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Planetary Biology and Microbial Ecology: Molecular Ecology and the Global Nitrogen Cycle

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Planetary Biology and Microbial Ecology: Molecular Ecology and the Global Nitrogen Cycle NASA Contractor Report 4497 Planetary Biology and Microbial Ecology: Molecular Ecology and the Global Nitrogen Cycle Edited by Molly Stone Nealson and Kenneth H. Nealson Center for Great Lakes Studies Milwaukee, Wiscon.4n Prepared for NASA Office of Space 4cience and Applications under Grant NAGW-2136 National Aeronautics arid Space Administration Office of Management Scientific and Technic _1 Information Program 1993 Table of Contents Introduction vi Schedule of Lectures arid Labs viii Faculty, Lecturer and S:udent Addresses xvi Protocols -- Molecular Techniques for the Identification and Study of Luminous Bacteria Quick Genomic DNA Isolation from Bacterial Colonies 2 Restriction Digest of Bacterial DNA 3 Southern Blots 5 Prehybridization of Southern Blots 7 Hybridizati,_n of Southern Blots 8 Washing a-_d Exposing Southern Blcts 9 Colony Hybridization 13 The Polymerase Chain Reaction (PCR) 15 Cloning PCR Products into Single-st:anded Bacteriophage 16 Preparatior_ of M13 Sequencing Templates 23 DNA Sequ,._ncing 24 Classification of Bacteria Using 16S rRNA 33 cDNA Cloning Protocols Extraction of Total RNA and Poly-(A)+-RNA 37 Construction of cDNA Library 43 Preparing High Specific Activity Sin!lie-Stranded 51 cDNA Probes Transfer ot Nucleic Acids to Nylon Membrane 53 and Conditions for Hybridization DNA Sequencing with T7 DNA Polymerase (Sequenase) 56 Polymeras,_ Chain Reaction Assay Conditions 61 Preparatior= of Single-Stranded cDNA Synthesis for PCR 64 Cloning of DNA from PCR Amplification 64 Strategies lor Cloning and Characterizing cDNA 66 Appendix I" RACE 86 Appendix ;__:Screening a cDNA Clone by 89 Immunochemical Methods Appendix 3: Amplification of the Lambda Zap-II Library 91 Appendix 4: Extraction of High Molecular Weight DNA 92 Molecular Approaches to the Analysis of Population Polymorphisms and Enzyme Expression RNA Isolation 94 Restriction Enzyme Digest 96 In Vitro- Produced mRNA and cRNA 97 RNA Gel 98 Northern Blot 98 ooo III PRE_EEH_tG P,_H3E BLANK NOT FILMED Fragment Purification: Agarose 99 Random Priming 100 mRNA to cDNA 101 Southern Blot 102 Hybridizations: RNA or DNA 103 RNA Dot Blot 104 T4 DNA Polymerase 105 Media for Bacteria 106 Preservation of Animal Tissue at Room Temperature for 107 DNA Analyses Geneclean: the Wonders of Glass and DNA 108 Boiling Minipreps for Plasmid DNA 109 iv Lecturers' Abstracts and References Edward DeLong Fluorescent Labeling of Amino-Oligonucleotides 111 Application of Molecular Genetic Techniques to 117 Microbial Ecology Ann Giblin Overview of the Importance of Subtidal Sediments 122 to Nitrogen Cycling in Coastal Ecosystems ]-he Importance of Benthic Macrofauna to Decom- 123 position and Nitrogen Cycting in Sediments Robert Haselkorn Heterocyst Differentiation and Nitrogen Fixation in 125 Cyanobacteria Thomas Hollocher Enzymes of Nitrification and Denitrification: Parts of 128 the Global NitrogenCycle Stephen Macko Use of Bulk Stable Isotopes for Environmental 133 Studies: Stable Isotopes at the Molecular Level Donal Manahan Energy Metabolism During Larval Development 138 and Animal/Chemical Interactions in the Sea Joseph Montoya Stable Isotopes and Their Use in Biogeochemical 139 Studies Daniel E. Morse Molecular Mechanisms Controlling Settlement and 141 Metamorphosis of Marine Invertebrate Larvae Kenneth H. Nealson Ecology of Luminous Bacteria 143 Sandra Nierzwicki- Molecular Methods for the Identification and 146 Bauer Analysis of Phylogenedc Relationships Among Subsurface Microorganisms Gene Expression in Symbiotically Associated and 147 Free-Living Cyanobacteria David L. Peterson Remote Sensing of Ecosystems and Simulation of 148 Ecosystem Processes Thomas Schmidt Molecular Phylogenies and Microbial Evolution 159 Identification and Quantilication of Microorganisms 160 in Natural Habitats Using rRNA-Based Probes Simon Silver Transport Across the Cell Surface: Good 163 Substrates and Bad Biochemistry and Molecular Biology of Bacterial 164 Resistances to Toxic Heavy Metals Metallothioneins: Small Cadmium-Binding Proteins 165 from Horses, Plants and Bacteria Mitchell L. Sogin Recent Developments in Phylogeny of Eukaryotes 1 67 Ivan Valiela An Overview of Nitrogen Cycling in Marine and 169 Aquatic Systems Appendix: Abbreviations 170 V Introduction In the summer of 1991, the NASA Planetary Biology and Molecular Ecology (PBME) program was conducted at the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts. The program used the facilities of the MBL, including the laboratories, dormitories, cafeteria, and laboratory support system. This was the fifth course in a series of PBME programs, and was only the second to be conducted at a site remote from the NASA Ames Research Center. The goals of this intensive summer program were twofold: (1) to examine, via lectures and discussions, several aspects of the biogeochemistry of the nitrogen cycle; and (2) to teach the application of modern methods of molecular genetics to field studies. In this sense, this year's program differed from some of the previous PBME courses, which centered more on microbiology. Here we focused on teaching modern approaches to environmental studies of organisms, including not only microbes, but also phylogenetically more advanced forms. To accomplish the first goal, a distinguished group of scientists was invited to lecture on various aspects of biogeochemistry, specifically the biogeochemistry of nitrogen. The lectures covered many areas, including the biogeochemical cycling of nitrogen in freshwater, estuarine, marine, and terrestrial ecosystems. The lecturers included ecologists, physiologists, biochemists, molecular biologists, and ecosystems scientists. Abstracts of and references for the individual talks are presented in this document. Of particular note for the goals of the course was the series of lectures given by Dr. David Peterson of the NASA Ames Research Center. These lectures, in combination with those involving stable isotope geochemistry and general biogeochemistry, form a basis for analysis of the nitrogen cycle, and how to relate many of the methods taught here to more broadly based problems. To accomplish the second goal, both lectures and laboratory exercises were utilized. Three laboratory modules were run consecutively, each an intensive training session of two weeks. These modules were originated and supervised by Dr. Charles Wimpee, Dr. Thomas Chen, and Dr. Dennis Powers, and dealt with molecular biology of bacteria, fish, and mitochondria, respectively. A list of those faculty members and their teaching assistants, directly involved in the laboratory teaching, is included below. Laboratory education consisted of extensive lecture periods discussing both techniques and approaches, coupled with "hands-on" laboratory exercises. vi Wimpee Module: Mol_cular Biology of Bioluminescent Bacteria Dr. Charles Wimpee, Univ. of Wisconsin-Milwaukee Assisted by Dr. Daad Saffarini Assisted by Denise Garvin Chen Module: cDNA Cloning Dr. Thomas Chen, Center of Marine Biotechnology, Univ. Maryland Assisted by Dr. Chun-Mean Lin Assisted by Michael Shamblott Assisted by Bih-Ying Yang Powers Module: Population Polymorphisms and Enzyme Expression Dr. Douglas Crawford, Univ. of Chicago Dr. Simona Ba_tl, Hopkins Marine Station, Stanford Univ. Assisted by Stephanie Clendennen Detailed descriptions of the laboratory projects and protocols for each of the three modules are contained in this report. Through this presentation, we have attempted to bring the material to others who might find it valuable. Previous NASA PBME courses have used somewhat different approaches. They a(e described in NASA Technical Memorandum #86043 (Carbon Cycling), NASA Technical Memorandum #87570 (Sulfur Cycling), and NASA Contractor Report #4295 (Metal Cycling). Because of the different goals of the laboratory work in this course, the format was quite different than used in previous years. As with the previous courses, however, the experience proved to be exceptionally valuable for the transfer of information and techniques between ecologists, geochemists, and NASA personnel. vii MARINE ECOLOGY/PLANETARY BIOLOGY AND MOLECULAR ECOLOGY Schedule of Lectures and Labs June 16 - July 27, 1991 WEEK I - Module 1 Sunday 1930 Meet in laboratory June 16th Get acquainted session, arrange student talks Monday 0830 3 Student Talks June 17th 1000 Dr. Charles Wimpee: Univ. Wisconsin - Milwaukee Nucleic Acid Hybridization 1300 Dr. Wimpee: Laboratory Technique Lecture Collection and plating of water samples 6 Student Talks Tuesday 0830 3 Student Talks June 18th 1000 Dr. Daad Saffarini: Univ. Wisconsin - Milwaukee Laboratory Techniques Lecture 1300 Isolation and purification of luminous bacteria Colony lifts, lysis, cross-linking Prehybridization of filters Direct PCR of colony lysates, End-polishing Gel purification of PCR products Wednesday 0830 3 Student Talks June 19th 1000 Dr. Wimpee: Southern blotting, application of molecular techniques to the study of luminescent organisms 1300 Inoculate liquid cultures from isolated colonies Make hybridization probes using PCR Hybridize colony lifts Thursday 0830 3 Student Talks June 20th 1000 Dr. Saffarini: RNA Isolation and Sequencing 1300 Restriction digestion, gel electrophoresis Wash primary colony filters and expose Isolate RNA Friday 0830 3 Student Talks June 21st 1000 Dr. Kenneth Nealson: Univ. Wisconsin - Milwaukee Biology and Ecology of Luminous Bacteria 1300 Develop colony films, blot gels, cross-link Prehybridize, hybridize, PCR on genomic DNA End-polishing, gel purification of PCR
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