ALBERT

Beschreibung: © The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Gosselin, K. M., Nelson, R. K., Spivak, A. C., Sylva, S. P., Van Mooy, B. A. S., Aeppli, C., Sharpless, C. M., O’Neil, G. W., Arrington, E. C., Reddy, C. M., & Valentine, D. L. Production of two highly abundant 2-methyl-branched fatty acids by blooms of the globally significant marine cyanobacteria Trichodesmium erythraeum. ACS Omega, 6(35), (2021): 22803–22810, https://doi.org/10.1021/acsomega.1c03196.

Beschreibung: The bloom-forming cyanobacteria Trichodesmium contribute up to 30% to the total fixed nitrogen in the global oceans and thereby drive substantial productivity. On an expedition in the Gulf of Mexico, we observed and sampled surface slicks, some of which included dense blooms of Trichodesmium erythraeum. These bloom samples contained abundant and atypical free fatty acids, identified here as 2-methyldecanoic acid and 2-methyldodecanoic acid. The high abundance and unusual branching pattern of these compounds suggest that they may play a specific role in this globally important organism.

Beschreibung: This work was funded with grants from the National Science Foundation grants OCE-1333148, OCE-1333162, and OCE-1756254 and the Woods Hole Oceanographic Institution (IR&D). GCxGC analysis made possible by WHOI’s Investment in Science Fund.

Beschreibung: Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Oceanographic Engineering at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution February 2019.

Beschreibung: Marine microbes are key drivers of biogeochemical transformations within the world’s oceans. Although seawater appears uniform at scales that humans often interact with and sample, the world that marine microbes inhabit can be highly heterogeneous, with numerous biological and physical processes giving rise to resource hotspots where nutrient concentrations exceed background levels by orders of magnitude. While the impact of this microscale heterogeneity has been investigated in the laboratory with microbial isolates and theoretical models, microbial ecologists have lacked adequate tools to interrogate microscale processes directly in the natural environment. Within this thesis I introduce three new technologies that enable interrogation of microbial processes at the microscale in natural marine communities. The IFCB-Sorter acquires images and sorts individual phytoplankton cells, directly from seawater, allowing studies exploring connections between the diversity of forms present in the plankton and genetic variability at the single-cell level. The In Situ Chemotaxis Assay (ISCA) is a field-going microfluidic device designed to probe the distribution and role of motility behavior among microbes in aquatic environments. By creating microscale hotspots that simulate naturally occurring ones, the ISCA makes it possible to examine the role of microbial chemotaxis in resource acquisition, phytoplankton-bacteria interactions, and host-symbiont systems. Finally, the Millifluidic In Situ Enrichment (MISE) is an instrument that enables the study of rapid shifts in gene expression that permit microbial communities to exploit chemical hotspots in the ocean. The MISE subjects natural microbial communities to a chemical amendment and preserves their RNA in a minute-scale time series. Leveraging an array of milliliter-volume wells, the MISE allows comparison of community gene expression in response to a chemical stimulus to that of a control, enabling elucidation of the strategies employed by marine microbes to survive and thrive in fluctuating environments. Together, this suite of instruments enables culture-independent examination of microbial life at the microscale and will empower microbial ecologists to develop a more holistic understanding of how interactions at the scale of individual microbes impact processes in marine ecosystems at a global scale.

Beschreibung: I’d like to thank the Gordon and Betty Moore Foundation, the National Science Foundation, and NSERC for funding portions of my research.

Beschreibung: I’d like to thank the Gordon and Betty Moore Foundation, the National Science Foundation, and NSERC for funding portions of my research.

Beschreibung: 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 June 2014

Beschreibung: Viral predation on bacteria in the ocean liberates carbon from the particulate fraction, where it is accessible to higher trophic levels, and redirects it to the dissolved fraction, where it supports microbial growth. Although viruses are highly abundant in the ocean little is known about how their interactions with bacteria are structured. This challenge arises because the diversity of both bacteria and viruses is exceedingly high and interactions between them are mediated by specific molecular interactions. This thesis uses heterotrophic bacteria of the genus Vibrio as a model to quantify virus-host interactions in light of host population structure and ecology. The methods developed in this thesis include streamlining of standard bacteriophage protocols, such as the agar overlay, and facilitate higher throughput in the isolation and characterization of novel environmental virus-host systems. Here, 〉1300 newly isolated Vibrio are assayed for infection by viral predators and susceptibility is found to be common, though total concentrations of predators are highly skewed, with most present at low abundance. The largest phylogenetically-resolved host range cross test available to date is conducted, using 260 viruses and 277 bacterial strains, and highly-specific viruses are found to be prevalent, with nearly half infecting only a single host in the panel. Observations of blocks of multiple viruses with nearly identical infection profiles infecting sets of highly-similar hosts suggest that increases in abundance of particular lineages of bacteria may be important in supporting the replication of highly specific viruses. The identification of highly similar virus genomes deriving from different sampling time points also suggests that interactions for some groups of viruses and hosts may be stable and persisting. Genome sequencing reveals that members of the largest broad host-range viral group recovered in the collection have sequence homology to non-tailed viruses, which have been shown to be dominant in the surface oceans but are underrepresented in culture collections. By integrating host population structure with sequencing of over 250 viral genomes it is found that viral groups are genomically cohesive and that closely-related and co-occurring populations of bacteria are subject to distinct regimes of viral predation.

Beschreibung: I also gratefully acknowledge the WHOI Ocean Ventures Fund, which provided funding for the sequencing of over 250 viral genomes of the Nahant Collection and thereby contributed immensely to the impact of the thesis work presented here. Work presented in this thesis was also made possible by support from National Science Foundation grant DEB 0821391, National Institute of Environmental Health Sciences grant P30- ES002109, the Moore Foundation and the Broad Institute’s SPARC program.

Beschreibung: 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 June 1984

Beschreibung: The distribution and feeding behavior of bacterivorous micro flagellates (2-20 μm protozoa) and their ingestion by copepods were examined in an attempt to assess the importance of these protozoa as a trophic link between planktonic bacteria and zooplankton. The abundance of microflagellates relative to other picoplankton (0.2-2.0 μm) and nanoplankton (2-20 μm) populations in water samples in the North Atlantic and in Lake Ontario and on macroaggregates in the North Atlantic was determined using direct microscopical and culture estimation techniques. Seasonal, vertical and geographical changes in the density of microflagellates were generally not greater than one order of magnitude. Microscopical counts of heterotrophic nanoplankton (presumably microflagellates) typically ranged from a few hundred to a few thousand m1-1 for a variety of planktonic environments. They constituted approximately 1/3 to 1/2 of the nanoplankton in the euphotic zone and dominated the nanoplankton in the aphotic zone. Most Probable Number (MPN) estimation of the density of bacterivorous protozoa indicated that microflagellates were, on average, an order of magnitude more abundant than bacterivorous ciliates and amoebae. MPN and direct microscopical counts of microflagellates differed by as much as 104. This discrepancy was smaller in eutrophic environments (e.g. Continental Shelf and Lake Ontario) and on macroscopic detrital aggregates. All microbial populations enumerated were highly concentrated on macroscopic detrital aggregates relative to their abundance in the water surrounding the aggregates. Enrichment factors (the ratio of abundance of a population on a macroaggregate to its abundance in the surrounding water) increased along a eutrophic-to-oligotrophic gradient because of the combined effects of an increased abundance of microorganisms on macroaggregates in oligotrophic environments and a decreased abundance in the surrounding water in these same environments. Average enrichment factors for direct microscopical counts of heterotrophic nanoplankton (range = 17-114) were not as large as enrichment factors observed for MPN estimates of the number of bacterivorous microflagellates (range = 273-18400). Microflagellates numerically dominated the bacterivorous protozoa cultured from macroaggregates by one to two orders of magnitude, but ciliates and amoebae were also highly enriched on macroaggregates. Microenvironments are therefore a potentially important aspect for the ecology of planktonic microorganisms. Observations on the microbial colonization of mucus sloughed by ctenophores and discarded appendicularian houses suggest that these materials may be important sources of macroaggregates. Batch and continuous culture experiments were conducted with clonal cultures of microflagellates to test their ability to grow on various types and densities of bacteria. The doubling time of Monas sp. 1 ranged from 43 hr (when fed the cyanobacterium Synechococcus Strain WH 8101) to 6.9 hr (when fed the heterotrophic bacterium Serratia marinorubra). Cell yields (i.e. the conversion of bacterial biomass into protozoan biomass) of Monas sp. 1 fed two species of heterotrophic bacteria were greater than yields for the microflagellate fed two species chroococcoid cyanobacteria (range = 7-68%). Cell yields of two other species of microflagellates (Monas sp. 2 and Cryptobia maris) were 48% and 61%, respectively, on the bacterium Pseudomonas halodurans. Microflagellates grew in continuous culture at concentrations of bacteria which were lower than bacterial densities required for the growth of ciliates as shown by other investigations. Therefore, microflagellates appear to be well-adapted for grazing bacterioplankton. Microflagellates were also investigated for their ability to graze bacteria attached to particles. Bodo nanorensis and Rhynchomonas nasuta both showed a marked ability to graze attached bacteria and a limited ability to graze unattached cells. These results suggest that microflagellates may also be important consumers of bacteria attached to particles in the plankton and may explain the highly elevated densities of microflagellates on macroaggregates. Grazing experiments performed with the copepod Acartia tonsa indicated that heterotrophic microflagellates were ingested by the copepods at rates comparable to the ingestion of phytoplankton of similar size. The presence of heterotrophic microflagellates did not depress filtration rates of the copepods, and one species (Cryptobia maris) appeared to be selectively grazed. Survival of A. tonsa on a diet of heterotrophic microflagellates was similar to survival on a diet of phytoplankton and was significantly longer than survival of starved Controls or copepods fed only bacteria. Due to their ability to grow at in-situ densities of planktonic bacteria, their relatively high cell yields, and their acceptability as food for zooplankton, it is concluded that bacterivorous microflagellates may constitute an important trophic link between bacteria and zooplankton. This link may provide a mechanism whereby organic material and energy from the detrital food chain can be returned to the classical phytoplankton-copepod-fish food chain.

Beschreibung: This research was supported by National Science Foundation grants OCE80-2444l and OCE82-l4928 and Ocean Industry Program grant 4473 awarded to Dr. Laurence P. Madin, NSF Doctoral Dissertation grant OCE8l-l299l, the Woods Hole Oceanographic Institution Education Program and the Wood Hole Oceanographic Institution Biology Department.

Beschreibung: 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 2009

Beschreibung: Trichodesmium 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.

Beschreibung: 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.

Beschreibung: 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 April 1978

Beschreibung: The structure of the membrane-free nucleoid of Escherichia coli and of unfolded chromosomal DNA was investigated by sedimentation on neutral sucrose gradients after irradiation with 60Co gamma-rays and ultraviolet light (2S4nm). Irradiation both in vivo and in vitro was used as a molecular probe of the constraints on DNA~packaging in the bacterial chromosome. The extremely gentle lysis and unfolding procedures which were developed yielded undamaged, replicating genomes, thus permitting direct measurement of the formation and repair of DNA double-strand breaks at biologically-significant doses of ionizing radiation. In vitro UV-irradiation of nucleoids resulted in an increase in the observed rate of sedimentation due to the formation of an unknown photo-product. In contrast, UV-irradiation of wild-type cells in vivo showed evidence of the formation of incision breaks which resulted in the relaxation of supercoiling in the nucleoid. Strand breakage was also observed following in vivo UV-irradiation of a uvrB-5 strain, but at a lower rate and also accompanied by considerable unfolding of the chromosome. Such lesions may have been the result of direct photochemical reactions in the nucleoid, or enzyme activity associated with a uvr-independent mode of repair. The number of domains of supercoiling was estimated at 170 per genome equivalent of DNA based on measurements of relaxation caused by single-strand break formation in in vivo- and in vitro-gamma-irradiated folded chromosomes. Similar estimates based on the target size of RNA molecules responsible for maintaining the compact packaging of the nucleoid predicted negligible unfolding due to the formation of RNA single-strand breaks at doses up-to 10 Krad, and were born out by experimental measurements. Unfolding of the nucleoid in vitro by limit-digestion with RNase or by heating at 70° resulted in DNA complexes with sedimentation coefficients of 1030±59S and 625±15S respectively. The difference in these rates was apparently due to more complete deproteinization and thus less mass in the heated material. These structures are believed to represent intact, replicating genomes in the form of complex-theta structures containing 2-3 genome equivalents of DNA. The rate of formation of double-strand breaks was determined from molecular weight measurements of thermally unfolded chromosomal DNA gamma-irradiated in vitro. Break formation was linear with dose up to 10 Krad, resulting in 0.27 double-strand breaks per kilorad per genome equivalent of DNA and requiring 1080 eV/double-strand break. The influence of possible non-linear DNA conformations of these calculations is discussed. Repair of ionizing radiation damage to folded chromosomes was observed within 2-3 hours of post-irradiation incubation in growth medium. A model based on recombinational repair is proposed to explain the formation of 2200-2300S material during early stages of incubation and subsequent changes in the gradient profiles. Such behavior is not observed for post-irradiation incubation of wild-type cells in buffer or for a recA-13 strain incubated in growth medium. Association of unrepaired DNA with plasma membrane is proposed to explain the formation of a peak of rapidly sedimenting material (〉〉3100S) during the later stages of repair. Direct evidence of repair of double-strand breaks during post-irradiation incubation in growth medium was obtained from gradient profiles of DNA from RNAse-digested chromosomes. The sedimentation coefficient of broken molecules was restored to the value of unirradiated DNA after 2-3 hours of incubation, and the fraction of the DNA repaired in this fashion was equal to the fraction of cells which survived at the same dose. An average of 2.7 double-strand breaks per genome per lethal event was observed, suggesting that 1-2 double-strand breaks per genome are repairable in this strain of E. coli.