CATABOLITE REPRESSION,INDUCER EXCLUSION,AND DIAUXIC GROWTH

HOW DOES A TRIPLE SUGAR IRON AGAR WORK?

What are the 3 sugars in TSI?

Which is least abundant?

What happens after 6-10 hours?

What happens next?

Why? How?

WILD TYPE E. COLI ON MINIMAL MEDIUM WITH GLYCEROL / IPTG

Addition β-galactosidase

activity

lac mRNA levels

none <1 <1

IPTG 100 100

IPTG + Glc 10 10

IPTG + Glc + cAMP

80 80

WHAT IS GLUCOSE DOING?

Inhibiting β-galactosidase?

Glucose, or it’s intermediates, repress lac expression?

Why does cAMP help? Is CRP affected?

Is Adenylate Cyclase affected?

HOW DOES GLUCOSE GET IN THE CELL?

Phosphotransferase system (PTS)Group Translocation

WHAT IS LEFT IF GLUCOSE IS COMING INTO THE CELL?

Glucose 6-phosphate

Enzyme IIA HPr Enzyme I Pyruvate

INDUCER EXCLUSION

Enzyme IIA glc (without P) acts as inhibitor of lac permease Lactose not taken into cell Lactose not converted to allolactose by β-

galactosidase Repressor Protein stays on operator

CATBOLITE REPRESSION

Enzyme IIA glc – P not available Adenylate cyclase not activated No cAMP to bind CRP CAP site unbound – no activation of operon

WHAT IS LEFT IF GLUCOSE IS NOT COMING INTO THE CELL?

Enzyme IIA - P HPr - P Enzyme I - P PEP

ABSENCE OF GLUCOSE

Enzyme IIA glc - P does not act as inhibitor of lac permease Lactose taken into cell Lactose converted to allolactose by β-

galactosidase Repressor Protein comes off operator -

INDUCTION Enzyme IIA glc – P available

Adenylate cyclase activated cAMP available to bind CRP CAP site bound – ACTIVATION of operon

WHAT WOULD THE MUTANTS BE LIKE?

Genotype Phenotype?

Enzyme I -

Enzyme II BC - –

Enzyme II A -

EFFECT OF MUTATIONS ON ADENYLATE CYCLASE (ABSENCE OF GLUCOSE)

Strain Adenylate Cyclase

Wild Type 100

Enzyme I minus

(HPr, E II A never phosphorylated)<5

Enzyme II BC minus

(Glucose doesn’t come in, E II A always phosphorylated

100 ± glucose

Enzyme II A minus

(Cannot activate Adenylate Cyclase)<5

DIAUXIC GROWTH RESPONSE

Log OD Log [S]

DIAUXIC GROWTH RESPONSE

Lag Phase Ribosome synthesis

DIAUXIC GROWTH RESPONSE

Exponential Phase Primary metabolism –

glucose fermented to mixed acid products

Enz IIA not phosphorylated Lac permease inhibited Lactose excluded No induction by

allolactose Adenylate cyclase inactive cAMP absent Lac operon not activated

DIAUXIC GROWTH RESPONSE

Entering Stationary Phase (aka secondary lag phase) Primary metabolism ebbs Enz IIA – P accumulates Lac permease inhibition

relieved Lactose enters Induction by allolactose Adenylate cyclase increasingly

activated cAMP begins to form Lac operon begins to be activated

DIAUXIC GROWTH RESPONSE

Leaving Stationary Phase (aka secondary lag phase) Primary metabolism resumes

with lactose fermented to mixed acids

Enz IIA – P predominates Lac permease inhibition

relieved Lactose enters rapidly Induction by allolactose Adenylate cyclase fully actived cAMP forms rapidly Lac operon is activated

DIAUXIC GROWTH RESPONSE

Second exponential phase Primary metabolism

continues with lactose fermented to mixed acids

Enz IIA – P predominates Lac permease inhibition

relieved Lactose enters rapidly Induction by allolactose Adenylate cyclase fully

activated cAMP forms rapidly Lac operon is activated

WHAT WOULD THE MUTANTS BE LIKE?

Genotype Behavior Relative to Diauxic Growth

lacZ-, lacY-

cya-

crp-