molecular microbiology bioenergetics bacteriology for 2001
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Molecular Microbiology
BioenergeticsBacteriology for 2001
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Molecular Microbiology 2
Outline
• Bacterial bioenergetics– What does this mean?– Mitchell’s hypotheses– Why G and mV do not appear in
these lectures
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Molecular Microbiology 3
Bacterial bioenergetics
• What I expect you to understand– electron transport chain and ATP
synthesis– How bacteria are adapted to pursue
life according to Mitchell’s hypotheses
• What is possible irrelevant– measuring PMF– the concept of Gibbs free energy
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Molecular Microbiology 4
How does bacterial metabolism work
• Central pathways of metabolism• Electron transport chain• ATP synthase
• Are these separate entities or do they work together?
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Molecular Microbiology 5
What is the point of the ETC?
• To reduce oxygen to water?• To transport electrons?• To move protons across a
membrane?• All of the above with a side
order of chips?
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Molecular Microbiology 6
Mitchell’s postulates
• Four postulates to explain– respiratory phosphorylation– transport of solutes– all membrane-based enzymology
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Molecular Microbiology 7Periplasmic space
Building a chemiosmotic organism
Cell membrane
ATP
ADP + Pi
H+
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Molecular Microbiology 8
Building a chemiosmotic organism
ATP
ADP + Pi
H+
H+ H+ H+
H+
H+
H+
H+
H+H+
ATP
ADP + Pi
H+
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Molecular Microbiology 9
Building a chemiosmotic organism
H+ H+ H+
H+
H+
H+
H+
H+H+
ATP
ADP + Pi
H+
H+Solute
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Molecular Microbiology 10
Mitchell’s postulates
• Four postulates to explain– respiratory phosphorylation– transport of solutes– all membrane-based enzymology
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Molecular Microbiology 11
Postulate 1
• The respiratory or photosynthetic electron transport chains should translocate protons– i.e. the electron transport chain
and other reactions should function as proton pumps
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Molecular Microbiology 12
Building a chemiosmotic organism
ATP
ADP + Pi
H+
H+ H+ H+
H+
H+
H+
H+
H+H+
ATP
ADP + Pi
H+
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Molecular Microbiology 13
Postulate 2
• The ATP synthase should function as a reversible proton-translocating ATPase– f1f0 ATPase– activity in the absence of a PMF– reverse flow in presence of PMF
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Molecular Microbiology 14
Building a chemiosmotic organism
ATP
ADP + Pi
H+
H+ H+ H+
H+
H+
H+
H+
H+H+
ATP
ADP + Pi
H+
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Molecular Microbiology 15
Postulate 3
• Energy-translocating membranes should have a low effective proton conductance– For a chemiosmotic organism to
survive,protons must only be able to move through proteins, and not through membranes
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Molecular Microbiology 16
Building a chemiosmotic organism
ATP
ADP + Pi
H+
H+ H+ H+
H+
H+
H+
H+
H+H+
ATP
ADP + Pi
H+
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Molecular Microbiology 17
Postulate 4
• Energy-transducing membranes should possess specific exchange carriers to permit metabolites to permeate, and high osmotic stability to be maintained, in the presence of a high membrane potential
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Molecular Microbiology 18
Ports, symports, antiports
Solute
Basic port
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Molecular Microbiology 19
Ports, symports, antiports
Solute
Symport
H+
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Molecular Microbiology 20
Ports, symports, antiports
Solute
Antiport
Solute H+or
H+ or
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Molecular Microbiology 21
So what drives ATP synthesis?
• What is the primary pump?• The electron transport chain
pumps 10 protons per NADH + H+
• The NADH + H+ produced during glycolysis etc. is used to create ATP chemiosmotically
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Molecular Microbiology 22
The ETC is the primary pump in aerobic respiratory
organisms
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Molecular Microbiology 23
Bacteria are chemiosmotic
organisms• Paracoccus denitrificans is an
obligately respiratory organism.
• Has an f1f0 ATPase which is coupled to a conventional electron transport chain.
• E. coli is similar but has a strange ETC and can ferment
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Molecular Microbiology 24
The bacterial f1f0 ATPase
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Molecular Microbiology 25
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Molecular Microbiology 26
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Molecular Microbiology 27
What can pH be used for?
• Solute transport– substrates– drug efflux– maintenance of ionic balance
• Bacterial flagellar motion
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Molecular Microbiology 28
What is the point of the ETC?
• To reduce oxygen to water?• To transport electrons?• To move protons across a
membrane?
Sometimes
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How does it all link in?
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Molecular Microbiology 30
H+ H+ H+
H+
H+
H+
H+
H+
H+
ATP
ADP + Pi
H+
H+NADH + H+
reduced FMNNADH + H+
reduced FMN
Reducedelectronacceptor
Carbon Source
GlycolyticPathway
TCA
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Molecular Microbiology 31
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Molecular Microbiology 32
Short questions for group work
• What other means are there of generating PMF?
• What other uses can a proton gradient be put to in bacterial cells?
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Molecular Microbiology 33
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Molecular Microbiology 34
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Molecular Microbiology 35
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Section IIChemiosmotic solutions
to environmental problems
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Molecular Microbiology 37
Aerobic neutrophiles
H+
H+
H+
H+
H+
H+
H+
H+
ATP
ADP + Pi
H+
H+Solute
External pH 6-8
Internal pH 6-8
--
--------
-
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Molecular Microbiology 38
Aerobic acidophilesH+ H+
H+
H+
H+
H+
H+
H+
ATP
ADP + Pi
H+
H+Solute
External pH 1-4
Internal pH 5.5
++
++++++++
+
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Molecular Microbiology 39
Aerobic alkaliphiles
ATP
ADP + Pi
H+
Na+Solute
External pH 10-11
Internal pH 8.2
--
--------
-
Na+
H+
++
+
++++++
++
H+
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Molecular Microbiology 40
Halophiles
ATP
ADP + Pi
H+
Na+Solute
--
--------
-
Na+
H+
++
+
++++++
++
H+
H+
Light
External pH 6-10
Internal pH 6-8
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Molecular Microbiology 41
Marine/halotolerant
ATP
ADP + Pi
Na + or H+
Na+Solute
--
--------
-
Na+
H+
++
+
++++++
++
Na+ or H+
External pH 6-10
Internal pH 6-8
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Section III
An example of a proton pump
Bateriorhodopsin
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Molecular Microbiology 43
Halobacterium salinarum
• A member of the Archaea• Also known as Halobacterium
halobium • When grown aerobically utilises a
normal respiratory chain
• In the light under very low O2, purple patches appear on their membranes
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Molecular Microbiology 44
Halobacterium salinarum
• Cells lack chlorophyll• Still can generate ATP from
light• Protons are pumped by a
simple (ish) to understand mechanism centered on bacteriorhodopsin
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Molecular Microbiology 45
Bacteriorhodopsin, crystal structure
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Molecular Microbiology 46
Bacteriorhodopsin, crystal structure, showing retinal
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Molecular Microbiology 47
Bacteriorhodopsin, crystal structure, showing retinal, top view
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Molecular Microbiology 48
Clever bacterium
• Halobacterium salinarum possesses four rhodopsins– bacteriorhodopsin (bR)
– halorhodopsin (HR)
– sensory rhodopsins I and II
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Molecular Microbiology 49
See overhead
• Mechanism p184 Nicholls & Ferguson
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Molecular Microbiology 50
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Molecular Microbiology 51
References
• Nichols and Ferguson - Bacterial Bioenergetics (Academic Press)
• Lehninger, Stryer or another good biochemistry text book
• ASM News