Mitchell L. Sogin, Ph.D. - US grants

Workshops in molecular evolution will be offered at the Marine Biological Laboratory at Woods Hole in 1991, 1992 and 1993. The goal is to provide an opportunity for organismal, evolutionary and population biologists to learn about the powerful tools of molecular biology while providing molecular/cell biologists with an enriched understanding of evolutionary theory. Lectures exploring multiple approaches to molecular evolution will be supplemented by a computer workshop for the analysis of protein and genetic sequences. The workshop is designed for established investigators, college and university professors, postdoctoral fellows, and graduate students from diverse biological fields. The theoretical basis of comparative sequence analysis and phylogenetic tree construction will be presented as well as lectures about the application of molecular techniques to problems in ecology and population packages for analysis of molecular data on different computer systems including workstations, personal computers and large mainframes. Guided by authors for phylogenetic software packages, the students will have the opportunity to compare different data analysis techniques for molecular data.

9317657 Sogin This proposal seeks to offer interdisciplinary training and promote collaborative efforts between molecular biologists, organismal biologists and theoreticians. Prominent scientists in molecular biology, ecology, population biology, organismal biology and biometrics will offer a high powered lecture series. Topics to be covered include but are not limited to the theory of phylogenetic (genealogical) reconstruction, macromolecular alignment techniques, current trends in molecular phylogenetics, molecular approaches to studies of population biology and biodiversity, comparative molecular biology, and comparative analysis of genome data bases. The lectures will be augmented by a computer laboratory. Students will have the opportunity to use state-of-the-art facilites to test theoretical concepts described in the lecture series. Guided by authors of phylogenetic analytic software programs and designers of data base retrieval software from the National Laboratory of Medicine, the students will have the opportunity to compare different analytical techniques for molecular data. The computer laboratory will be equipped with diverse computer platforms and will offer each student the opportunity to work in an environment that is relevant to hardware configurations available at their home institution. The opportunity to implement concepts described in the lecture series using diverse up-to-date computer hardware is a distinguishing feature of this course. The course is designed for established investigators, postdoctoral fellows, and graduate students from diverse biological fields.

This is a group proposal to acquire an automated DNA sequencing machine for shared use by investigators at the Marine Biological Laboratory and the Woods Hole Oceanographic Institution. The equipment to be purchased includes a DNA sequencing machine and an auxiliary workstation for analyzing data produced by the sequencing machine. Comparative molecular biology has transformed studies of evolution, ecology, biogeography and biodiversity into experimental sciences. Using measures of similarity for ribosomal RNAs, microbiologists can measure genetic differences between members of the same species or reconstruct phylogenies for organisms that span the largest evolutionary distances. Mitochondrial gene sequences provide quantitative genetic tools for biogeography studies in both microbial and multicellular systems. With the advent of polymerase chain reaction techniques, almost any gene family can be incorporated into biodiversity investigations. For any one of these enterprises, the major expense in terms of financial and human resources is that required to determine DNA sequences. Numerous research projects in the Molecular Evolution Program, the Ecosystems Center at the Marine Biological Laboratory and the Biology Department at the Woods Hole Oceanographic Institution rely upon comparative analyses of ribosomal RNA sequences, mitochondrial genes or other nuclear coding regions. Despite excellent computational facilities for integrating results of these studies, no automated equipment is available for sequencing DNA in the Woods Hole Scientific Community. If laboratories at the Marine Biological Laboratory and the Woods Hole Oceanographic Institution are to remain competitive in studies of molecular evolution/systematics, molecular ecology and emerging programs in biodiversity, they must have access to shared instrumentation for rapid, automated DNA sequence determinations. The principal benefit will be a dramatic reduction in human resources required for DNA sequence analyses. In addition, this multi-user instrument will further encourage interdisciplinary approaches to studies of molecular ecology, biodiversity and molecular systematics/evolution. Five of the laboratory's represented in this application will form a consortium to sequence ribosomal RNAs from prokaryotes and eukaryotes. Their research objectives include studies of molecular systematics, molecular ecology, biodiversity, and biogeography. Through the common administration and use of this instrumentation, it will be possible to share many costly reagents including a library of primers for rapidly sequencing rRNA coding regions from both prokaryotes and eukaryotes. Other groups will use the machine to study structure and functional constraints in coding regions such as cytochrome P450, actins, and receptors. Most of these projects represent existing collaborations within the Woods Hole Scientific Community. The automated sequencer will also be available to visiting scientists in the Woods Hole Scientific Community including summer investigators at the Marine Biological Laboratory and the Woods Hole Oceanographic Institution.

In one extreme freshwater environment, a long-term research site in arctic Alaska, a large amount of information about microbial communities and their ecology has been accumulated. The least known member of this, and most other microbial communities, is the single cell eukaryotes. This LExEN project will investigate the ecology, physiology, and evolution of nanoflagellates, and important member of the aquatic microbial system. There will be extensive cooperation with two funded projects, one on freshwater ecology and one on flagellate evolution. * With the large ecological project this new research will extend knowledge of nanaflagellates in their natural habitat of arctic lakes and ponds with an emphasis on their adaptation to cold and freezing conditions. * Diversity of protists and of their evolutionary relationships will be studies. In addition, microaerophilic environments will be examined to look for amitochondriates flagellates, basic knowledge of lineages basal to all other eukaryotes. LExEN additions to this existing understanding of the microbial system will focus on the biology and ecology of the nanoflagellates, the main predator of the aquatic bacteria. Protists from a shallow pond that freezes solid each year and from a deep arctic lake with a 9-month ice cover will be intensively sampled during the winter, spring, summer and fall. Results will include: * The identity of the nanoflagellates present, their seasonal abundance, distribution, and succession with microscopic and molecular methods (including probes targeting ribosomal RNA for species in samples from the field). * The reconstruction from rRNA sequences of the evolutionary relationships among the flagellates present in these extreme environments including the microaerophilic layers of sediment. The data base will enable further research on these virtually unknown organisms. * The physiology of organisms cultured from this extreme environment with emphasis on the effects of cold and freezing on their feeding behavior and on their respiration and survival during starvation. The experiments will examine flagellate adaptations for survival in cold waters and in frozen sediments.

9977897
Hobbie

This Microbial Observatory at the Plum Island Long Term Ecological Research (LTER) site in northeastern Massachusetts identifies microbes and microbial communities in the salt marsh and determines their role in controlling major ecosystem processes. The observatory project, staffed by scientists from the Marine Biological Laboratory (MBL), the Woods Hole Oceanographic Institution, and Northwestern University, adds knowledge of the microbes and their ecology to the LTER research on the effects of changing inorganic nutrient supply and the quality and quantity of organic matter on estuarine trophic structure. The LTER research gains detailed knowledge about the microbial communities underlying the processes and control parameters which it investigates; the Microbial Observatory uses LTER data to place microbial communities into the larger context of their physical and chemical environment. In the salt marsh sediments, nitrogen and sulfur cycles influence all of the salt marsh biogeochemical cycles while sulfate reduction dominates decomposition. Therefore, the Observatory Project focuses on the microbes involved with the nitrogen and sulfur cycle with special attention to nitrification and sulfate reduction. This focus is possible because these investigators bring a suite of tools for culturing, molecular detection, and identification of nitrifying and sulfate reducing bacteria. The Microbial Observatory carries out the following activities: 1. identifies the microbes and microbial communities by means of culture and sequencing techniques, 2. investigates the seasonal changes in the community structure with culture methods and with denaturing gradient gel electrophoresis (DGGE) of nucleic acids, 3. links functional genes and microbial populations responsible for specific processes with rRNA hybridization techniques, and 4. correlates the presence and activity of various populations with ecological measurements of major processes in the salt marsh. Molecular surveys using functional genes and rRNA hybridization techniques are correlated with LTER measures of sulfate reduction and the nitrogen cycle. Carbon sources are identified with techniques using the delta 13C of phospholipid fatty acids specific to bacteria. Salt marshes provide one of the best examples of a site where microbes build and shape an entire ecosystem. Cyanobacteria are the pioneer organisms in salt marshes; they initiate microbial mat communities on mudflats that prevent erosion, trap sediment particles, and elevate the surface of the sediments. When higher plants colonize the site, the microbes fix nitrogen, recycle organic matter and nutrients, oxidize sulfides, and provide food for higher trophic levels. Salt marshes continue to be important models for basic microbiological and biogeochemical research. These systems are easily accessible. Their high process rates, large and diverse populations of functionally distinct microbes, and steep chemical and microbial gradients allow scientists to understand how microbial interactions and chemical cycles work. For example, many of our insights into the process and microbiology of sulfate reduction in the marine environment have come from the study of salt marshes. The value of salt marshes in teaching and education is well established. Students of the Microbial Diversity Course at the MBL in Woods Hole, associated with this Microbial Observatory, use the salt marsh as a "Microbial Rainforest" for the isolation and identification of a wide diversity of microbes.

This award provides support for purchase of equipment to establish an integrated laboratory for high-throughput processing of DNA samples in environmental research. Establishment of the laboratory will give scientists at the Marine Biological Laboratory (MBL) and the Woods Hole Oceanographic Institution (WHOI) access to high-throughput capabilities that are typically restricted to biomedical or industrialized genomic research centers. Equipment to be purchased include several robotic workstations, a real-time quantitative polymerase chain reaction device and a pulsed field gel electrophoresis) system. This new equipment will permit scientists at both institutions to achieve optimum use of an existing high capacity DNA sequencing and microarray facility. This robotic equipment will completely automate management and processing of DNA samples, and thus enable scientists to sample genetic diversity in the environment at a level previously unimaginable. Research questions to be pursued address issues in genome evolution, biodiversity, and environmental influences on gene expression. The equipment will allow precise measurement of changes in gene expression in response to external factors (e.g. toxins, other chemicals) and as a result of symbiosis, and will also be used to measure specific DNA sequences in uncultivable environmental populations. The new high-throughput capabilities will also directly impact the training and education of visiting and year-round scientists, students, and postdoctoral fellows at the two institutions in Woods Hole, MA.

Except for a small number of human pathogens, microbial genome efforts have steered clear of the diverse world of protozoans, many of which are important agricultural parasites. Of particular interest are the Microsporidia, which by eukaryotic standards have remarkably small genomes. Since they infect most invertebrate taxa and all classes of vertebrates, their impact on agriculture and fisheries is significant. They either cause disease or can serve as bio-control agents. These same organisms have attracted the interests of biologists and molecular evolutionists. They offer excellent models for studying interactions between the genomes of a host and an obligate, intracellular parasite. But molecular phylogenetic analyses point towards a confused evolutionary history. To advance knowledge of these unusual microbes and to address the ever-increasing demand for researchers broadly trained in genome sciences, the genome of the microsporidian Nosema locustae will be sequenced to closure and a comprehensive course in genome science will be established to provide broad-based training in high throughput sequencing techniques and bioinformatics. By integrating this course with the N. locustae project, nearly 100 students will be trained in all aspects of genomics. Although this model for research and training could be applied to other microbial groups, Microsporidia were selected because of their impact upon agriculture, their enigmatic character in molecular evolution studies and what they may reveal about interactions between the genomes of hosts and obligate intracellular parasites. Sequence data (3X and higher coverage, monthly updates and annotation) will be available to the research community on the Marine Biological Laboratory's web server (www.mbl.edu). This is a Microbial Genome Sequencing Award funded through a collaborative program between the National Science Foundation and the Department of Agriculture Initiative for Future Agriculture and Food Systems

environmental adaptation; eukaryote; biomedical resource; acidity /alkalinity;

[unreadable] DESCRIPTION (provided by applicant): Eukaryotes existed exclusively as microorganisms for a minimum of 500,000 to one billion years prior to the evolution of plants, animals and fungi. Understanding the evolutionary history of microbial eukaryotes, especially the protists, is key to understanding the origins of eukaryotes, the emergence of parasitism, and the evolution of this remarkably diverse assemblage into plants, animals, fungi and other recently derived groups. Protists represent an eclectic group of organisms marked by numerous innovations in body plans, ecology, biochemistry, and molecular processes. The transition from prokaryotic life-forms to eukaryotic cellular architecture, and the origins of multi-cellular eukaryotes are rooted in the world of protists. [unreadable] [unreadable] This proposal describes the collection of new molecular data and the construction of a large-scale, multi-gene data set for exploring evolutionary relationships for diverse eukaryotes. Our experimental strategy seeks to capture the diversity of eukaryotes by exploring relationships within major clades, and by placing taxa of uncertain position on genealogies. We will construct normalized cDNA libraries for as many as 30 key protists and sequence 10,000-20,000 cDNA clones from each library. Using computer-generated alignments we will use high-throughput phylogenomics to address the following questions: What is the composition and relative branching order of major eukaryotic lineages? To what extent has lateral gene transfer affected the evolution of eukaryotic genomes? Is there a genomic core that is refractory to horizontal gene transfer? Does the pattern of metabolic evolution mirror environmental changes in earth's history? Does adaptation to parasitic life-styles impose major changes in genome architecture and its transcriptome? And finally, what are the key ultrastructural and morphological innovations that led to modem eukaryotic complexity? These phylogenetic frameworks generated by the project will become the basis for interpreting the origin and evolution of genes and genome organization patterns that are responsible for novel phenotypes that underpin organismal complexity and disease. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This proposal to the NCRR Shared Instrumentation Grant Program requests funds to acquire an automated DNA sequencing machine and data storage equipment for shared use by NIH-funded investigators at the Marine Biological Laboratory. The equipment to be purchased includes an Applied Biosystems 3730XL DNA Analyzer and a 3.4 tera-byte data storage system based upon Apple Computer's Xserve RAID and Xserve G5 architecture. The equipment will accommodate new DNA sequencing requirements for five new NIH programs (funded within the last year or projects to be activated by May 1, 2004). These NIH projects include several Serial Analysis of Gene Expression (SAGE) studies in parasitic protists, a large-scale evolutionary analysis of cDNAs from as many as thirty parasitic protists, sequence and Serial Analysis of Ribosomal Sequence Tags (SARST) analyses of environmental PCR amplicons for the new Woods Hole Center for Oceans and Human Health (COHH) program, and small-scale genome studies of symbiotic prokaryotes. These projects will impact our understanding of patterns of gene expression in human pathogens, the evolution of protists that cause disease, the dispersal of pathogenic microrganisms in marine environments, and adaptive mechanisms that lead to parasitic lifestyles. This equipment will nearly double our current sequencing capacity, which is currently fully subscribed. The equipment will operate within the W.M. Keck Ecological and Evolutionary Genetics Facility within the Bay Paul Center at the Marine Biological Laboratory. The facility is a shared-use resource for investigators in the Woods Hole Scientific Community and is equipped with robotic systems for colony picking, template production, preparation of sequencing reactions, automated DNA sequencing and DNA microarraying. Since 1997, a users' committee has guided use of the facility, which is managed by a faculty member in the Bay Paul Center. Foundation funds have been reserved to pay for five years of maintenance. [unreadable] [unreadable]

nucleic acid quantitation /detection

nucleic acid quantitation /detection

nucleic acid quantitation /detection

DESCRIPTION: The indigenous microbiota of the gastrointestinal tract is the richest of the human associated microbial communities in terms of absolute biomass and relative diversity. Although the gut microbiota play critical roles in the maintenance of gut homeostasis, evidence is accumulating that disturbances in the gut microbial community ("dysbiosis") underlie a number of gastrointestinal disease states including antibiotic-associated diarrhea and inflammatory bowel disease (IBD). To address our hypothesis we propose to determine the relationship between the gut microbiome and the development of mucosal inflammation in patients with ulcerative colitis. We will coordinate and integrate existing resources for the study of IBD patients, metagenomic analysis, molecular microbial diversity analysis and targeted sequence-aided bacterial culture into a research pipeline for the study of complex host-associated microbial communities. We will characterize the gut microbiota in human patients who have undergone total colectomy with the construction of an ileal pouch anal anastomosis for treatment of ulcerative colitis. These patients will be followed temporally after surgery to monitor for the development of mucosal inflammation in the pouch anastomosis ("pouchitis"). Comparison within patients over time and between patients will be done to associate specific ecologic and functional characteristics of the gut microbiome with the development of pouchitis. To accomplish these goals, we have assembled an experienced, integrated team of researchers from a variety of disciplines. We will study a carefully selected group of patients with ulcerative colitis that will permit us to make inter- and intra-patient comparisons over time. Though the use of high-throughput, cost effective technologies, which members of this team have played a key role in developing, we intend to demonstrate a critical role for the gut microbiome in the development of the dysregulated mucosal immune response that is the hallmark of IBD. This knowledge will lead the way to novel ways to monitor patients with IBD and to the development of novel preventative and therapeutic interventions. PUBLIC HEALTH RELEVANCE: The bacteria that live in the gut are felt to play a key role in the health of the gastrointestinal tract. We will determine the role of the gut bacteria in the development of human inflammatory bowel disease through the use of state-of-the-art molecular biology techniques and advanced culture techniques.

This Integrative Graduate Education and Research Training (IGERT) award supports a novel graduate program in Reverse Ecology, training Ph.D. students at the interface of computational biology, genomics and environmental science. It leverages new education and research collaborations between Brown University and the Marine Biological Laboratories.

Reverse Ecology is the application of genomic approaches to living systems to uncover the genetic bases of functional variation in nature. The revolution in high-throughput DNA sequencing and gene expression technologies redefines the notion of a "model" organism. Interrogation of genomes from animals, plants and microbial communities can identify genetic markers of processes at multiple scales: ecological, physiological, developmental, and transcriptional. The full interpretation of these powerful datasets demands intellectual dialogue between ecosystems ecologists, microbial geneticists, biogeochemists, and computational biologists. The program will train a cohort of Ph.D.s who can apply these technologies creatively to convert genomic and computational power into novel insights of organismal function in nature. Program highlights include 1) a year-long immersion course focused at Long Term Ecological Research sites where students design an experiment, use high-throughput genomic and computational strategies to test hypotheses and prepare multi-authored manuscripts for publication; 2) jointly mentored research rotations where students and faculty cross disciplinary boundaries; and 3) career training integrating grant writing, public speaking, ethics, diversity and international perspectives on science. This graduate program's research themes include 1) microbial and comparative genomics; 2) genetic responses to environmental stressors; 3) assembling genomes from environmental samples, and will engage IBM and the J. Craig Venter Institute in creating a new model for graduate education. Students will be trained in the context of university, institutional, and corporate environments to become leaders in the identification and integration of scientific questions across formerly distant disciplines.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

This proposal describes the acquisition of an Illumina GAIIx nucleic acid sequencer and related instrumentation to enhance microbial ecology and genomics research programs at the Marine Biological Laboratory and the broader Woods Hole scientific community. The instrumentation will dramatically reduce the cost of microbial profiling, genome sequencing and RNA-Seq profiles while leveraging existing experience with advanced DNA sequencing platforms. The requested equipment would provide a daily throughput capacity of ~5 billion nt/day, sufficient for the ~1755 billion nucleotides (nt) of sequencing needs identified by major and minor users of the equipment under existing NSF and NIH funded programs; these estimates do not include projected use by MBL high school, undergraduate, and graduate level courses as described below.

Intellectual Merit: The massively parallel DNA sequencer and a supporting DNA sample preparation instrument requested for shared use in this MRI proposal will serve NSF-funded and other investigators at the Marine Biological Laboratory in Woods Hole and collaborators at the Woods Hole Oceanographic Institution inclusive of Massachusetts Institute of Technology scientists who participate in the Woods Hole Center for Oceans and Human Health funded by the NSF. The instruments to be purchased include an Illumina GAIIx massively parallel DNA sequencing machine plus an accessory Covaris S-2 DNA acoustic shearer. The equipment will accommodate new DNA sequencing requirements for at least eight funded projects falling within two major themes: microbial community profiling and genomics/transcriptomics. Microbial ecology projects include profiling of human-impacted fresh water supplies, long-term ecological research sites, controlled mesocosms, and the human gut; genomics projects include studies of genome evolution that focus on mechanisms and consequences of deleterious evolution in bacterial symbionts, the role of microRNAs in asexual rotifers, and genomic variation in natural communities, including the phyllosphere and the subseafloor. This equipment will dramatically increase our massively parallel sequencing capacity while at the same time reducing sequencing costs by many fold. Acquisition of an Illumina GAIIx will additionally enable investigators to increase the scope and depth of their ongoing research and consider sequence-based approaches to new questions in ecology and evolutionary biology. The equipment will operate within the W. M. Keck Ecological and Evolutionary Genetics Facility within the Josephine Bay Paul Center for Comparative Molecular Biology and Evolution (BPC) at the Marine Biological Laboratory (MBL), a shared-use resource for investigators in the Woods Hole scientific community operational since 1997.

Broader Impacts: The new instrumentation will provide exciting opportunities for students and researchers at all levels. The PIs currently mentor postdoctoral scholars and Brown-MBL graduate students who will immediately benefit from the addition of a GAIIx to our sequencing facility. Additionally, MBL laboratories will host undergraduates in the MBL?s NSF Research Experience for Undergraduates and Semester in Environmental Sciences programs. These students will benefit from the development of a new curriculum module, From Seawater to Sequences, which will incorporate use of the GAIIx data. This curriculum module will also serve upper-level high school students through our collaboration with the Zephyr Education Foundation, a local non-profit marine education group in Woods Hole. In addition, through the integration of the GAIIx sequencing facility into MBL summer courses, we will extend the training of a diverse population of graduate and postdoctoral students next generation sequencing methods that will have national and international impact. Finally, visiting and summer faculty who might usually outsource their sequencing will have access to the instrument while in residence, learning first hand how sequence datasets are generated and processed, what protocols are possible, and perhaps equally importantly, the limitations and sources of error in next-generation sequencing. This combination of new curriculum development geared towards high school and undergraduate students with rigorous training for summer and visiting faculty, graduate students, and postdocs will allow for a strong integration of the GAIIx with both new and established training opportunities at the MBL.