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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 + other 
Regulation
metabolites

E Enzymes
E= Enzymes
E E E E E E E
amino acids
organelle/structures
biomass anabolism

survival
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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

3
double‐stranded DNA
5
5
3’

3
replication
5

5’
3

regulation
5’ 3’ newly synthesized DNA

transcription

n
mRNA
mRNA

translation

protein
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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 b c DNA
P=promoter
O=operator
a,b,c=gene
polycistronic mRNA
a b c

Protein 
A B C (enzymes)

X Y Z 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 DNA
repressor
No transcription

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

Effector present

gene B

effector protein
transcription of gene B
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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

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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 Y‐ lac Z+ / lac Y+ lac Z‐ : Lac+

F’lac Y+ lac Z‐ / lac Y‐ lac Z+ : Lac+
F’lac+/lac‐
F’lac Y‐ lac Z+ / lac Y‐ lac Z+ : Lac‐
F’lac‐/lac+ F’lac Y+ lac Z‐ / lac Y+ lac Z‐ : 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 pprotein
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
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Negative Regulations of Lac Operon

Regulatory gene Structural gene

i p o z y a

Repressor gene operator Normal Condition

promoter

i p o z y a

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

repressor
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Negative Regulations of Lac Operon

i p o z y a

mRNA

In the presence of 
repressor
Inducer
 galactosidase

Lac permease
Inducer
(lactose) transasetilase
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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

Medium mRNA lac
i p o z y + glucose synthesis
‐ lactose 
NO
CAMP
repressor active

i p o z y ‐ glucose
CAMP‐CRP ‐ lactose  NO
CAMP
repressor active

i p o z y ‐ glucose
+ lactose
+ lactose YES
CAMP‐CRP
CAMP
lactose repressor inactive
repressor
+ glucose
+ lactose
i p o z y CAMP NO
repressor inactive
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Regulation of Bacterial Anabolism

Trp Operon and Tryptophan Synthesis

Trp D Trp O Trp L Trp E Trp D Trp C Trp B Trp A

E D C B A

chorismic anthranilic PRA CDRP INGP Trp

acid acid

INGP : indolglycerolphosphate
CDRP: carboxylphenylamino ‐ deoxyrybosil phosphate
PRA : phospho ribosyl anthranylate

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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
RNA  transcription occurs
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 L Trp E D C B A

RNA  No transcription occurs
polymerase
Represor inactive
[Aporepresor]

+
Trp present 
[corepressor]

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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

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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

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Stem-loop configuration

Formed when repressor binds to operator at bases 120‐136
110 120 130 140

AUACC AGCCCGC CUAAUGA GCGGGCU UUUUU

A

A U
U G
C A
GCGGGCU
G
AGCCCGC

transcription stops

AUACC 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
D G

A B C E H I

_ F

Sequential Feedback Regulation
_
C D

A B
_
E F
_

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Feedback Regulation

Cumulative Feed  25%
D G
back Control 
A B C E H

F
25%
50%

Isoenzyme Feed 
back Control
back Control  —
D G
Enz A
A B C E H
Enz B
— F

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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

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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
the inducer E (inducible

E(const.)
E(const.)
S P
Low MW
transported in

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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)

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Q and A

E. coli grown in lactose :
Will produce  galactosidase and lac permease because of
A Will produce ‐galactosidase and lac permease because of 
A.
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. Shows that microorganisms are economical
B. Only in catabolisms
C. Only in anabolism
D. ,
A,B correct
E. 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

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