CATABOLITE REPRESSION INDUCER EXCLUSION AND DIAUXIC GROWTH HOW

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CATABOLITE REPRESSION, INDUCER EXCLUSION, AND DIAUXIC GROWTH

CATABOLITE REPRESSION, INDUCER EXCLUSION, AND DIAUXIC GROWTH

HOW DOES A TRIPLE SUGAR IRON AGAR WORK? What are the 3 sugars in

HOW DOES A TRIPLE SUGAR IRON AGAR WORK? What are the 3 sugars in TSI? � Which is least abundant? � What happens after 610 hours? � What happens next? � Why? How?

WILD TYPE E. COLION MINIMAL MEDIUM WITH GLYCEROL / IPTG Addition β-galactosidase activity lac

WILD TYPE E. COLION MINIMAL MEDIUM WITH GLYCEROL / IPTG Addition β-galactosidase activity lac m. RNA levels none <1 <1 IPTG 100 IPTG + Glc 10 10 IPTG + Glc + c. AMP 80 80

WHAT IS GLUCOSE DOING? Inhibiting β-galactosidase? � Glucose, or it’s intermediates, repress lac expression?

WHAT IS GLUCOSE DOING? Inhibiting β-galactosidase? � Glucose, or it’s intermediates, repress lac expression? � Why does c. AMP help? � Is CRP affected? � Is Adenylate Cyclase affected?

HOW DOES GLUCOSE GET IN THE CELL? Phosphotransferase system (PTS) Group Translocation

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

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 �

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

CATBOLITE REPRESSION Enzyme IIA glc – P not available � Adenylate cyclase not activated � No c. AMP to bind CRP � CAP site unbound – no activation of operon

WHAT IS LEFT IF GLUCOSE ISNOT COMING INTO THE CELL? Enzyme IIA - P

WHAT IS LEFT IF GLUCOSE ISNOT 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

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 � c. AMP available to bind CRP � CAP site bound – ACTIVATION of operon

WHAT WOULD THE MUTANTS BE LIKE? Genotype Enzyme I - Enzyme II BC –

WHAT WOULD THE MUTANTS BE LIKE? Genotype Enzyme I - Enzyme II BC – Enzyme II A - Phenotype?

EFFECT OF MUTATIONS ON ADENYLATE CYCLASE (ABSENCE OF GLUCOSE) Strain Adenylate Cyclase Wild Type

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 Log OD Log [S]

DIAUXIC GROWTH RESPONSE Lag Phase � Ribosome synthesis

DIAUXIC GROWTH RESPONSE Lag Phase � Ribosome synthesis

DIAUXIC GROWTH RESPONSE Exponential Phase � Primary metabolism – glucose fermented to mixed acid

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 � c. AMP absent � Lac operon not activated

DIAUXIC GROWTH RESPONSE Entering Stationary Phase (aka secondary lag phase) � Primary metabolism ebbs

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 � c. AMP begins to form � Lac operon begins to be activated

DIAUXIC GROWTH RESPONSE Leaving Stationary Phase (aka secondary lag phase) � Primary metabolism resumes

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 � c. AMP forms rapidly � Lac operon is activated

DIAUXIC GROWTH RESPONSE Second exponential phase � Primary metabolism continues with lactose fermented to

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 � c. AMP forms rapidly � Lac operon is activated

WHAT WOULD THE MUTANTS BE LIKE? Genotype lac. Z-, lac. Ycyacrp- Behavior Relative to

WHAT WOULD THE MUTANTS BE LIKE? Genotype lac. Z-, lac. Ycyacrp- Behavior Relative to Diauxic Growth