Photosynthetic organisms have recently gained considerable attention for a role in development of renewable energy sources. Genome-enabled systems biology methods, coupled with functional and synthetic genomics, present opportunities to develop sustainable and economical applications such as fuel production within the next 10 to 15 years. However, optimization of light-driven metabolism for biomass or biofuel production will require a detailed systems biology understanding of photosynthetic processes and cellular metabolism. Genome-scale metabolic models (GEMs) are at the core of systems analysis of cellular processes and form a common organizational framework for analyses of data resulting from functional genomics experimental work and computational studies. Therefore, there is a clear demand for high quality photosynthetic model organisms and the appropriate computational tools that enable systems analysis of light-driven metabolism. Through research conducted we expanded the currently available repertoire of photosynthetic GEMs to include the commercially valuable model diatom Phaeoctylum tricornutum. Diatoms have a peculiar and distinct evolutionary footprint and represent a major eukaryotic lineage that is taxonomically and functionally distinct from green and red algae and vascular plants. Therefore, the true potential for light-driven metabolism aimed at biofuel production remains poorly understood at a systems level for a large subset of the global diversity of photosynthetic organisms. The metabolic capabilities of P. tricornutum were comparatively modeled with those from other photosynthetic groups in order to elucidate the occurrence of metabolic traits within and between phototrophs. Additionally, this research resulted in significant extension of the COnstraints Based Reconstruction and Analysis (COBRA) Toolbox to accommodate the crucial need for infrastructure required for ‘omics data integration and analysis in the context of genome-scale models. Therefore, the proposed research achieved two important goals. First, within the broad scope of photosynthetic organisms, we functionally compared and, as a result, identified cellular processes that require optimization in order to enable deployment as biofuel feedstock. Second, the proposed research resulted in development of key computational infrastructure, which can be further extended to other biological systems, that is currently lacking but necessary for multiple ‘omics data integration. Development of new genome scale experimental and genetic manipulation techniques for elucidation of gene and metabolic module function in diatom represent additional priorities of the research conducted. Highlighting the collaborative aspects and scope of the research, transcriptomc, proteomic, metabolite, metabolite flux and protein localization and interaction data were incorporated into integrated multi-omic genome scale models designed to pinpoint key chokepoints that will become targets for manipulation with synthetic genomic modules. To augment our genetic work with Phaeodactylum we also developed novel synthetic genomic techniques in this species. To apply this technology to photosynthetic systems, we developed technologies that will result in maintenance of large, >100-kb DNA regions as extra-chromosomal units in diatoms. Using yeast and E. coli as a platform, we created synthetic, extra-chromosomal elements for installation of new biological function in diatoms. Ultimately, we seek to design, construct, and transplant in vivo synthetic modules that will effectively reprogram regulatory and metabolic networks to result in enhanced flux of energy and materials into key lipid precursors. Fusing cutting-edge techniques in genome manipulation with genome-scale metabolic modeling, functional genomics, and associated physiological studies represents an exciting leap forward in exploring next generation biofuels.
Diatoms are major contributors to global primary production and their populations in the modern oceans are affected by availability of iron, nitrogen, phosphate, silica, and other trace metals, vitamins, and infochemicals. However, little is known about the role of phosphorylation in diatoms and its role in regulation and signaling. We report a total of 2759 phosphorylation sites on 1502 proteins detected in Phaeodactylum tricornutum. Conditionally phosphorylated peptides were detected at low iron (n = 108), during the diel cycle (n = 149), and due to nitrogen availability (n = 137). Through a multi-omic comparison of transcript, protein, phosphorylation, and protein homology, we identify numerous proteins and key cellular processes that are likely under control of phospho-regulation. We show that phosphorylation regulates: (1) carbon retrenchment and reallocation during growth under low iron, (2) carbon flux towards lipid biosynthesis after the lights turn on, (3) coordination of transcription and translation over the diel cycle and (4) in response to nitrogen depletion. We also uncover phosphorylation sites for proteins that play major roles in diatom Fe sensing and utilization, including flavodoxin and phytotransferrin (ISIP2A), as well as identify phospho-regulated stress proteins and kinases. These findings provide much needed insight into the roles of protein phosphorylation in diel cycling and nutrient sensing in diatoms.
Abstract The relative prevalence of endemic and cosmopolitan biogeographic ranges in marine microbes, and the factors that shape these patterns, are not well known. Using prokaryotic and eukaryotic amplicon sequence data spanning 445 near-surface samples in the Southern California Current region from 2014 to 2020, we quantified the proportion of taxa exhibiting endemic, cosmopolitan, and generalist distributions in this region. Using in-situ data on temperature, salinity, and nitrogen, we categorized oceanic habitats that were internally consistent but whose location varied over time. In this context, we defined cosmopolitan taxa as those that appeared in all regional habitats and endemics as taxa that only appeared in one habitat. Generalists were defined as taxa occupying more than one but not all habitats. We also quantified each taxon’s habitat affinity, defined as habitats where taxa were significantly more abundant than expected. Approximately 20% of taxa exhibited endemic ranges, while around 30% exhibited cosmopolitan ranges. Most microbial taxa (50.3%) were generalists. Many of these taxa had no habitat affinity (> 70%) and were relatively rare. Our results for this region show that, like terrestrial systems and for metazoans, cosmopolitan and endemic biogeographies are common, but with the addition of a large number of taxa that are rare and randomly distributed.
We investigated phytoplankton dynamics in the southern California Current System (SCCS) in August 2014 during the early phase of the 2014-15 marine heat wave (MHW). Multi-day experiments were conducted at three inshore and two offshore sites, with daily depth profiles of dilution incubations on a drifting array to determine growth and grazing rates and shipboard assessments of nutrient effects. Picophytoplankton populations were analyzed by flow cytometry and eukaryotic phytoplankton by 18 S sequencing. Mixed-layer nutrients were low across the region, but inshore sites had substantial nitrate concentrations and prominent Chla maxima in the lower euphotic zone. Shoreward transport of warm-stratified waters from the offshore suppressed coastal upwelling and shifted picophytoplankton distributions toward increased onshore abundance of Prochlorococcus and decreased Synechococcus and picoeukaryotes. These trends were reinforced by higher-than-average growth of Prochlorococcus at inshore sites and higher grazing of Synechococcus and picoeukaryotes. Prasinophytes (Chlorophyceae) were notably important among eukaryotic taxa, and pennates replaced centric taxa as the dominant diatoms in inshore waters compared to normal upwelling. Despite substantial spatial variability in community composition, offshore and inshore experimental locations both showed growth-grazing balances, with microzooplankton consuming similar percentages (80%) of primary production. We thus confirm expectations that the 2014-15 MHW resulted in greater trophic flow through the microbial food web at the expense of reduced direct phytoplankton (Chla) consumption by mesozooplankton. However, impacts on mesozooplankton energy budgets were partially offset by increased trophic flow through protistan microzooplankton and higher phytoplankton C:Chla.
