advancing science with dna sequence vii. context: the organism

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

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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

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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

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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

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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

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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

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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

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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

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Egon T. Degens

Courtesy of Karl Stetter

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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)

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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

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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%

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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)