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Advancing Science with DNA Sequence
VII. CONTEXT: THE ORGANISM
Advancing Science with DNA Sequence
CONTEXT: THE ORGANISM
• Isolated from a neutral volcanic hot spring• Thermophilic, gram negative bacterium
(optimal temp 77ºC)• Chemolithoautotroph
Huber et al., 1992
Advancing Science with DNA Sequence
Context as Bridge
• Connections to other parts of the curriculum (autotrophy, redox/electron transport, evolution, phylogeny, etc.)
Advancing Science with DNA Sequence
CONTEXT: Contents
• Extremophiles
• Chemolithoautotrophy
• A. degensii isolation and characterization
• Horizontal gene transfer
Advancing Science with DNA Sequence
Extremophiles
• -20 to 121 C
• P < 120 Mpo
• -0.2 to 11 pH
• salinities up to 6 M
• Basically our idea of normal is pretty narrow
Advancing Science with DNA Sequence
Extremophiles
• Thermophile 45° C - 117° C
• Psychrophile -10° C - 10° C
• Barophile
• Acidophile
• Halophile
• alkaphile
Advancing Science with DNA Sequence
Comparing the genomes of thermophiles and mesophiles
• Singer and Hickey (2003) compare three characteristics of genomes: nucleotide content, codon usage and amino acid composition: 2-fold decrease in the amount of Q in thermophiles (thermolabile)?
• Investigated in: Das and Gerstein. 2000. The stability of thermophilic proteins: a study based on comprehensive genome comparison. Funct. Integr. Genomics 1:76-88
Advancing Science with DNA Sequence
Why study extremophiles?
• To learn limits of life on earth
• To learn what conditions (& planets/moons) to look for life in solar system
• To obtain biocatalysts (enzymes) which work under extreme conditions
Advancing Science with DNA Sequence
Extreme Applications
• Important extremophile enzyme applications• 1. Washing machine: Protease at alkaline pH,
bleach and high temperature• 2. Starch hydrolysis industrial process: α-
amylase active and stable at 104° C• 3. Biomining: Bacteria which reduce or
oxidize metals at pH 1.0 during mining operations
• To obtain extremophilic enzymes, look in extreme environments
Advancing Science with DNA Sequence
Advancing Science with DNA Sequence
The Context is the goal
Scott et al., 2006
Advancing Science with DNA Sequence
Autotrophy
• CO2 biomass carbon – Carbohydrates, amino acids, nucleic acids, etc.
• Many pathways for carbon fixation– Calvin Benson Bassham cycle– Rev. TCA cycle– Acetyl CoA pathway– Hydroxypropionate cycle
• All have in common: Energetically expensive – High demand for ATP, NAD(P)H
Advancing Science with DNA Sequence
Energy Electrons Carbon Practitioners
photo litho auto trophs plants, algae
chemo organo hetero trophs humans, E. coli
chemo litho auto trophs protagonists of this presentation
Chemolithoautotrophs by Comparison
Courtesy of KM Scott
Advancing Science with DNA Sequence
Chemolithoautotrophy
• “…something from almost nothing…”--J. Shively
• Ammonifex degensii (and other chemolithoautotrophs) can generate all their lipids, proteins, nucleic acids, cofactors, cell wall components from CO2, NO3, PO4 (!!)
Courtesy of KM Scott
Advancing Science with DNA Sequence
How do cells obtain energy from lithotrophic electron transport?
• Electrons are fed into electron transport chains
• As the electrons travel down the electron transport chain, protons are pumped out of the cell at “coupling sites”
• The proton motive force that is generated is used for ATP synthesis etc.
Advancing Science with DNA Sequence
Formation of Ammonium from Nitrate During Chemolithoautotrophic Growth of the Extremely Thermophilic Bacterium Ammonifex degensii
System. Appl. Microbiol. 19, 40-49 (1996)
R. Huber, P. Rossnagel, C. R. Woese, R. Rachel, T. A. Langworthy, and K.
O. Stetter
Advancing Science with DNA SequenceFrom Karl Stetter’s notebook, used by permission
Advancing Science with DNA Sequence
Courtesy of Karl Stetter
Advancing Science with DNA Sequence
Ammonifex degensii
Ammonifex: Ammonium maker (Ammon, ammonium salt; L. facere)
degensii: named after Egon T. Degens
Advancing Science with DNA Sequence
Egon T. Degens
Courtesy of Karl Stetter
Advancing Science with DNA Sequence
Egon T. Degens
• April 15, 1928- February 19, 1989• Aries• Geologist and geochemist at Hamburg
University• Established first organic geochemistry labs• Worked also at PSU and CalTech• Cycles of elements, SCOPE/UNEP (the
scientific committee on problems of the environment/United nations Environment Program
Advancing Science with DNA Sequence
Egon T. Degens
• 300 articles and 19 books
• (Co-)Author of Biogeochmistry of Major World Rivers (1991)
• Perspectives on Biogeochemistry (1989)
• Structural Molecular Biology of Phosphates (1971)
Advancing Science with DNA Sequence
Courtesy of Karl Stetter
Advancing Science with DNA Sequence
Courtesy of Karl Stetter
Advancing Science with DNA Sequence
Courtesy of Karl Stetter
•“At a nitrate-containing volcanic hot spring in Java,
•10ml of anaerobic modified medium was inoculated
•with 1ml of original sample material….”
Advancing Science with DNA Sequence
Courtesy of Karl Stetter
Advancing Science with DNA Sequence
Advancing Science with DNA Sequence
University of Regensberg, photo by Cheryl Kerfeld
Advancing Science with DNA Sequence
University of Regensberg, photo by Cheryl Kerfeld
Advancing Science with DNA Sequence
University of Regensberg, photo by Cheryl Kerfeld
Advancing Science with DNA Sequence
Courtesy of Karl Stetter
Advancing Science with DNA Sequence
Presenting the paper….Isolation
After 3 days…
sample KC4 showed growth of rod-shaped cells.
Huber et al., 1996
Advancing Science with DNA Sequence
Phylogenetic analysis 16s rRNA (1996)
A. degensii is related to
Desulfotomaculum, a sulfate
reducing species
A. degensii has a distant relationship
with thermophilic clostridia
ref
Huber et al., 1996
Advancing Science with DNA Sequence
DNA base composition
* Melting point analysis → 53% GC
• Chromatographic base analysis → 55.5% GC
• Results from sequencing?
Advancing Science with DNA Sequence
Lipids
* Glycerol diethers---------------85%
* Glycerol monoethers-----------9%
* Fatty acid methyl esters-------8%
Advancing Science with DNA Sequence
Lipids
Huber et al., 1996
Advancing Science with DNA Sequence
• “New” biospheres• Mid-ocean crust (300
meter deep borehole)• Small subunit rRNA from
organisms collected on filters sequenced
• The most abundant class of clones is most closely related to A. degensii
• Science, 299: 120-123 (2003)