- Slides: 19
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 610 hours? � What happens next? � Why? How?
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? � Why does c. AMP 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 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 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 � c. AMP available to bind CRP � CAP site bound – ACTIVATION of operon
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 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 � c. AMP 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 � c. AMP 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 � c. AMP 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 � c. AMP forms rapidly � Lac operon is activated
WHAT WOULD THE MUTANTS BE LIKE? Genotype lac. Z-, lac. Ycyacrp- Behavior Relative to Diauxic Growth