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