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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
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
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
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
SterilizationWhy sterilize?
Methods: AutoclavingFiltration
ChemicalsRadiation
?
e.g. disposable medical supplies
Plastics
Gas-permeable windows/covers
CH2-CH2
OEthylene oxide
What is “Pasteurization?
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
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
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
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 (?)
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
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”
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” (?)
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
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
Site-specific DNA resolution
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
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”
Caulobacter crescentus
Stays and makes more Swarmers
Asymmetric strategies
Swarmer
Stalked cell
Swims to better home
Changes into Stalked cell
Hold-fast
chromosome
Stalked cell
new flagellum Swarmer cell
Developmental detour
Cell cycle with swarmer differentiation
Symmetric and Asymmetric protein distribution
Che Antibodies
CtrA Antibodies
C. crescentus early division
Caulobacter applications
“bio-reactors”
S-layer protein secretion and “live” surface vaccines
Recombinant S-layer
Fish farming
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 ?
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