Diversity of the marine cyanobacterium Trichodesmium : characterization of the Woods Hole culture collection and quantification of field populations

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution September 2009Trichodesmium is a colonial, N2-fixing cyanobacterium found in tropical oceans. Species of Trichodesmium are genetically similar but several species exist together in the same waters. In order to coexist, Trichodesmium spp. may occupy different niche spaces through differential utilization of resources such as nutrients and light, and through responses to physical characteristics such as temperature and turbulence. To investigate niche differentiation in Trichodesmium, I characterized cultured strains of Trichodesmium, identified and enumerated Trichodesmium clades in the field, and investigated P stress and N2 fixation in field populations. Species of Trichodesmium grouped into two clades based on sequences from 16S rDNA, the internal transcribed spacer (ITS), and the heterocyst differentiation gene hetR. Clade I contained Trichodesmium erythraeum and Trichodesmium contortum, and clade II contained Trichodesmium thiebautii, Trichodesmium tenue, Trichodesmium hildebrandtii, and Trichodesmium pelagicum. Each clade was morphologically diverse, but species within each clade had similar pigmentation. I developed a quantitative polymerase chain reaction (qPCR) method to distinguish between these two clades. In field populations of the Atlantic and Pacific Oceans, the qPCR method revealed that clade II Trichodesmium spp. were more prominent than clade I in the open ocean. Concentrations of Trichodesmium did not correlate with nutrient concentrations, but clade I had wider temperature and depth distributions than clade II. Temperature and light are physical characteristics that may define niche spaces for species of Trichodesmium. Clade I and II concentrations correlated with each other in the Pacific but not in the Atlantic, indicating that the two clades were limited by the same factors in the Pacific while different factors were limiting the abundance of the two clades in the Atlantic. Trichodesmium populations in the North Atlantic were more P stressed and had higher N2 fixation rates than populations in the western Pacific. While nutrient concentrations didn’t directly correlate with Trichodesmium concentrations, the contrasting nutrient regimes found in the Atlantic and Pacific Oceans might influence distributions of the two clades differently. Unraveling the differences among species of Trichodesmium begins to explain their coexistence and enables us to understand factors controlling global N2 fixation.National Science Foundation (NSF) Biocomplexity Program Grant (OCE-0323332); the Center for Microbial Oceanography Research and Education (C-MORE), an NSF Science and Technology Center (EF-0424599); the Woods Hole Oceanographic Institution (WHOI) Ocean Life Institute (OLI) grant to J. Waterbury, and the WHOI Academic Programs Office

Cross-Basin Comparison of Phosphorus Stress and Nitrogen Fixation in Trichodesmium

We investigated the phosphorus (P) status and N2 fixation rates of Trichodesmium populations from the North Pacific, western South Pacific, and western North Atlantic. Colonies of Trichodesmium were collected and analyzed for endogenous alkaline phosphatase (AP) activity using enzyme-labeled fluorescence ( ELF) and for nitrogenase activity using acetylene reduction. AP hydrolyzes dissolved inorganic phosphate (DIP) from dissolved organic phosphorus and is active in Trichodesmium colonies experiencing P stress. Across multiple stations in the subtropical North and South Pacific, there was low to moderate ELF labeling in Trichodesmium, although labeling was present in other taxa. In contrast, Trichodesmium ELF labeling in the North Atlantic ranged from low to high. Low ELF labeling corresponded with high DIP concentrations while high ELF labeling occurred only at North Atlantic stations with DIP concentrations \u3c = 40 nmol L-1, indicating that Trichodesmium was not experiencing dramatic P stress in the Pacific Ocean while P stress was evident in the western North Atlantic. However, nitrogenase activity was significantly higher in the P-stressed western North Atlantic than in the Pacific Ocean (0.40-1.30 compared to 0.01-0.46 nmol C2H4 h-1 colony-1. These data underscore the differential basin-level importance of P availability to Trichodesmium and suggest that factors other than P are constraining their N2 fixation rates in the Pacific

SeaFlow Data V1, High-Resolution Abundance, Size and Biomass of Small Phytoplankton in the North Pacific

SeaFlow is an underway flow cytometer that provides continuous shipboard observations of the abundance and optical properties of small phytoplankton (\u3c5 μm in equivalent spherical diameter, ESD). Here we present data sets consisting of SeaFlow-based cell abundance, forward light scatter, and pigment fluorescence of individual cells, as well as derived estimates of ESD and cellular carbon content of picophytoplankton, which includes the cyanobacteria Prochlorococcus, Synechococcus and small-sized Crocosphaera (\u3c5 μm ESD), and picophytoplankton and nanophytoplankton (2–5 μm ESD). Data were collected in surface waters (≈5 m depth) from 27 oceanographic cruises carried out in the Northeast Pacific Ocean between 2010 and 2018. Thirteen cruises provide high spatial resolution (≈1 km) measurements across 32,500 km of the Northeast Pacific Ocean and 14 near-monthly cruises beginning in 2015 provide seasonal distributions at the long-term sampling site (Station ALOHA) of the Hawaii Ocean Time-Series. These data sets expand our knowledge of the current spatial and temporal distributions of picophytoplankton in the surface ocean

SeaFlow data v1, high-resolution abundance, size and biomass of small phytoplankton in the North Pacific

SeaFlow is an underway flow cytometer that provides continuous shipboard observations of the abundance and optical properties of small phytoplankton (<5 mu m in equivalent spherical diameter, ESD). Here we present data sets consisting of SeaFlow-based cell abundance, forward light scatter, and pigment fluorescence of individual cells, as well as derived estimates of ESD and cellular carbon content of picophytoplankton, which includes the cyanobacteria Prochlorococcus, Synechococcus and small-sized Crocosphaera (<5 mu m ESD), and picophytoplankton and nanophytoplankton (2-5 mu m ESD). Data were collected in surface waters (approximate to 5 m depth) from 27 oceanographic cruises carried out in the Northeast Pacific Ocean between 2010 and 2018. Thirteen cruises provide high spatial resolution (approximate to 1 km) measurements across 32,500 km of the Northeast Pacific Ocean and 14 near-monthly cruises beginning in 2015 provide seasonal distributions at the long-term sampling site (Station ALOHA) of the Hawaii Ocean Time-Series. These data sets expand our knowledge of the current spatial and temporal distributions of picophytoplankton in the surface ocean

Nitrogen Fixation in Mesoscale Eddies of the North Pacific Subtropical Gyre: Patterns and Mechanisms

Mesoscale eddies have been shown to support elevated dinitrogen (N2) fixation rates (NFRs) and abundances of N2-fixing microorganisms (diazotrophs), but the mechanisms underlying these observations are not well understood. We sampled two pairs of mesoscale cyclones and anticyclones in the North Pacific Subtropical Gyre in 2017 and 2018 and compared our observations with seasonal patterns from the Hawaii Ocean Time-series (HOT) program. Consistent with previous reports, we found that NFRs were anomalously high for this region (up to 3.7-fold above previous monthly HOT observations) in the centers of both sampled anticyclones. In 2017, these elevated rates coincided with high concentrations of the diazotroph Crocosphaera. We then coupled our field-based observations, together with transcriptomic analyses of nutrient stress marker genes and ecological models, to evaluate the role of biological (via estimates of growth and grazing rates) and physical controls on populations of Crocosphaera, Trichodesmium, and diatom symbionts at the mesoscale. Our results suggest that increased Crocosphaera abundances in the 2017 anticyclone resulted from the alleviation of phosphate limitation, allowing cells to grow at rates exceeding grazing losses. In contrast, distributions of larger, buoyant taxa (Trichodesmium and diatom symbionts) appeared less affected by eddy-driven biological controls. Instead, they appeared driven by physical dynamics along frontal boundaries that separate cyclonic and anticyclonic eddies. No examined controls were able to explain our 2018 findings of higher NFRs in the anticyclone. A generalized explanation of elevated NFRs in mesoscale eddies remains challenging due to the interplay of eddy-driven bottom-up, top-down, and physical control mechanisms.This work was funded by the Simons Foundation (Award # 721252 to DMK, 721256 to AEW, 721223 to EFD, 721221 to MJC, 721244 to EVA, 721225 to STD, 329108 to SJ, and 724220 to JPZ) and expedition funding from the Schmidt Ocean Institute for R/V Falkor Cruise FK180310 in 2018.Peer reviewe

Illuminating the life of GPCRs

The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. G-protein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented