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Transcript
Microbiology and Molecular Biology
for Engineers
IGEM, 20 June 2006
There are three types of cell
• A: Archaea
• B: Bacteria (Gram positive, Gram negative)
• E: Eukarya (Animals, plants, yeasts, others)
Eschericha coli
• Kingdom/Division Proteobacteria
• Class/Subdivision Gammaproteobacteria
• Family Enterobacteriaceae
sensors
communications
hull
propulsion
control systems
power plant
Cell envelope
• Typical of Gram
negative bacteria
• Outer membrane:
repels hydrophobic
molecules.
• Peptidoglycan
sacculus: resists
osmotic pressure.
• Cell membrane:
main permeability
barrier.
Energy generation
• Energy generated by oxidation or disproportionation of
organic molecules.
• Stored as non-equilibrium ATP/ADP ratio and transmembrane proton gradient.
Photon capture
Respiration
Motility
Trans-membrane
proton gradient
Transport processes
F1F0 ATPase
Fermentation
ATP/ADP ratio
DNA, RNA and
protein synthesis
Motility
• Swimming motility: rotating flagella powered by proton influx.
• Helical filaments: 20 nm diameter, 5 to 20 mm long.
filament
hook
MS-ring
switch protein
(Gram negative bacteria only)
L-ring
OM
P-ring
PG
CM
motor protein
EM of flagellar base structure
Two-component sensor systems
• Sense external stimuli.
• External stimulus causes modification of internal protein.
ligand
PERIPLASM
Cell membrane
Sensor
Kinase
Response
Regulator
CYTOPLASM
P
Chemotaxis
• Swimming towards an attractant or away from a repellant
is accomplished by a biased random walk – variable
length runs interspersed with random changes of
direction (tumbles).
Mechanism of chemotaxis
• Attractant / repellent chemicals are detected by
chemotaxis receptors (MCPs).
• Phosphorylation state of CheY alters frequency of tumbles.
• Methylation of MCPs decreases sensitivity.
methylase
Glu-Me
CheY
phosphatase
CheY P promotes tumbling
ligand
MCP
CheA
CheW
CheB
phosphatase
CheB P demethylase:
increases MCP sensitivity
Intercellular communication
• Cells sense population density by
‘quorum sensing’.
• Detect critical density of an
autoinducer, usually a homoserine
lactone (LuxI/LuxR-type system)
Picture of squid
O
N
O
OHHL
O
O
luxE
luxG
LuxR
lux promoter
luxI
luxC
luxD
luxA
luxB
Life cycle 1: shaken broth cultures
• Exponential growth phase followed by stationary phase
with different genes expressed.
Biomass/
Optical density
lag
exponential
phase
phase
stationary
phase
decline
phase
Life Cycle 2: in nature
• Cells prefer to grow attached at solid-liquid interfaces
(biofilm).
swimming cells
detachment
swimming
motility,
chemotaxis
surfaceassociated
motility
attached cells
quorum
sensing
microcolonies
mature biofilm
Stochastic vs. mean field models
• Simplistic models often treat biomass as a single
compartment.
• More realistically, billions of individual cells which may be
in quite different states.
• Therefore, oscillators etc. must include a cell
synchronization mechanism unless individual cells are to
be monitored (eg by FACS or fluorescence microscopy).
Hosts other than E. coli
• Salmonella: related to E. coli but better
secretion of proteins into the medium.
Problem: pathogen.
• Bacillus: good secretion of proteins,
forms highly stable resting state
(endospores).
• Budding yeast (Saccharomyces
cerevisiae): eukaryotic cells.
• Plant, insect and mammalian cells.
endospores
yeast
Coming up…
• What the stuff inside the cell is made of, and how it
works.
• How to modify it.