C ATABOLITE R EPRESSION, I NDUCER E XCLUSION, AND D IAUXIC G ROWTH

H OW DOES A T RIPLE S UGAR I RON A GAR WORK ? What are the 3 sugars in TSI? Which is least abundant? What happens after hours? What happens next? Why? How?

W ILD T YPE E. COLI ON MINIMAL MEDIUM WITH G LYCEROL / IPTG Additionβ-galactosidase activity lac mRNA levels none<1 IPTG100 IPTG + Glc10 IPTG + Glc + cAMP80

W HAT 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?

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

W HAT IS LEFT IF GLUCOSE IS COMING INTO THE CELL ? Glucose 6-phosphate Enzyme IIA HPr Enzyme I Pyruvate

I NDUCER E XCLUSION 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

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

W HAT IS LEFT IF GLUCOSE IS NOT COMING INTO THE CELL ? Enzyme IIA - P HPr - P Enzyme I - P PEP

A BSENCE 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

W HAT WOULD THE MUTANTS BE LIKE ? GenotypePhenotype? Enzyme I - Enzyme II BC - – Enzyme II A -

E FFECT OF MUTATIONS ON A DENYLATE C YCLASE (A BSENCE OF GLUCOSE ) StrainAdenylate Cyclase Wild Type100 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

D IAUXIC GROWTH R ESPONSE Log OD Log [S]

D IAUXIC GROWTH R ESPONSE Lag Phase Ribosome synthesis

D IAUXIC GROWTH R ESPONSE 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

D IAUXIC GROWTH R ESPONSE 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

D IAUXIC GROWTH R ESPONSE 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

D IAUXIC GROWTH R ESPONSE 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

W HAT WOULD THE MUTANTS BE LIKE ? GenotypeBehavior Relative to Diauxic Growth lacZ-, lacY- cya- crp-