Edited Fertech_lect 3_microbial Regulation.pdf

9/3/2015

ITP 321 Lecture Note 3

Microbial Regulation

Ratih Dewanti-Hariyadi Department of Food Science and Technology Bogor Agricultural University

microorganisms

catabolism Source of C Source of C  Source of Energy

E

E

E

E

E

E

E

ATP

Regulation

amino acids organelle/structures biomass

E

E

E

E

E

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+ other  metabolites

E Enzymes E= Enzymes

E

anabolism

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Keys in Metabolisms • In addition to ATP, enzymes (proteins) are keys in  metabolisms • Enzyme synthesis in microbes  is tightly regulated,  microorganisms are economical : ‐

Constitutive Enzymes (Enzim Konstitutif) : always  present in microbial cells at  constant amount ,  independent of metabolic state of microorganisms

‐

Inducible Enzymes (Enzim Terinduksi) :  present/synthesized at different amount  depending on factors such as  metabolic state of  microorganisms, availability of substrates etc

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Strategy of Microbial Regulation • Generally microorganisme regulates the synthesis of inducible protein (enzymes) at trancriptional level • Control is “On/Off” : Off is basic level, 1 transcription per generation • Model is well developed for bacteria

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3 5

double‐stranded DNA 3’

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replication

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5’

5’

3’

newly synthesized DNA transcription mRNA mRNA

regulation n

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Regulation in Bacteria : Coordinate Regulation • Definition : Coordinate regulation is control of synthesis of polycistronic mRNA • Polycistronic mRNA is an mRNA that encodes for several gene products, i.e. enzymes that will work in a sequence in one pathway

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Coordinate Regulation on polycistronic mRNA P

O

a

a

b

c

DNA P=promoter O=operator a,b,c=gene

polycistronic

A X

b

c

B

C

Y

Z

mRNA Protein  (enzymes) V

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Types of Regulation Negative Regulation : a regulation in which under normal  condition an inhibitor (= repressor) is present thus inhibit  transcription; an inducer is needed to allow transcription to  occur Normal

gene A No transcription

DNA repressor

Inducer present inducer

gene A repressor inactive Transcription of A

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Types of Regulation Positive Regulation : a regulation in which under normal condition no transcription occurs; an effector is needed to  activate the promoter and allow transcription to occur Normal

gene B No transcription

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Regulation of Bacterial Catabolism Lac Operon and Lactose Catabolism

• To metabolize lactose To metabolize lactose in the medium, in the medium bacteria need (and  need (and therefore, synthesize) 2 proteins : ‐ Lactose Permease  ‐ produced/encoded for  by gene lac Y ‐ to carry and transport lactose through cytoplasmic  membrane to cytoplasm ‐ ‐galactosidase enzyme  ‐ produced/encoded for by gene lac Z ‐ to convert lactose into glucose and galactose 03/09/2015

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Regulation of Bacterial Catabolism Lac Operon and Lactose Catabolism • Lactose Regulation is studied from mutants Lac–, which do not  produce lactose permease and/or b‐galaktosidase and can not  metabolize lactose  • These mutants may have mutation in their chromosome and/or  their plasmid

F’lac+/lac‐ F’lac‐/lac+

F’lac Y‐ lac Z+ / lac Y+ lac Z‐ F’lac Y+ lac Z‐ / lac Y‐ lac Z+ F’lac Y‐ lac Z+ / lac Y‐ lac Z+ F’lac Y+ lac Z‐ / lac Y+ lac Z‐

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: Lac+ : Lac+ : Lac‐ : Lac‐

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Lac Operon Model (Jacob & Monod) •

Lac Operon that metabolizes lactose consists of 2 components : - Structural genes to transport and cleave lactose, lacY and lacZ (lacA=transasetilase, functions is unknown) - Regulatory genes, i.e. lacI, lacO dan lacP

•

Gene products of lacY and lacZ are encoded for by a polycistronic mRNA. Promoter (early transcription area) of lacY and lacZ is located besides lac O (Operator)

•

lacI ac product p oduct is s a repressor ep esso p protein ote tthat at b binds ds to a u unique que sequence of DNA called operator

•

When repressor binds to operator, no transcription occurs

•

Presence of an inducer will cause inducer to bind the repressor thus allow induction or de-repression

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Regulation of Lac Operon 1. When E. coli Lac+ is grown in medium containing no lactose,  [ gal] & [lac permease] low, 1‐2 molecules/cell.  When  lactose concentration is high , [ gal] & [lac permease  i increases 1000x 1000 2. When lactose is   gal Z added to lactose‐ lac permease Y free medium, the  lac mRNA behavior of E coli  (Lac+) is as follows  + lactosa 3. In Lac+, genes encoding for proteins for lactose catabolism is  inducible; their transcription is induced by an inducer (lactose) 4. Mutans sythesize the two proteins in the presence/absence of  lactose 13

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Negative Regulations of Lac Operon Regulatory gene

i

p

Structural gene

o

Repressor gene

z

y

a

operator

Normal Condition

promoter

i mRNA

p

o

z

y

a

repressor binds to  operator Transcriptions of gene z, y, a do not occur

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Negative Regulations of Lac Operon

i

p

o

z

y

a

mRNA

repressor

In the presence of  Inducer  galactosidase Lac permease

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transasetilase 15

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Positive Regulations of Lac Operon • Fact : When E. coli is grown in a medium containing lactose  and glucose, the bacterium does not metabolize lactose until  all glucose is used up :

-gal, lac permease is low in the presence of inducer, why? • It turns out that Lac operon is also regulated indirectly by  glucose : presence of glucose influence the concentration of glucose : presence of glucose influence the concentration of  cAMP (cyclic‐AMP) in the cell that plays a role in the positive  regulation of Lac operon

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Positive Regulations of Lac Operon • Medium with high [glucose] results in low level of cAMP level within cells • Medium without glucose (starved) increases the cAMP level C source

[cAMP] within cell

Glucose

low

y Glycerol

high g

Lactose

high

Lactose+glucose

low

Lactose+glycerol

high

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Positive Regulations of Lac Operon • E. coli produces CRP (cAMP Receptor Protein) that binds to cAMP • CRP-cAMP has to be associated with the promoter to allow RNA polymerase to bind to lac p • Binding of cAMP-CRP to the Promoter is required for lac Y, Y lac Z synthesis (positive regulation)

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Positive Regulations of Lac Operon i

p

o

z

y

i

p

o

z

y

i

p

o

z

y

mRNA lac Medium synthesis + glucose ‐ lactose  NO CAMP repressor active ‐ glucose ‐ lactose  NO CAMP repressor active

CAMP‐CRP

‐ glucose YES + lactose + lactose CAMP repressor inactive

CAMP‐CRP lactose

i

repressor

o

p

+ glucose + lactose NO CAMP repressor inactive

y

z

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Regulation of Bacterial Anabolism Trp Operon and Tryptophan Synthesis

Trp D

Trp O

chorismic acid

Trp L

Trp E

Trp D

Trp C

Trp B

Trp A

E

D

C

B

A

anthranilic acid

PRA

CDRP

INGP

Trp

INGP : indolglycerolphosphate CDRP: carboxylphenylamino ‐ deoxyrybosil phosphate PRA : phospho ribosyl anthranylate 03/09/2015

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Regulation of Bacterial Anabolism Trp Operon and Tryptophan Synthesis When tryptophan is Wh h i absent or present in low amount in the cell, b i l i h ll bacteria will synthesize Trp

Repressor gene

P

O

L

Trp E

D

C

B

A

transcription transcription occurs

RNA  polymerase Represor inactive [Aporepresor]

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Regulation of Bacterial Anabolism • When Trp is present in large amount in the cell, Trp is not  synthesized

Repressor gene

P

O

RNA  polymerase

L

Trp E

D

C

B

A

No transcription occurs

Represor inactive [Aporepresor]

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Trp Operon regulatory

structural

L P

O

Trp E

D

C

B

A

leader/ attenuator

E

D

C

B

A Tryptophan yp p

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Trp Operon Repressor Gene Trp R

R = aporepresor

• When [Trp] is low        Aporepresor is present, co‐repressor is absent, no  repression, transcription of mRNA trp A  E occurs • When [Trp] is high Aporepressor binds Trp, repressor active, repressor  binds to operator at the L sequence, resulting in  inactive operator and no transcription occurs 03/09/2015

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Trp Operon Wild Type • In the presence of [Trp]

:  transcription stops at bases  number 123 ‐ 150 Trp + aporepresor binds to operator  such that attenuator  forms a stem‐loop configuration

Mutants • In some mutants synthesis of Trp increases to up to 6X • Mutants do synthesize Trp in the presence of Trp • Mutants has base deletion at bases # 123‐150, the bases that  form stem‐loop configuration in wild type 25

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Stem-loop configuration Formed when repressor binds to operator at bases 120‐136 120

110

130

AGCCCGC

AUACC

CUAAUGA

140

GCGGGCU

UUUUU

A A

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U G A GCGGGCU G

AUACC

AGCCCGC

U C

transcription stops UUUUU

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General Scheme of Anabolism Regulation : Feedback Regulation

_

A

_

_

B

C

D

_

inhibition repression

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Feedback Regulation Concerted Feedback Regulation A

B

D

G

C

E

H

_

F

I

Sequential Feedback Regulation _

C

D

B

A _

E

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Feedback Regulation 25%

Cumulative Feed  back Control  A

C

B

G

E

H

F

25% 50%

Isoenzyme Feed  back Control back Control 

D

—

D

G

E

H

Enz A

A

Enz B

B

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Other Example Of Regulation : Environmental Control In Extracellular Enzyme Production Constitutive Enzymes :  Synthesis rate is constant at any medium e.g.  Amylase of B.licheniformis,  protease of B.amyloliquefaciens

Inducible Enzymes y Synthesis rate increases dramatically when substrate is  available  When substrate is not available, enzyme is synthesized at basal  level 03/09/2015

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How Do Bacteria “Sense” The Presence Of Substrate For Extracellular Enzyme?  Substrate for extracellular enzyme is usually  high MW   substance which is too large for bacteria to take up substance  which is too large for bacteria to take up  Bacteria detect the presence of the macromolecule in their  environment because of the constitutive enzymes  The degradation products are E (inducible the inducer E(const.)

E(const.) S

P Low MW

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Examples of Inducible Enzymes

1. Pectinase and Pectic Lyase y of Erwinia carotovora are induced by digalacturonic acid : Pectin  Digalacturonic acid (inducer, when concentration in cell is high it acts as repressor) 2. -amylase of Bacillus stearothermophilus and Bacillus licheniformis are induced by maltotetraose and maltopentaose, respectively : Starch  Maltotetraose (B. stearothermophilus ) Maltopentaose (B. licheniformis) 03/09/2015

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Q and A E. coli grown in lactose : A Will produce ‐galactosidase and lac permease because of  A. Will produce  galactosidase and lac permease because of high cAMP that result in cAMP‐CRP binding to promoter B. Will produce ‐galactosidase and lac permease because  repressor is removed by lactose C. Are regulated both negatively and positively to metabolize  lactose D. A,B correct E. A,B,C correct

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Q and A Regulation of microbial metabolism : A. B. C. D. E.

Shows that microorganisms are economical Only in catabolisms Only in anabolism , A,B correct A,B,C correct

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Q and A A mutant is found to produce tryptophan 10 times  more than the wild type The following may have more than the wild type.  The following may have  occurred : A.The structural genes in the Trp operon is blocked B. The Trp R gene no longer produce aporepressor  C. The promoter lose the sequences for repressor  attachment h D. A,B correct E. B, C correct 03/09/2015

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