Was ist eigentlich "Thermophilie"?

Was ist eigentlich "Thermophilie"?
Hitzeschock
Funktionen von molekularen Chaperonen (Hitzeschockproteinen)
Erkennung von entfalteten Proteinen, Faltung von Proteinen,
Proteinabbau
10-15% aller Proteine,
Und
denaturierte
Proteine
1
Radiation
The mutation rate correlates directly
with the dose of mutagenic radiation
(kR: kiloRöntgen)
Gamma radiation
160 kV X-Ray
Beta-radiation
HO and O
Radicals,
Hydroxylperoxide
Are formed
DNA-damage caused by ionised radiation
Crosslinks
(covalent bond of
Opposing bases)
Double-strand
Breaks
Single strand break
Destruction of bases
2
Anschalten der Reparatur-Mechanismen:
Die SOS Response in Bakterien
uvr: Nucleotide Excision Repair
sfiA: hemmt Zellteilung, schafft Zeit für Reparatur
Pol B: DNA polymerase II
dinB: DNA polymerase IV
umuC/umuD: - DNA pol V (umuD‘+umuC)
Natural radiation resistance
3
Deinococcus radiodurans :
Extrem Stahlungsresistent: 15,000 grays (humans: 10 grays)
Austrocknungsresistent
Gram+ bacterium mit outer membrane (ohne lipid A)
4
Deinococcus wächst in Tetraden
5
Fragmented chromosomal DNA of Deinococcus radiodurans
1: Not I digest of intact DNA
2/6: photo-fragmented DNA
3-5: repaired DNA
Proposed DNA repair for Deinococcus radiodurans:
Use of a second Chromosome
6
Resistance to radiation evolved in many different lineages
7
Microbes and Oxygen
The effects of oxygen
“Janus”
8
Die gute Seite von O2
• Hohe Energieausbeute bei Atmungskette mit O2
• Wichtig fuer enzymatische Katalyse (Oxygenasen)
• Bildung von Ozon in der Atmosphäre: UV-Schutz
Die schlechte Seite von O2
• toxische Wirkung --> Zellgift
• ‘Reactive oxygen species’ ROS
9
Erdgeschichtliche Entwicklung der atmosphaerischen O2
Konzentration
http://www.fas.org/irp/imint/docs/rst/Sect19
Mikroorganismen mussten sich erst ‘spät’ auf O2 einstellen
Entgiftung vor Atmungskette
Cyanobacteria
• Oxygenic photosynthesis led to development
of banded iron formations, an oxic
environment, and great bursts of biological
evolution (Figure 11.8).
10
Respiration: very attractive!
• Mitochondria and chloroplasts, the principal
energy-producing organelles of eukaryotes,
arose from the symbiotic association of
prokaryotes of the domain Bacteria within
eukaryotic cells, a process called
endosymbiosis (Figure 11.9).
•Mitochondria arose from the Proteobacteria,
a major group of Bacteria.
11
12
13
•Energieausbeute mit Glucose als Substrat:
•Glycolyse (Fermentation): 2 ATP
•bei Atmungskette mit O2: 36 ATP
Oxygen classes of microorganisms
14
• Aerobes require oxygen to live, whereas
anaerobes do not and may even be killed by
oxygen.
• Facultative organisms can live with or
without oxygen. Aerotolerant anaerobes can
tolerate oxygen and grow in its presence even
though they cannot use it.
• Microaerophiles are aerobes that can use
oxygen only when it is present at levels
reduced from that in air.
15
a) Obligate aerobe
16
a) Obligate aerobe
b) Obligate anaerobe
c) Facultative aerobe
(can respire)
d) Microaerophile
e) Aerotolerant anaerobe
Cultivation
Of
anaerobes
17
Anaerobic chamber
Fakultativ anaerobe Mikroorganismen:
Warum ist Sauerstoffmessung so wichtig?
80°C
pH 2.5
White Island, New Zealand
Photo: Arnulf Kletzin
18
Acidianus ambivalens
80°C
pH 2.5
1m
•Hyperthermopilic and chemolithoautotrophic Archaeon
•facultative anaerobic
Foto: W. Zillig
aerobic:
S0 + O2 + 2H2O --> SO42- + 2H+
anaerobic:
H2 + S0 --> H2S
10cm
Foto: A. Kletzin
White Island, New Zealand
Photo: Arnulf Kletzin
The sulfur oxidation pathway in Acidianus ambivalens
SOR: sulfur oxygenase reductase
TQO: Thiosulfate:quinone oxidoreductase
Several enzymes, > 10% proteins in cell
19
Sulfur oxygenase reductase
24 monomers form a highly symmetrical particle
Mr 870 kDa (1/3 of 70S ribosome)
A self-compartmentalizing hollow sphere
20
Anaerobic conditions: short anaerobic electron transport chain
Electron donor: Hydrogen
Electron acceptor: Sulfur
Mechanism of O2 sensing unclear in Archaea.
Life at oxic-anoxic interfaces
21
Alexander Winogradsky
Discoverer of AOB
The concept of chemoautotrophy
Bacterial GIANTS
100m
“Gigantische Schwefelbakterien”
Beggiatoa, Thioploca, Thiomargarita
22
Filaments on a crustacean (Isopode) leg
23
Steep gradient of oxygen and sulfide
•Sulfide oxidizers
depth (mm)
•Energy source H2S, S
•Electron acceptors: Oxygen,
Nitrate
•actice movement between oxic
and anoxic areas
•Storage of Sulfur and nitrate
•“holding the breath”
concentration
Vacuolated Bacteria
24
mutualistische symbiose
25
Messung der O2 Konzentration in fakultativ
anaeroben Bakterien
Escherichia coli: 2 komplementäre Systeme, streng
kontrolliert.
1)
2)
ArcBA (2-Komp.Syst)
FNR
aerobe Atmung
anaerobe Atmung
Fermentation
ArcBA (aerobic respiratory control)
•reguliert Gene des aeroben Stoffwechsels (terminale Oxidase,
Citrat cyclus, etc)
aerob
O2
Oxidase
QH2
Q
HS
HS
QH2
ArcB
Q
S
S
ArcB
Sensor inaktiv
2 H2 O
anaerob
Oxidase
QH2
HS
ArcB
HS
P
ArcA
P
Repression d. Transcription
d. Zielgene (>50)
Aktivierung d. Pyruvat-Formiat Lyase
26
FNR (Fumarat-Nitrat-Reductase Regulator)
•dimeres DNA-Bindeprotein
•transkriptioneller Aktivator
•aktiviert anaerobe Atmungskette
aussen
O2
innen
1-4M
4Fe4S
FNR
FNR
2Fe2S
FNR
2 Fe
2S
+
FNR
Aktivierung d. Transcription
d. Zielgene (>100)
Figure 1 Predicted structure of an FNR monomer
Biochemical Society Transactions
www.biochemsoctrans
.org
www.biochemsoctrans.org
(2008) 36, 1144-1148
Biochem.
Biochem. Soc. Trans.
27
Figure 2 Schematic representation of the reaction of transcriptionally active [4Fe-4S]2+ FNR with oxygen
Biochemical Society Transactions
www.biochemsoctrans.org
(2008) 36, 1144-1148
Biochem. Soc. Trans.
Toxic Forms of Oxygen
• Several toxic forms of oxygen can be
formed in the cell, but enzymes are present
that can neutralize most of them .
•Superoxide in particular seems to be a
common toxic oxygen species.
28
Four electron reduction of O2 to H2O
Aerobic Respiration: Cytochrome oxidase / Oxygen reductase
Photosynthesis- Oxygen evolving complex (PSII)
Makrophages
Decontamination of oxygen species
29
Simple Test for Catalase:
Mix cells with drop of 30% H2 O2
Bubbles appear (O2 developed)
Catalase: H2 O2 + H2O2 = 2 H2 0 + O2
Defense against ROS in Cyanobacteria
Enzymatic
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Erkennung von oxidativem Stress
Defense against ROS in Cyanobacteria
Non-Enzymatic: ‘Anti-oxidants’
Carotenoids
Vitamin E (alpha-Tocopherol)
Vitamin C (ascorbate)
Tocopherols can undergo two oxidation reactions: they may
be oxidized by ROS to a tocopheryl radical and convert
singlet oxygen to hydroperoxide. Both reactions can be
reversed by ascorbate, recycling tocopherol.
31
Molecular Oxygen as a
Reactant in Biochemical
Processes
• In addition to its role as an electron acceptor,
oxygen is also a chemical reactant in certain
biochemical processes. Enzymes called
oxygenases introduce O2 into a biochemical
compound.
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• There are two classes of oxygenases:
dioxygenases, which catalyze the
incorporation of both atoms of O2 into the
molecule, and monooxygenases, which
catalyze the transfer of only one of the two
oxygen atoms in O2 to an organic compound;
the second atom of O2 is reduced to water,
H2O
33
• Many microorganisms can degrade aliphatic
and aromatic hydrocarbons. Aerobic
catabolism involves the activity of oxygenase
enzymes
34
Methanotrophy
• Methanotrophy is the use of CH4 as a
carbon and energy source.
•Key enzyme is methane monooxygenase
(MMO)
A new oxygenic metabolism
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