The logic of bacterial regulatory networks

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

β

(10)

σ



(11)

(12)

(13)

(14)

(15)

β

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(A) Random network (B) Scale-free network

(25)

(26)

DNA Systems

Devices

Parts

GTGGATTTCAGTTAATCAATTGGTTAATCTTTCAGGACCACCTAAGCAAATGCTAAAGTGGCAGAT

-10

-35 -35 -10

Pr1 Pr2

+1 +1

UAS UAS

IHF xylR

Pr XylR

XylS upper CH₃

CH₃

CH₃ O OH

meta TCA XylR

CH₃ CH₃

CH₃ O

OH TCA

XylR upper

XylS meta

NH₂ HO

OCH₃

OH CH₃

Biolo gic al Di gita l

(27)

(28)

Site Habitat

Niche Community

Population Cell Genome Operon/pathway

Gene/enzyme

Complexity

(29)

(30)

(31)

ATG……TAA

P

UP element -35 Ext -10Dis + 1

RBS Ter

Activation

Repression

-24 -12 +

1

Transcription Translation

Degradation Degradation

Sigma70 family dependent promoters

Sigma54 dependent promoters Small molecule binding

Oligomerization Protein-protein interaction

Post-translational modification Translational

regulation

(I) (II) (III)

(32)

(33)

Activation

Y

₁

Y

₂

Y

_N

… X

Single Input Module (SIM)

X Y

Feedback Loop

Z W

X Y

Bifan

X Y Z

Feed Forward Loop (FFL)

Multiple Input Module (MIM)

Y

₂

Y

₃

… Y

_N

X

₁

Y

₁

X

₂

X

₃

X

_N

(A) (B) (C)

(D) (E) (F)

X Y Z

S

_X

S

_Y

crp araC araBAD

cAMP Arabinose Type-1 coherent FFL

: Repression

(34)

(35)

(36)

(37)

(38)

(39)

(A) Amplifier (YES)

(B) NOT

(C) AND

P

P -35 -10

-35 -10 B A

P

-35 -10 A

B

A P 0 0 P 1 1

A

A P

A P B

A P 0 1 1 0

A B P 0 0 0 1 0 0 0 1 0 1 1 1

(D) OR

P

-35 -10 A

A P

B B

A B P 0 0 0 1 0 1 0 1 1 1 1 1

RNAP Symbology:

-35 -10 P

Promoter TF binding site

B A

TFs Cooperative interaction Degradation

(40)

(41)

(A) NOT AND (NAND)

P

-35 -10 A B

A P B

A B P 0 0 1 1 0 1 0 1 1 1 1 0 (B) AND NOT (ANDN)

P

-35 -10 A B

A B P 0 0 0 1 0 1 0 1 0 1 1 0 A P

B

(42)

(C) XOR

P’ P

-35 -10 B -35

-10 A A P

B

P P’

-35 -10 B -35

-10

B A (D) XNOR

A P B

(A) NOT OR (NOR)

P

A B P 0 0 1 1 0 0 0 1 0 1 1 0 -35 -10

A B A P

B

(B) OR NOT (ORN)

P

-35 -10 B A

A B P 0 0 1 1 0 1 0 1 0 1 1 1 A P

B

A B P 0 0 0 1 0 1 0 1 1 1 1 0

A B P 0 0 1 1 0 0 0 1 0 1 1 1

(43)

(44)

(45)

(46)

(47)

(48)

(49)

(50)



(51)

β

(52)



 λ

α

β

(53)

Δ Δ

λ λ

λ

Δ

(54)

(

(55)



ori p15A Km

T1 gfplva

T0 pRV2

mcherry P_lexA

-10

-35 +1

-10 -35

+1 P_recA

lexO lexO lexO

lexO (A)

ori pBR322 Ap

Ter lexA

pTrc-lexA lacI^q

P_trc (B)

(56)

(57)

xylR Pr

ori pUC Km

E B H

T7ter Pr

Sm oriT oriR6K

T0 T1

Tn7R Tn7LE H Gm

ori pUC Km

E B H

T7ter lacZ

Sm oriT oriR6K

T0 T1

Tn7R Tn7L

B Gm

Pr H

E

Tn7R

Tn7L Pr Gm

glmS PP5408

(I) ^(II)

(III) P. putida genome

P. putida genome

xylR’-’lacZ xylR’-’lacZ

xylR’-’lacZ

pUJ9 pNBTN7

pUJ9-Pr pTN7-PrZ

(58)

β

(59)

β

( ) ( )

θ θ

( ) ( )

θ θ

(60)

( ) [ ( ) ( )]

θ θ

⁄

θ θ

(61)

θ

(62)

⁄ ( ) ( )

⁄ ( )

( ) ( ) ( )

⁄

⁄ ( ) ⁄

⁄ ⁄

( ) ⁄

⁄

⁄ ( ) ⁄

⁄

(63)

(64)

(65)

(66)

(67)

(68)

(69)

SOS Promoter RecA*

LexA

Nalidixic acid

LacI

^q

(A) (B)

IPTG

SOS Promoter LexA RecA*

Nalidixic acid

(C)

Nalidixic acid

IPTG

ssDNA RecA*

LacI

^q

GFP LexA

(D)

Nalidixic acid

IPTG

GFP

0

1 0 1

0

(E)

1

(70)



(71)

(A) (B)

(C) (D)

(72)

(C) (D)

ORN-P

_lexA

ORN-P

_lexA

(A) (B)

ORN-P

_recA

ORN-P

_recA

(73)

(A) ITPG (B) Nalidixic acid

(74)

ORN-P_lexA ORN-P_recA

(A) (B)

(75)

ORN-P

_lexA

ORN-P

_recA

(A) 75 min (D) 75 min

(B) 120 min (E) 120 min

(C) 165 min (F) 165 min

(76)

(77)

(78)

(79)

XylR

_a

XylR

_i

XylS

_i

XylS

_h

meta operon upper operon

Pm

Pu

XylS

_a

CH

₃

CH

₃

CH

₃

COOH

m-xylene 3MBz

Meta pathway TCA Upper pathway

Metabolism Gene regulation

xylS xylR

Pr

Ps

(80)

(81)

σ σ

(82)

(83)

σ

NOT A O

A O 0 1 1 0

A O B

AND

A B O 0 0 0 1 0 0 0 1 0 1 1 1

A O B

OR

A B O 0 0 0 1 0 1 0 1 1 1 1 1 (A)

XylR m-xyl

(C)

XylR_a

XylR_i

XylSh

Pr xylR xylS

(B)

m-xyl

XylS_i Ps2

upper

XylS

XylS_h XylSa

CH3

COOH 3MBz

CH₃

CH3

meta

TCA Ps1

(84)

(85)

σ

max k⁰+k¹/g k¹/g k⁰/g θ

zero s1 s5 s9 s11 s12 max

k⁰/g θ

zero s1 s5 s9 s11 s12 max

k⁰/g θ

zero s1 s5 s6

max k⁰+k¹/g θ² k⁰/g θ¹

zero s1 s5 s6

max k⁰/g θ

zero s1 s5 s6 max

k⁰+k¹/g k¹/g k⁰/g θ

zero s1 s5 s6

XylR

zero s1 s5 s9 s11 s12 XylS

max k⁰/g θ

zero s1 s5 s9 s11 s12 upper

rmeta XylR

XylS

upper

rmeta (A)

(B)

stead-state

(86)

θ θ

θ XylR

_a

upper

XylS

_h

X

?

(A)

Single-input module like

zero s1 s5 s9 s11 s12 XylS max

k⁰/g θ

zero s1 s5 s9 s11 s12 upper (B)

(87)

(88)

σ σ

(C) (D)

(A)

Km oriT oriV trfA

T1 T0

pSEVA226 luxCDABE

SD

-24/-12

UAS -24/-12

-35/-10 XylS sites

Pu Ps

Pm UAS

P

(B) Control Induced

(89)

(A) XylR

_i

XylR

_a

upper

m-xyl

3MBz

TCA

XylS

_i

XylS

_a

meta XylS

_h

XylR

_a

XylS

_h

meta XylS

_a

XylR

_a

XylS

_h

meta meta XylS

_a

Wild type

No XylS activation No XylS hyper-expression

(B)

max k

⁰

+k

¹

/g k

¹

/g k

⁰

/g θ zero

s1 s5 s9 s11 s12

Wild type max

k

⁰

+k

¹

/g k

¹

/g k

⁰

/g θ

zero s1 s5 s7 s9 s10 No XylS activation max

k

⁰

+k

¹

/g k

¹

/g k

⁰

/g θ

zero s1 s5 s7 s9 s10 No XylS hyper-

expression

(C)

(90)

(91)

Wild type (A)

XylR

_a

XylS

_h

meta XylS

_a

CH₃ HO

3MBA ^CH³

O O^-

upper

3MBz

XylR

_a

XylS

_h

meta XylS

_a

CH₃ O

O^-

3MBz

XylR

_a

XylS

_h

meta XylS

_a

CH₃ CH₃

o-xyl

(B) (C)

Cnt

o-xyl m-xyl

40 36 32 28 24 20 16

Time (h)

0 0.5 1 1.5 2 2.5

(D) Pu-lux fusion (E)

No XylS hyper-expression

No XylS activation

(92)

(93)

Cnt

o-xyl

m-xyl

0 0.5 1 1.5 2

Time (h)

240 200 160 120 80

2.5 40

(A) (B)

(C) (D)

z

(94)

(95)

σ

(96)

(97)

σ σ σ σ

(98)



σ

(99)

β

β β

IHF -70

UASd

UASp -10

-35 +1

-10

-35 +1 0

1 2 3

0 90 180 270 360

OD600

Time (min)

(A) (B)

(I)

(II)

(III) (IV)

0 20 40 60 80 100 120

- / - IHF/ - - /XylR IHF/XylR

Relative protmoer activity

0 20 40 60 80 100 120

(C) (D)

(E) (F)

0 20 40 60 80 100 120

Pr1

Pr2

(100)



β

(101)

β

0 200 400 600 800 1000 1200

1 3 5 7 9 11

Time (h) wt

ihf

0 2 4 6 8 10

OD600

Time (h)

(A) 1 (B)

0.1

0.01

β-galactosidase activity (M.U.)

(102)

β

(A) Type-4 incoherent FFL (I4-FFL) X

Y

Z

S_X

S_Y

IHF

XylR

Pu

m-xyl Stationary

Phase (B) TOL I4-FFL motif

-1 -0.5 0 0.5 1 1.5 2 2.5

0 0.5 1

-1 -0.5 0 0.5 1 1.5 2 2.5

0 0.5 1

-1 -0.5 0 0.5 1 1.5 2 2.5

0 0.5 1

Time IHF

XylR

Pu (C)

(103)

β

0 200 400 600 800

lag early exp mid exp latter exp

β-galactosidase activity(M.U.)

Suc Glu

(A) (B)

0 2 4 6 8 10

Succinate

Glucose 1

0.1

0.01 Time (h)

OD600

lag early exp

mid exp

latter exp

(104)

β



β

(105)

β

σ

0 500 1000 1500 2000 2500 3000 3500

wt crc

β-Galactosidase activity (M.U.)

Suc Glu

(106)

(107)

(108)

(109)



(110)

(111)

   

 



(112)

  Type I coherent FFL

X

Y

Z S_X

S_Y

Metabolic Amplifier Motif X

Y

Z S_X

S_Y

W XylR

XylS

meta m-xyl

3MB

upper crp

araC

araBAD cAMP Arabinose

(A) (B)

(113)

(114)

β (A) I4-FFL stabilization by Crc

IHF

Pu

XylR

Stationary Phase

Crc m-xyl

Glu

^Prote

in levels

OD₆₀₀ XylR (B)

(C) OD₆₀₀

XylR prodution rate

IHF Crc

(115)

(116)

(117)

(118)

(119)

(120)

(121)

(122)

(123)

σ σ

(124)

σ

(125)

σ

(126)

σ

σ σ

(127)

(128)

(129)

(130)

(131)

σ

(132)

(133)

(134)

σ

(135)

(136)

(137)

(138)

(139)

(140)

(141)

(142)

(143)

(144)

(145)