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Molecular Microbiology
BioenergeticsBacteriology for 2001
Molecular Microbiology 2
Outline
• Bacterial bioenergetics– What does this mean?– Mitchell’s hypotheses– Why G and mV do not appear in
these lectures
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
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?
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?
Molecular Microbiology 6
Mitchell’s postulates
• Four postulates to explain– respiratory phosphorylation– transport of solutes– all membrane-based enzymology
Molecular Microbiology 7Periplasmic space
Building a chemiosmotic organism
Cell membrane
ATP
ADP + Pi
H+
Molecular Microbiology 8
Building a chemiosmotic organism
ATP
ADP + Pi
H+
H+ H+ H+
H+
H+
H+
H+
H+H+
ATP
ADP + Pi
H+
Molecular Microbiology 9
Building a chemiosmotic organism
H+ H+ H+
H+
H+
H+
H+
H+H+
ATP
ADP + Pi
H+
H+Solute
Molecular Microbiology 10
Mitchell’s postulates
• Four postulates to explain– respiratory phosphorylation– transport of solutes– all membrane-based enzymology
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
Molecular Microbiology 12
Building a chemiosmotic organism
ATP
ADP + Pi
H+
H+ H+ H+
H+
H+
H+
H+
H+H+
ATP
ADP + Pi
H+
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
Molecular Microbiology 14
Building a chemiosmotic organism
ATP
ADP + Pi
H+
H+ H+ H+
H+
H+
H+
H+
H+H+
ATP
ADP + Pi
H+
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
Molecular Microbiology 16
Building a chemiosmotic organism
ATP
ADP + Pi
H+
H+ H+ H+
H+
H+
H+
H+
H+H+
ATP
ADP + Pi
H+
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
Molecular Microbiology 18
Ports, symports, antiports
Solute
Basic port
Molecular Microbiology 19
Ports, symports, antiports
Solute
Symport
H+
Molecular Microbiology 20
Ports, symports, antiports
Solute
Antiport
Solute H+or
H+ or
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
Molecular Microbiology 22
The ETC is the primary pump in aerobic respiratory
organisms
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
Molecular Microbiology 24
The bacterial f1f0 ATPase
Molecular Microbiology 25
Molecular Microbiology 26
Molecular Microbiology 27
What can pH be used for?
• Solute transport– substrates– drug efflux– maintenance of ionic balance
• Bacterial flagellar motion
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
How does it all link in?
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
Molecular Microbiology 31
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?
Molecular Microbiology 33
Molecular Microbiology 34
Molecular Microbiology 35
Section IIChemiosmotic solutions
to environmental problems
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
--
--------
-
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
++
++++++++
+
Molecular Microbiology 39
Aerobic alkaliphiles
ATP
ADP + Pi
H+
Na+Solute
External pH 10-11
Internal pH 8.2
--
--------
-
Na+
H+
++
+
++++++
++
H+
Molecular Microbiology 40
Halophiles
ATP
ADP + Pi
H+
Na+Solute
--
--------
-
Na+
H+
++
+
++++++
++
H+
H+
Light
External pH 6-10
Internal pH 6-8
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
Section III
An example of a proton pump
Bateriorhodopsin
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
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
Molecular Microbiology 45
Bacteriorhodopsin, crystal structure
Molecular Microbiology 46
Bacteriorhodopsin, crystal structure, showing retinal
Molecular Microbiology 47
Bacteriorhodopsin, crystal structure, showing retinal, top view
Molecular Microbiology 48
Clever bacterium
• Halobacterium salinarum possesses four rhodopsins– bacteriorhodopsin (bR)
– halorhodopsin (HR)
– sensory rhodopsins I and II
Molecular Microbiology 49
See overhead
• Mechanism p184 Nicholls & Ferguson
Molecular Microbiology 50
Molecular Microbiology 51
References
• Nichols and Ferguson - Bacterial Bioenergetics (Academic Press)
• Lehninger, Stryer or another good biochemistry text book
• ASM News
