Metabolite diversity among representatives of divergent Prochlorococcus ecotypes.

IMPORTANCE: Approximately half of the annual carbon fixation on Earth occurs in the surface ocean through the photosynthetic activities of phytoplankton such as the ubiquitous picocyanobacterium Prochlorococcus. Ecologically distinct subpopulations (or ecotypes) of Prochlorococcus are central conduits of organic substrates into the ocean microbiome, thus playing important roles in surface ocean production. We measured the chemical profile of three cultured ecotype strains, observing striking differences among them that have implications for the likely chemical impact of Prochlorococcus subpopulations on their surroundings in the wild. Subpopulations differ in abundance along gradients of temperature, light, and nutrient concentrations, suggesting that these chemical differences could affect carbon cycling in different ocean strata and should be considered in models of Prochlorococcus physiology and marine carbon dynamics.

Chitin utilization by marine picocyanobacteria and the evolution of a planktonic lifestyle.

Marine picocyanobacteria Prochlorococcus and Synechococcus, the most abundant photosynthetic cells in the oceans, are generally thought to have a primarily single-celled and free-living lifestyle. However, while studying the ability of picocyanobacteria to supplement photosynthetic carbon fixation with the use of exogenous organic carbon, we found the widespread occurrence of genes for breaking down chitin, an abundant source of organic carbon that exists primarily as particles. We show that cells that encode a chitin degradation pathway display chitin degradation activity, attach to chitin particles, and show enhanced growth under low light conditions when exposed to chitosan, a partially deacetylated soluble form of chitin. Marine chitin is largely derived from arthropods, which underwent major diversifications 520 to 535 Mya, close to when marine picocyanobacteria are inferred to have appeared in the ocean. Phylogenetic analyses confirm that the chitin utilization trait was acquired at the root of marine picocyanobacteria. Together this leads us to postulate that attachment to chitin particles allowed benthic cyanobacteria to emulate their mat-based lifestyle in the water column, initiating their expansion into the open ocean, seeding the rise of modern marine ecosystems. Subsequently, transitioning to a constitutive planktonic life without chitin associations led to cellular and genomic streamlining along a major early branch within Prochlorococcus. Our work highlights how the emergence of associations between organisms from different trophic levels, and their coevolution, creates opportunities for colonizing new environments. In this view, the rise of ecological complexity and the expansion of the biosphere are deeply intertwined processes.

Microbial metabolites in the marine carbon cycle.

One-quarter of photosynthesis-derived carbon on Earth rapidly cycles through a set of short-lived seawater metabolites that are generated from the activities of marine phytoplankton, bacteria, grazers and viruses. Here we discuss the sources of microbial metabolites in the surface ocean, their roles in ecology and biogeochemistry, and approaches that can be used to analyse them from chemistry, biology, modelling and data science. Although microbial-derived metabolites account for only a minor fraction of the total reservoir of marine dissolved organic carbon, their flux and fate underpins the central role of the ocean in sustaining life on Earth.

Microbial feedbacks optimize ocean iron availability.

Iron is the limiting factor for biological production over a large fraction of the surface ocean because free iron is rapidly scavenged or precipitated under aerobic conditions. Standing stocks of dissolved iron are maintained by association with organic molecules (ligands) produced by biological processes. We hypothesize a positive feedback between iron cycling, microbial activity, and ligand abundance: External iron input fuels microbial production, creating organic ligands that support more iron in seawater, leading to further macronutrient consumption until other microbial requirements such as macronutrients or light become limiting, and additional iron no longer increases productivity. This feedback emerges in numerical simulations of the coupled marine cycles of macronutrients and iron that resolve the dynamic microbial production and loss of iron-chelating ligands. The model solutions resemble modern nutrient distributions only over a finite range of prescribed ligand source/sink ratios where the model ocean is driven to global-scale colimitation by micronutrients and macronutrients and global production is maximized. We hypothesize that a global-scale selection for microbial ligand cycling may have occurred to maintain "just enough" iron in the ocean.

Evolution of cellular metabolism and the rise of a globally productive biosphere.

Metabolic processes in cells and chemical processes in the environment are fundamentally intertwined and have evolved in concert for most of Earth's existence. Here I argue that intrinsic properties of cellular metabolism imposed central constraints on the historical trajectories of biopsheric productivity and atmospheric oxygenation. Photosynthesis depends on iron, but iron is highly insoluble under the aerobic conditions produced by oxygenic photosynthesis. These counteracting constraints led to two major stages of Earth oxygenation. After a cyanobacteria-driven biospheric expansion near the Archean-Proterozoic boundary, productivity remained largely restricted to continental boundaries and shallow aquatic environments where weathering inputs made iron more accessible. The anoxic deep open ocean was rich in free iron during the Proterozoic, but this iron was largely inaccessible, partly because an otherwise nutrient-poor ocean was limiting to photosynthesis, but also because a photosynthetic expansion would have quenched its own iron supply. Near the Proterozoic-Phanerozoic boundary, bioenergetics innovations allowed eukaryotic photosynthesis to overcome these interconnected negative feedbacks and begin expanding into the deep open oceans and onto the continents, where nutrients are inherently harder to come by. Key insights into what drove the ecological rise of eukaryotic photosynthesis emerge from analyses of marine Synechococcus and Prochlorococcus, abundant marine picocyanobacteria whose ancestors colonized the oceans in the Neoproterozoic. The reconstructed evolution of this group reveals a sequence of innovations that ultimately produced a form of photosynthesis in Prochlorococcus that is more like that of green plant cells than other cyanobacteria. Innovations increased the energy flux of cells, thereby enhancing their ability to acquire sparse nutrients, and as by-product also increased the production of organic carbon waste. Some of these organic waste products had the ability to chelate iron and make it bioavailable, thereby indirectly pushing the oceans through a transition from an anoxic state rich in free iron to an oxygenated state with organic carbon-bound iron. Resulting conditions (and parallel processes on the continents) in turn led to a series of positive feedbacks that increased the availability of other nutrients, thereby promoting the rise of a globally productive biosphere. In addition to the occurrence of major biospheric expansions, the several hundred million-year periods around the Archean-Proterozoic and Proterozoic-Phanerozoic boundaries share a number of other parallels. Both epochs have also been linked to major carbon cycle perturbations and global glaciations, as well as changes in the nature of plate tectonics and increases in continental exposure and weathering. This suggests the dynamics of life and Earth are intimately intertwined across many levels and that general principles governed transitions in these coupled dynamics at both times in Earth history.

Emergence of trait variability through the lens of nitrogen assimilation in Prochlorococcus.

Intraspecific trait variability has important consequences for the function and stability of marine ecosystems. Here we examine variation in the ability to use nitrate across hundreds of Prochlorococcus genomes to better understand the modes of evolution influencing intraspecific allocation of ecologically important functions. Nitrate assimilation genes are absent in basal lineages but occur at an intermediate frequency that is randomly distributed within recently emerged clades. The distribution of nitrate assimilation genes within clades appears largely governed by vertical inheritance, gene loss, and homologous recombination. By mapping this process onto a model of Prochlorococcus' macroevolution, we propose that niche-constructing adaptive radiations and subsequent niche partitioning set the stage for loss of nitrate assimilation genes from basal lineages as they specialized to lower light levels. Retention of these genes in recently emerged lineages has likely been facilitated by selection as they sequentially partitioned into niches where nitrate assimilation conferred a fitness benefit.

Single cell genomes of Prochlorococcus, Synechococcus, and sympatric microbes from diverse marine environments.

Prochlorococcus and Synechococcus are the dominant primary producers in marine ecosystems and perform a significant fraction of ocean carbon fixation. These cyanobacteria interact with a diverse microbial community that coexists with them. Comparative genomics of cultivated isolates has helped address questions regarding patterns of evolution and diversity among microbes, but the fraction that can be cultivated is miniscule compared to the diversity in the wild. To further probe the diversity of these groups and extend the utility of reference sequence databases, we report a data set of single cell genomes for 489 Prochlorococcus, 50 Synechococcus, 9 extracellular virus particles, and 190 additional microorganisms from a diverse range of bacterial, archaeal, and viral groups. Many of these uncultivated single cell genomes are derived from samples obtained on GEOTRACES cruises and at well-studied oceanographic stations, each with extensive suites of physical, chemical, and biological measurements. The genomic data reported here greatly increases the number of available Prochlorococcus genomes and will facilitate studies on evolutionary biology, microbial ecology, and biological oceanography.

Metabolic evolution and the self-organization of ecosystems.

Metabolism mediates the flow of matter and energy through the biosphere. We examined how metabolic evolution shapes ecosystems by reconstructing it in the globally abundant oceanic phytoplankter Prochlorococcus To understand what drove observed evolutionary patterns, we interpreted them in the context of its population dynamics, growth rate, and light adaptation, and the size and macromolecular and elemental composition of cells. This multilevel view suggests that, over the course of evolution, there was a steady increase in Prochlorococcus' metabolic rate and excretion of organic carbon. We derived a mathematical framework that suggests these adaptations lower the minimal subsistence nutrient concentration of cells, which results in a drawdown of nutrients in oceanic surface waters. This, in turn, increases total ecosystem biomass and promotes the coevolution of all cells in the ecosystem. Additional reconstructions suggest that Prochlorococcus and the dominant cooccurring heterotrophic bacterium SAR11 form a coevolved mutualism that maximizes their collective metabolic rate by recycling organic carbon through complementary excretion and uptake pathways. Moreover, the metabolic codependencies of Prochlorococcus and SAR11 are highly similar to those of chloroplasts and mitochondria within plant cells. These observations lead us to propose a general theory relating metabolic evolution to the self-amplification and self-organization of the biosphere. We discuss the implications of this framework for the evolution of Earth's biogeochemical cycles and the rise of atmospheric oxygen.

Metabolic evolution of a deep-branching hyperthermophilic chemoautotrophic bacterium.

Aquifex aeolicus is a deep-branching hyperthermophilic chemoautotrophic bacterium restricted to hydrothermal vents and hot springs. These characteristics make it an excellent model system for studying the early evolution of metabolism. Here we present the whole-genome metabolic network of this organism and examine in detail the driving forces that have shaped it. We make extensive use of phylometabolic analysis, a method we recently introduced that generates trees of metabolic phenotypes by integrating phylogenetic and metabolic constraints. We reconstruct the evolution of a range of metabolic sub-systems, including the reductive citric acid (rTCA) cycle, as well as the biosynthesis and functional roles of several amino acids and cofactors. We show that A. aeolicus uses the reconstructed ancestral pathways within many of these sub-systems, and highlight how the evolutionary interconnections between sub-systems facilitated several key innovations. Our analyses further highlight three general classes of driving forces in metabolic evolution. One is the duplication and divergence of genes for enzymes as these progress from lower to higher substrate specificity, improving the kinetics of certain sub-systems. A second is the kinetic optimization of established pathways through fusion of enzymes, or their organization into larger complexes. The third is the minimization of the ATP unit cost to synthesize biomass, improving thermodynamic efficiency. Quantifying the distribution of these classes of innovations across metabolic sub-systems and across the tree of life will allow us to assess how a tradeoff between maximizing growth rate and growth efficiency has shaped the long-term metabolic evolution of the biosphere.

The compositional and evolutionary logic of metabolism.

Metabolism is built on a foundation of organic chemistry, and employs structures and interactions at many scales. Despite these sources of complexity, metabolism also displays striking and robust regularities in the forms of modularity and hierarchy, which may be described compactly in terms of relatively few principles of composition. These regularities render metabolic architecture comprehensible as a system, and also suggests the order in which layers of that system came into existence. In addition metabolism also serves as a foundational layer in other hierarchies, up to at least the levels of cellular integration including bioenergetics and molecular replication, and trophic ecology. The recapitulation of patterns first seen in metabolism, in these higher levels, motivates us to interpret metabolism as a source of causation or constraint on many forms of organization in the biosphere. Many of the forms of modularity and hierarchy exhibited by metabolism are readily interpreted as stages in the emergence of catalytic control by living systems over organic chemistry, sometimes recapitulating or incorporating geochemical mechanisms.We identify as modules, either subsets of chemicals and reactions, or subsets of functions, that are re-used in many contexts with a conserved internal structure. At the small molecule substrate level, module boundaries are often associated with the most complex reaction mechanisms, catalyzed by highly conserved enzymes. Cofactors form a biosynthetically and functionally distinctive control layer over the small-molecule substrate. The most complex members among the cofactors are often associated with the reactions at module boundaries in the substrate networks, while simpler cofactors participate in widely generalized reactions. The highly tuned chemical structures of cofactors (sometimes exploiting distinctive properties of the elements of the periodic table) thereby act as 'keys' that incorporate classes of organic reactions within biochemistry.Module boundaries provide the interfaces where change is concentrated, when we catalogue extant diversity of metabolic phenotypes. The same modules that organize the compositional diversity of metabolism are argued, with many explicit examples, to have governed long-term evolution. Early evolution of core metabolism, and especially of carbon-fixation, appears to have required very few innovations, and to have used few rules of composition of conserved modules, to produce adaptations to simple chemical or energetic differences of environment without diverse solutions and without historical contingency. We demonstrate these features of metabolism at each of several levels of hierarchy, beginning with the small-molecule metabolic substrate and network architecture, continuing with cofactors and key conserved reactions, and culminating in the aggregation of multiple diverse physical and biochemical processes in cells.

The emergence and early evolution of biological carbon-fixation.

The fixation of CO2 into living matter sustains all life on Earth, and embeds the biosphere within geochemistry. The six known chemical pathways used by extant organisms for this function are recognized to have overlaps, but their evolution is incompletely understood. Here we reconstruct the complete early evolutionary history of biological carbon-fixation, relating all modern pathways to a single ancestral form. We find that innovations in carbon-fixation were the foundation for most major early divergences in the tree of life. These findings are based on a novel method that fully integrates metabolic and phylogenetic constraints. Comparing gene-profiles across the metabolic cores of deep-branching organisms and requiring that they are capable of synthesizing all their biomass components leads to the surprising conclusion that the most common form for deep-branching autotrophic carbon-fixation combines two disconnected sub-networks, each supplying carbon to distinct biomass components. One of these is a linear folate-based pathway of CO2 reduction previously only recognized as a fixation route in the complete Wood-Ljungdahl pathway, but which more generally may exclude the final step of synthesizing acetyl-CoA. Using metabolic constraints we then reconstruct a "phylometabolic" tree with a high degree of parsimony that traces the evolution of complete carbon-fixation pathways, and has a clear structure down to the root. This tree requires few instances of lateral gene transfer or convergence, and instead suggests a simple evolutionary dynamic in which all divergences have primary environmental causes. Energy optimization and oxygen toxicity are the two strongest forces of selection. The root of this tree combines the reductive citric acid cycle and the Wood-Ljungdahl pathway into a single connected network. This linked network lacks the selective optimization of modern fixation pathways but its redundancy leads to a more robust topology, making it more plausible than any modern pathway as a primitive universal ancestral form.