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Transcript of Lecture 6: Bacterial Growth Reading assignments in Text: Lengeler et al. 1999 Text: pages 88-90,...
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Lecture 6: Bacterial Growth
Reading assignments in Text: Lengeler et al. 1999Text: pages 88-90, 95-103, 108-109 Growth and nutritionText: pages 541-544 E. coli symmetric divisionText: pages 571-574 Caulobacter asymmetric cell divisionText: pages 882-884 Making MSG
Lecture 5Text: pages 205-214, 229-232 Examples of organotrophyText: pages 234-244, 267-268 Lithotrophs in generalText: pages 245-259, 911-12 Lithotroph specificsText: pages 327-340 PhototrophsText: pages 67 PhotophosphorylationLecture 4Text: pages 116-122 AssimilationText: pages 177-182 Assimilation reactionsText: pages 155-157 Storage compounds
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Last lecture
All organisms
Chemotroph Phototroph
1 Organotroph 2 Lithotroph 3 Anoxygenic 4 Oxygenic
(get their energy)
Assimilate C ? ? ?Fix CO2
Calvin cycleFix CO2
Reverse TCA cycle CM
12 MP’s
ATP
NAD(P)HBiosyn.
??
ATP
NAD(P)H
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Lecture Overview
Bacterial populations (lab conditions)
Applications: Making MSG
Food/ Media
Sterilization
Growth measurement
Metabolism
GROWTH
Bacteria as single cells (“cell cycles”)
E. coli divides symmetrically
Caulobacter crescentus divides asymmetrically
Changes during growth
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Food/ MediaRich Minimal
“undefined” from organisms “defined” from chemicals
?
E. coli LB (Luria + Bertani) M9
per literwater
10 g “Tryptone”
5 g “Yeast Extract”10 g NaCl
6 g Na2 HPO4
3 g KH2 PO4
0.5 g NaCl1 g NH4Cl
Autoclave (steam & pressure)
(15 g Agar for plates)
Done Done ?Add: Sugar (0.2% w/v)
CaCl2 0.1 mM
MgSO4 1 mM
Fe water*Done ?
CoZn Cu
Se
LB vs M9 grown E. coli, same or different,
composition, growth rate ?
“The truth is seldom simple, but never pure.”
-Oscar Wilde
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SterilizationWhy sterilize?
Methods: AutoclavingFiltration
ChemicalsRadiation
?
e.g. disposable medical supplies
Plastics
Gas-permeable windows/covers
CH2-CH2
OEthylene oxide
What is “Pasteurization?
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Bacterial growth and measurement
time
N
t = 0
“Exponential growth”
time
Log(N)
t = 0
“Log growth”
2x N
DT = Doubling Time
Bacterium DT max
Bacillus stearothermophilus 8 minE. coli 23 min
Caulobacter crescentus 90 min
Mycobacterium tuberculosis 6 hours
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Measuring “N”
1 Viable cell counting: SampleDilute (note dilution factor)
Incubate / grow
One cell = One colony ~106 cells
S.D. = N colonies
2 Direct counts:
side
coverslip
bottom
Microscopic chamber
Spread on plate (0.1 ml)
“Petri” Agar
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Measuring “N”
3 Automated flow cell/ cytometry:Counter
laser
detector
Volume = (flow) x (time)
4 Absorbency or “Optical Density (OD)”
Red light(~600 nm)
Scatter by cells
I (+cells)
OD = log[I0/I(+cells)]
OD = log[10/1]
= 1.0 ~ 109 cells / ml
Useful linear range OD = 0.1 to 1.0
Must calibrate
1 cm
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Changes during growth
time
Log(N)
lag
log
stationary
??inoculate
1 Induce survival genes
s New mRNA ~30 proteins
2 Fewer % ribosomes
3 Smaller cells (>8-fold range)
4 Modified fatty acids in P-lipids CH
HC
Log Stationary phase changes of E. coli
CH
HCCH2
5 High mutation rates in sub-population (?)
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Making Mono-Sodium Glutamate (MSG)(see Course Pack lecture 6, Text pages 882-4)
~800,000 tons / year What is MSG ?
Industrial production principles
Food =Glucose
AmmoniumSaltsBiotin
Starting materials
Reactions Products
Central metabolism
Synthetic PW=Glutamate
Dehydrogenase(GD)
MSG(secreted)
Byproducts
Optimum concentration
Growth control ?with bio- strain
?Bacteriaetc.
Altered membranes / high secretion / deregulation
Bacterium = Corynebacterium glutamicumGm(+) rods
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Making MSG from Central Metabolism
Glucose-6-P (6C)
Fructose-6-P
Triose 3-P (2x 3C)
3-Phosphoglycerate
Phosphoenolpyruvate
Pyruvate
Pentose 5-P (5C)
Erythrose 4-P (4C)
MalateFumarate
Succinyl~CoA
Succinate
-KetoglutarateCitrate
Acetyl~CoA
Oxaloacetate
BiosynthesisGrowing bacteria + BiotinGlucose
Membrane P-lipids
NH3GD NADPH
Glutamate
export
low
Fatty Acids
Biotin
Blocked demand
A
PEP Carboxylase
Cells = MSG “Factories”
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Single cell growth “Cell division cycles”E. coli
Continuous processes:
Most biosynthesis (including cell wall)Ribosome assembly
Punctuated events:
Chromosome replication
Cell/wall division
Big assembly projects
Start / middle / end
Chromosome Replication:
1 Commitment (?): Critical cell mass trigger
2 Start (assemble) replication factories: Timing (?)
Place = oriCoriC
DnaA proteins bindAT-DNA
Unwind oriC
Load DnaB ( ) helicases
Assemble replication “factories” (?)
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E. coli Chromosome replication (continued)
3a DNA synthesis: 2 x ~2x106 bp Accurate and processive
oriC
3b Polar orientation of oriC and chromosome movement (?)
E. coli
4 Termination 6 “ter sites” = pause sites
ter
balance replication
Mechanism:ter
Anti-helicase
5 Resolution and separation: Topological DNA linkage
Covalent linkage problems
Gyrase Topo IV unlink
a
bc
a
bc
a
a
b
cb
cJoin
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oriC
Covalent circular DNA5 Resolution and separation:
oriCHomologous recombination (RecA, BCD)
Even X-over
Odd X-over
dif
dif
XerC,D site-specific recombination
+1 X-over
Steps 1-5 “C period” 40 min = best time
Paradox: ?Best DT = 23 min
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Site-specific DNA resolution
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E. coli cell division1 Commitment (?)
2 Pick site:
Potential sites
MinCDE restriction
X X minCDE
Chromosome-free“mini-cell”
MinCD
MinCD
~20 sec dynamic gradients restrict MinE ring
FtsZ mono-mer (GTP) tubulin-like protein3 form FtsZ ring
FtsZ polymer
Blocked by SulA binding “SOS” DNA damage“check point”
PBP3
mutant pbp3 ts
4 Septum cell wall synthesis “Fts” proteins
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5 Cell wall separation
20 min proceed without protein synthesis
Staggered projects2, 4, 8, 16 chromosomes
inside one large cell
23 min DT ?
Steps 1-5 “D period” = 60 min best time
D40 min C periodC
“Feast or famine strategy”
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Caulobacter crescentus
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Stays and makes more Swarmers
Asymmetric strategies
Swarmer
Stalked cell
Swims to better home
Changes into Stalked cell
Hold-fast
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chromosome
Stalked cell
new flagellum Swarmer cell
Developmental detour
Cell cycle with swarmer differentiation
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Symmetric and Asymmetric protein distribution
Che Antibodies
CtrA Antibodies
C. crescentus early division
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Caulobacter applications
“bio-reactors”
S-layer protein secretion and “live” surface vaccines
Recombinant S-layer
Fish farming
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C = Conc. of limiting nutrient
R = growth rate
0
0
R max
R = (R max)x(C)
(Km + C)
Km
R max / 2
Michaelis-Menton -like analysis of growth
Interpretations ?
R max ?
Km ? Measures affinity for and uptake of nutrient
Measures the metabolic processing of nutrient
Where on the curve ?
In batch cultures ?
In a “turbido-stat” ? a “chemo-stat” ?
In nature ?
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culture
time
Log(N)
lag
log
stationary
inoculate
Controlled growth conditions
“Batch cultures” or “Fermentation cultures”
Continuous cultures:
Mediaflow
Outflow
Turbido-stat: C >> Km
Cell conc. (point on curve )set by start conc.
Dilution rate must = growth rate(needs extra feedback control)
Chemo-stat: C ~ Km, C < Km
Cell conc. = constant x C
Growth rate R = Media flow rate
(i.e. adjust Y-axis and slope)
Reflect natural growth-limiting environments
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