional CCM (Badger and Price, 2003; Badger et al., 2006). In typical cyanobacteria, the CCM is composed of HCO₃⁻ transporters, CO₂ uptake systems, and the carboxysome, a protein microcompartment in which Rubisco and carbonic anhydrase (CA) are enclosed. HCO₃⁻ is accumulated in the cytoplasm by direct import from the environment and by the active conversion of CO₂ to HCO₃⁻ via an NADH-dependent process, which constitutes the CO₂ uptake mechanism (Shibata et al., 2001). The accumulated HCO₃⁻ then diffuses into the carboxysome, where CA converts it to CO₂, elevating the concentration of CO₂ around Rubisco (Reinhold et al., 1987; Price and Badger, 1989).Whereas some cyanobacteria have up to three different families of HCO₃⁻ transporters with differing affinities for use under different environmental conditions, Prochlorococcus spp. has only one or two families (Badger et al., 2006)_. Most cyanobacteria have low-affinity and high-affinity CO₂ uptake systems, but no CO₂ uptake systems are apparent in Prochlorococcus spp. genomes. The carboxysome of Prochlorococcus spp. and other α-cyanobacteria has apparently been laterally transferred from chemoautotrophs, but all of the required components of the carboxysome are present and it is functional (Badger et al., 2002; Roberts et al., 2012). Despite its simplicity, this CCM is likely functional. HCO₃⁻ can be accumulated in the cytoplasm by the HCO₃⁻ transporters and then diffuse into the carboxysome for conversion to CO₂ and subsequent fixation by Rubisco. However, the functionality of the CCM in Prochlorococcus spp. has not yet been tested. Prochlorococcus spp. is a representative of the α-cyanobacteria, a group with distinct CCMs, which have been much less well studied than the CCMs of β-cyanobacteria (Rae et al., 2011, 2013; Whitehead et al., 2014).We characterized inorganic carbon (C_i) acquisition and processing in Prochlorococcus spp. MED4, examined the effect of long-term acclimation to different CO₂ concentrations on CCM physiology and gene expression, and searched metagenomes for Prochlorococcus spp. CCM genes to determine if CCMs in the natural populations are similar to cultured strains.