Lecture 3: Bacterial Fueling Metabolism

Lecture 2Text: pages 343-352 DNA replicationText: pages 362-368, 441 RNA transcriptionText: pages 369-376 Translation

Lecture 1 Text: pages 110-113 Metabolic overviewText: pages 568-569, 573-574 Pili (Fimbriae) and flagellaText: pages 825-829 Surface virulence factors

Reading assignments in Text: Lengeler et al. 1999Text: pages 114-116, 123-128 Central metabolismText: pages 52-58 Substrate level phosphorylationText: pages 62-67 Electrontransport-coupled phosphorylationText: pages 296-307 FermentationText: pages 263-266 Oxygen and metabolismText: pages 524 Energy generation

Recap

AssemblyFuelling Biosyn. Polymer.

Lecture 1

Pili

Flagella

Lecture 2Metabolism =

Replication

DNA RNA Protein

Transcription Translation

Nalidixic acidNovobiocin

Rifampicim Streptomycin

Tetracycline

Chloramphenicol

Erythromycin

What makes a good Antibiotic ?

1 Distinguish

2 Effective Block

3 Cause danger

Major assemblies

Minor assemblies

General Biosyn.Unique genes (most, e.g. biosyn.)

Target heirarchy

Good

BactericidalBacteristatic

Great

Gene families (PBP’s, Topo’s)

?

RNA and Protein synthesis, “run out experiment”

E. coli

3-H radio-label uridine

14-C radio-label Amino acids

RNA

Protein

Measurements: Sample culture

Precipitate polymer (TCA)

Collect, place in fluoro-phore

Scintillation counter cpm

0 time

cpm

RNA

Protein

Add radio-label

RNA and Protein synthesis, “run out experiment”

E. coli

3-H radio-label uridine

14-C radio-label Amino acids

RNA

Protein

0 min

cpm

RNA

Protein

5 10 15

Add radio-labelPlus Rifampicin

Stable RNA

Un-stable RNAProtein uses mRNA

~ 50% rRNA, tRNA

Rapid1-2 min

half-lives

Polymerization without a nucleus

DNA

RNA

protein

membrane

Who needs a nucleus ?

“prokaryotes” vs “eukaryotes”

Rapid response

Lectures 3,4

= Glycolysis (EMP Pw)

Pentose phosphate cycle (PPC)

Citric acid [Kreb’s] cycle (TCA)

CM Central Metabolism

Strangefoods:oils,

benzene, pesticides, ...

PM Peripheral Met.

12 MP’s Met. Precursors ( 2C - 6C units)

“substrate-level”

ATP

NADHReducing Power NADPH eTS (electron-

Transp. Sys.)F0F1

ATPasePMF

(Proton Motive Force)

“oxidative phosphorylation”

Foods: glucose,

ribose, acetate, ...

AssemblyFuelling Biosyn. Polymer.

Lecture 1

Pili

Flagella

Lecture 2

DNA, RNAProtein

Overview of Metabolism

“Polyhedral organelles”

150 nm

Salmonella enterica

1,2-propane-diol B12-dependent degradationProtein shells encasing four enzymesPossibly sequester toxic propion-aldehyde intermediateFound in 30 of 209 sequenced bacterial genomes

“Strange food”

Central Metabolism / 12 Metabolic Precursors

Glycolysis (EMP Pw)

Citric acid cycle (TCA)

Glucose

Glucose-6-P (6C)

Fructose-6-P

Triose 3-P (2x 3C)

Fructose 1,6-P

3-Phosphoglycerate

2-Phosphoglycerate

Phosphoenolpyruvate

Pyruvate

1, 3-Diphosphoglycerate

Pentose 5-P (5C)

Erythrose 4-P (4C)

Pentose phosphate cycle

Malate

Fumarate

Succinyl~CoA

Succinate

-Ketoglutarate

IsocitrateCitrateAcetyl~CoA

Oxaloacetate

Biosynthesis (see Text: page 115)

Glycolysis (EMP)

Glucose

Glucose-6-P (6C)

Fructose-6-P

Triose 3-P (2x 3C)

Fructose 1,6-P

3-Phosphoglycerate

2-Phosphoglycerate

Phosphoenolpyruvate

Pyruvate

1, 3-Diphosphoglycerate

Pentose phosphate cycle

Pentose 5-P (5C)

Erythrose 4-P (4C)

Citric acid cycle (TCA)

Acetyl~CoA

OxaloacetateSuccinyl~CoA

-Ketoglutarate

MalateFumarate

Succinate

CitrateIsocitrate

-ATP

+ATP

+ATP

NADH

NADH

NADH

Central Metabolism / ATP and Reducing Power

“substrate-level”

NADPH

Biosynthesis

NADH

NADH

FADH 2

NADH+GTP

ATP

NADH

ATPF0F1

mitochondria

electron Transport Systems (eTS) Generate Proton Motive Force (PMF)

NADH

2 H+ 2 H+

2 H+

2e-

1/2 O2

2 H+ H2ONADH

H+

2 [H+ e- ]

NAD+

NAD+

2e-eTS

acceptor

Anaerobic respirationNitrate, sulfate, fumerate, dyes, legnins, etc...

= O2

H2O

G

PMF

H+

eTS = Flavine proteins, FeS-proteins, Cytochromes /heme-proteins, Quinones

in

out

E. coli

PMF

ATP from PMF

PMF

in

out

H+

F0

F1

ADP + P i ATP

H+

eTS

Rotary engine

Metabolic flexibility

Ability to grow on just one C-source.

Ability to grow without oxygen.

“Anaerobic respiration”

“Fermentation”

E. coli growth on Glucose as sole C-source plus oxygen

Pentose phosphate cycle

Citric acid /Kreb’s “cycle” ?

Glucose-6-P (6C)

Fructose-6-P

Triose 3-P (2x 3C)

Fructose 1,6-P

3-Phosphoglycerate

2-Phosphoglycerate

Phosphoenolpyruvate

Pyruvate

1, 3-Diphosphoglycerate

Acetyl~CoA

OxaloacetateSuccinyl~CoA

-Ketoglutarate

Pentose 5-P (5C)

Erythrose 4-P (4C)

Malate

FumarateSuccinate

Citrate Isocitrate

+ATP

+ATP

NADPH

A

NADH eTS

NADH eTS

NADH eTS

NADH

eTS

CO2ATP

Phosphoenolpyruvate Oxaloacetate

A = anapleurotic Rxn “PEP carboxylase”

?

Fueling without oxygen / Fermentation (Abstract)

S P

ATP NADH

NAD+

PH2

Bug

S = sugarOften: P = [ pyruvate] acid / alcohol

Simplest: glucose EMP Pw pyruvate Lactate dehydrogenase

Lactic acid Yogurt

glucose EMP Pw pyruvate Ethanol Beer/breadCO2

Points: ATP “substrate level” poor energy poor growth yield

~redox balance, C e-donors / C e-acceptors

Most C passes through cell without incorporation

More process than consumption (Food/ Industrial applications)

?

E. coli growth on Glucose as sole C-source but No oxygen

No electron acceptors Fermentation

Anapleurotic reaction strategies:

Reach all 12 Metabolic precursors

Produce less NADH

Balance NADH production with consumption

E. coli growth on Glucose as sole C-source but No oxygen

Pentose phosphate cycle

Citric acid Kreb’s “cycle”

Glucose-6-P (6C)

Fructose-6-P

Triose 3-P (2x 3C)

Fructose 1,6-P

3-Phosphoglycerate

2-Phosphoglycerate

Phosphoenolpyruvate

Pyruvate

1, 3-Diphosphoglycerate

Acetyl~CoA

OxaloacetateSuccinyl~CoA

-Ketoglutarate

Pentose 5-P (5C)

Erythrose 4-P (4C)

Malate

FumarateSuccinate

Citrate Isocitrate

A1 = Pep carboxylase (as before)

A1

Anapleurotic reactions:

A2 = Pyruvate formate lyase (no NADH)

A2

No synthetase, no NADH

NADH

A4 = Asp deaminase

A4

NADH

A3 = Asp synthetase

AspartateA3

A5 = Fumarate reductase

A5

CM

Non-fermentable C-sources

Fermentable C-sources

Non-fermentable C-sources

E.g. fermentation products

Other anaerobic metabolisms

E.g. methanogens

Dumping excess electrons (NAD+ from NADH)

S P

ATP NADH

NAD+

PH2

Bug?

eTS

Dissimulatory reduction

e.g. nitrate > nitrite > ammonium > nitrogen gas

Assimilatory reduction

Fermentation

H2

CO2

= anaerobic respiration

AssemblyFuelling Biosyn. Polymer.

Lecture 1

Pili

Flagella

Lecture 2Lectures 3,4

DNA, RNAProtein

CM Central Metabolism = Glycolysis (EMP Pw)

Pentose phosphate cycle (PPC)

Citric acid [Kreb’s] cycle (TCA)PM Peripheral Met.

Foods: glucose,

ribose, acetate, ...

Strangefoods:oils,

benzene, pesticides, ...

12 MP’s Met. Precursors ( 2C - 6C units)

Reducing Power NADPH

“substrate-level”

ATP

[1-C units]

NADH eTS (electron-

Transp. Sys.)

PMF

F0F1

ATPase (Proton Motive Force)

“oxidative phosphorylation”

Overview of Metabolism