Kimberly H. Halsey

Evolved physiological responses of phytoplankton to their integrated growth environment

Phytoplankton growth and productivity relies on light, multiple nutrients and temperature. These combined factors constitute the ‘integrated growth environment’. Since their emergence in the Archaean ocean, phytoplankton have experienced dramatic shifts in their integrated growth environment and, in response, evolved diverse mechanisms to maximize growth by optimizing the allocation of photosynthetic resources (ATP and NADPH) among all cellular processes. Consequently, co-limitation has become an omnipresent condition in the global ocean. Here we focus on evolved phytoplankton populations of the contemporary ocean and the varied energetic pathways they employ to solve the optimization problem of resource supply and demand. Central to this discussion is the allocation of reductant formed through photosynthesis, which we propose has the following three primary fates: carbon fixation, direct use and ATP generation. Investment of reductant among these three sinks is tied to cell cycle events, differentially influenced by specific forms of nutrient stress, and a strong determinant of relationships between light-harvesting (pigment), photosynthetic electron transport and carbon fixation. Global implications of optimization are illustrated by deconvolving trends in the 10-year global satellite chlorophyll record into contributions from biomass and physiology, thereby providing a unique perspective on the dynamic nature of surface phytoplankton populations and their link to climate.

One Carbon Metabolism in SAR11 Pelagic Marine Bacteria

The SAR11 Alphaproteobacteria are the most abundant heterotrophs in the oceans and are believed to play a major role in mineralizing marine dissolved organic carbon. Their genomes are among the smallest known for free-living heterotrophic cells, raising questions about how they successfully utilize complex organic matter with a limited metabolic repertoire. Here we show that conserved genes in SAR11 subgroup Ia (Candidatus Pelagibacter ubique) genomes encode pathways for the oxidation of a variety of one-carbon compounds and methyl functional groups from methylated compounds. These pathways were predicted to produce energy by tetrahydrofolate (THF)-mediated oxidation, but not to support the net assimilation of biomass from C1 compounds. Measurements of cellular ATP content and the oxidation of 14C-labeled compounds to 14CO2 indicated that methanol, formaldehyde, methylamine, and methyl groups from glycine betaine (GBT), trimethylamine (TMA), trimethylamine N-oxide (TMAO), and dimethylsulfoniopropionate (DMSP) were oxidized by axenic cultures of the SAR11 strain Ca. P. ubique HTCC1062. Analyses of metagenomic data showed that genes for C1 metabolism occur at a high frequency in natural SAR11 populations. In short term incubations, natural communities of Sargasso Sea microbial plankton expressed a potential for the oxidation of 14C-labeled formate, formaldehyde, methanol and TMAO that was similar to cultured SAR11 cells and, like cultured SAR11 cells, incorporated a much larger percentage of pyruvate and glucose (27–35%) than of C1 compounds (2–6%) into biomass. Collectively, these genomic, cellular and environmental data show a surprising capacity for demethylation and C1 oxidation in SAR11 cultures and in natural microbial communities dominated by SAR11, and support the conclusion that C1 oxidation might be a significant conduit by which dissolved organic carbon is recycled to CO2 in the upper ocean.

Phytoplankton Strategies for Photosynthetic Energy Allocation

Phytoplankton physiology is dynamic and highly responsive to the environment. Phytoplankton acclimate to changing environmental conditions by a complex reallocation of carbon and energy through metabolic pathways to optimize growth. Considering the tremendous diversity of phytoplankton, it is not surprising that different phytoplankton taxa use different strategies to partition carbon and energy resources. It has therefore been satisfying to discover that general principles of energetic stoichiometry appear to govern these complex processes and can be broadly applied to interpret phytoplankton distributions, productivity, and food web dynamics. The expectation of future changes in aquatic environments brought on by climate change warrants gathering knowledge about underlying patterns of photosynthetic energy allocation and their impacts on community structure and ecosystem productivity.

Synergistic metabolism of a broad range of C1 compounds in the marine methylotrophic bacterium HTCC2181

The 1.3 Mbp genome of HTCC2181, a member of the abundant OM43 clade of coastal bacterioplankton, suggested it is an obligate methylotroph. Preliminary experiments demonstrated that methanol and formaldehyde, but not other common C1 compounds such as methylamine, could support growth. Methanol concentrations in seawater are reportedly < 100 nM, suggesting either that the flux of methanol through plankton pools is very rapid, or that methanol may not be the primary growth substrate for HTCC2181. Therefore, we investigated the apparent extreme substrate range restriction of HTCC2181 in greater detail. Growth rate and maximum cell density of HTCC2181 increased with methanol concentration, yielding a K(s) value of 19 µM. In contrast, no growth was observed in the presence of the methylated (C1) compounds, methyl chloride, trimethylamine-oxide (TMAO) or dimethylsulfoniopropionate (DMSP) when they were the sole substrates. However, growth rate, maximum cell density and cellular ATP content were significantly enhanced when any of these methylated compounds were provided in the presence of a limiting concentration of methanol. These observations fit a model in which the metabolic intermediate formaldehyde is required for net carbon assimilation, allowing C1 substrates that do not produce a formaldehyde intermediate to be oxidized for energy, but not assimilated into biomass. Rates of methanol and TMAO oxidation and assimilation were measured with (14)C-radiolabelled compounds in cultures of HTCC2181 and seawater microbial communities collected off the Oregon coast. The results indicated that in nature as well as in culture, C1 substrates are partitioned between those that are mainly oxidized to produce energy and those that are assimilated. These findings indicate that the combined fluxes of C1 compounds in coastal systems are sufficient to support significant populations of obligate methyltrophs by a metabolic strategy that involves the synergistic metabolism of multiple C1 compounds.

LINKING TIME-DEPENDENT CARBON-FIXATION EFFICIENCIES IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) TO UNDERLYING METABOLIC PATHWAYS1

The chl‐specific short‐term 14C‐based production (Pb) measurement is a widely used tool to understand phytoplankton responses to environmental stresses. However, among the metabolic consequences of these stresses is variability in lifetimes of newly fixed carbon that cause Pb to range between chl‐specific net primary production (NPP*) and chl‐specific gross photosynthetic electron flow that is available for carbon reduction () depending on growth rate. To investigate the basis for this discrepancy, photosynthate utilization was characterized in Dunaliella tertiolecta Butcher grown at three different growth rates in N‐limited chemostats. Pb was measured throughout a 2 min to 24 h time course and showed clear growth‐rate‐dependent differences in lifetimes of newly fixed carbon. 14C pulse‐chase experiments revealed differences in patterns of carbon utilization between growth rates. At high growth rate, the majority of 14C was initially fixed into polysaccharide and lipid, but the relative contribution of each labeled biochemical pool to the total label changed over 24 h. In fast‐growing cells, labeled polysaccharides decreased 50%, while labeled lipids increased over the first 4 h. At low growth rate, 14C was initially incorporated primarily into protein, but the contribution of labeled protein to the total label increased over the next 24 h. Together, time‐resolved measurements of Pb and cellular NAD and NADP content suggest an enhanced role for alternative dissipation pathways at very low growth rate. Findings of this study contribute to an integrated understanding of growth‐rate‐dependent shifts in metabolic processes from photosynthesis to net growth.

A common partitioning strategy for photosynthetic products in evolutionarily distinct phytoplankton species

· We compare the nutrient-dependent photosynthetic efficiencies of the chlorophyte, Dunaliella tertiolecta, with those of the marine diatom, Thalassiosira weissflogii. Despite considerable evolutionary and physiological differences, these two species appear to use nearly identical growth strategies under a wide range of nutrient limitation. · Using a variety of physiological measurements, we find that, for both species and across all growth rates, 75% of the gross photosynthetic electron flow is invested in carbon fixation and only 30% is retained as net carbon accumulation. A majority of gross photosynthesis (70%) is ultimately used as reductant for biosynthetic pathways and for the generation of ATP. · In both species, newly formed carbon products exhibit much shorter half-lives at slow growth rates than at fast growth rates. We show that this growth rate dependence is a result of increased polysaccharide storage during the S phase of the cell cycle. · We present a model of carbon utilization that incorporates this growth rate-dependent carbon allocation and accurately captures (r(2) = 0.94) the observed time-resolved carbon retention. Together, our findings suggest a common photosynthetic optimization strategy in evolutionarily distinct phytoplankton species and contribute towards a systems-level understanding of carbon flow in photoautotrophs.

The abundant marine bacterium Pelagibacter simultaneously catabolizes dimethylsulfoniopropionate to the gases dimethyl sulfide and methanethiol

Marine phytoplankton produce ∼109 tonnes of dimethylsulfoniopropionate (DMSP) per year1,2, an estimated 10% of which is catabolized by bacteria through the DMSP cleavage pathway to the climatically active gas dimethyl sulfide3,4. SAR11 Alphaproteobacteria (order Pelagibacterales), the most abundant chemo-organotrophic bacteria in the oceans, have been shown to assimilate DMSP into biomass, thereby supplying this cells unusual requirement for reduced sulfur5,6. Here, we report that Pelagibacter HTCC1062 produces the gas methanethiol, and that a second DMSP catabolic pathway, mediated by a cupin-like DMSP lyase, DddK, simultaneously shunts as much as 59% of DMSP uptake to dimethyl sulfide production. We propose a model in which the allocation of DMSP between these pathways is kinetically controlled to release increasing amounts of dimethyl sulfide as the supply of DMSP exceeds cellular sulfur demands for biosynthesis.

Site-Directed Amino Acid Substitutions in the Hydroxylase Subunit of Butane Monooxygenase from Pseudomonas butanovora: Implications for Substrates Knocking at the Gate

Butane monooxygenase (BMO) from Pseudomonas butanovora has high homology to soluble methane monooxygenase (sMMO), and both oxidize a wide range of hydrocarbons; yet previous studies have not demonstrated methane oxidation by BMO. Studies to understand the basis for this difference were initiated by making single-amino-acid substitutions in the hydroxylase alpha subunit of butane monooxygenase (BMOH-alpha) in P. butanovora. Residues likely to be within hydrophobic cavities, adjacent to the diiron center, and on the surface of BMOH-alpha were altered to the corresponding residues from the alpha subunit of sMMO. In vivo studies of five site-directed mutants were carried out to initiate mechanistic investigations of BMO. Growth rates of mutant strains G113N and L279F on butane were dramatically slower than the rate seen with the control P. butanovora wild-type strain (Rev WT). The specific activities of BMO in these strains were sevenfold lower than those of Rev WT. Strains G113N and L279F also showed 277- and 5.5-fold increases in the ratio of the rates of 2-butanol production to 1-butanol production compared to Rev WT. Propane oxidation by strain G113N was exclusively subterminal and led to accumulation of acetone, which P. butanovora could not further metabolize. Methane oxidation was measurable for all strains, although accumulation of 23 microM methanol led to complete inhibition of methane oxidation in strain Rev WT. In contrast, methane oxidation by strain G113N was not completely inhibited until the methanol concentration reached 83 microM. The structural significance of the results obtained in this study is discussed using a three-dimensional model of BMOH-alpha.

Contrasting strategies of photosynthetic energy utilization drive lifestyle strategies in ecologically important picoeukaryotes.

The efficiency with which absorbed light is converted to net growth is a key property for estimating global carbon production. We previously showed that, despite considerable evolutionary distance, Dunaliella tertiolecta (Chlorophyceae) and Thalassiosira weissflogii (Bacillariophyceae) share a common strategy of photosynthetic energy utilization and nearly identical light energy conversion efficiencies. These findings suggested that a single model might be appropriate for describing relationships between measures of phytoplankton production. This conclusion was further evaluated for Ostreococcus tauri RCC1558 and Micromonas pusilla RCC299 (Chlorophyta, Prasinophyceae), two picoeukaryotes with contrasting geographic distributions and swimming abilities. Nutrient-dependent photosynthetic efficiencies in O. tauri were similar to the previously studied larger algae. Specifically, absorption-normalized gross oxygen and carbon production and net carbon production were independent of nutrient limited growth rate. In contrast, all measures of photosynthetic efficiency were strongly dependent on nutrient availability in M. pusilla. This marked difference was accompanied by a diminished relationship between Chla:C and nutrient limited growth rate and a remarkably greater efficiency of gross-to-net energy conversion than the other organisms studied. These results suggest that the cost-benefit of decoupling pigment concentration from nutrient availability enables motile organisms to rapidly exploit more frequent encounters with micro-scale nutrient patches in open ocean environments.

The CAFE Model: A Net Production Model for Global Ocean Phytoplankton

The Carbon, Absorption, and Fluorescence Euphotic-resolving (CAFE) net primary production model is an adaptable framework for advancing global ocean productivity assessments by exploiting state-of-the-art satellite ocean color analyses and addressing key physiological and ecological attributes of phytoplankton. Here, we present the first implementation of the CAFE model that incorporates inherent optical properties derived from ocean color measurements into a mechanistic and accurate model of phytoplankton growth rates (μ) and net phytoplankton production (NPP). The CAFE model calculates NPP as the product of energy absorption (QPAR), and the efficiency (φμ) by which absorbed energy is converted into carbon biomass (CPhyto), while μ is calculated as NPP normalized to CPhyto. The CAFE model performance is evaluated alongside 21 other NPP models against a spatially robust and globally representative set of direct NPP measurements. This analysis demonstrates that the CAFE model explains the greatest amount of variance and has the lowest model bias relative to other NPP models analyzed with this dataset. Global oceanic NPP from the CAFE model (52 Pg C m-2 yr-1) and mean division rates (0.34 d-1) are derived from climatological satellite data (2002-2014). This manuscript discusses and validates individual CAFE model parameters (e.g. QPAR, φμ), provides detailed sensitivity analyses, and compares the CAFE model results and parameterization to other widely cited models.