1

Gene Essentialities of Bacterial Systems

TOMOYA BABAResearch Organization of Information and Systems

Transdisciplinary Research Integration Center

National Institute of Genetics, Misima, Japan

Joint work with: Barry L. Wanner (Purdue Univ., US)

Masaru Tomita (Keio Univ., Japan)

Hirotada Mori (NAIST, Japan)

ISSSB’11, Nov. 16th 2011, Shonan Village Centre, Japan

Introduction of following two talks:

Takehide Soh “Predicting Gene Knockout Effects by Minimal Pathway Enumeration”

Takeyuki Tamura “Predicting essential genes via impact degree on metabolic networks”

2

Genes (Genome)Genes (Genome) ProteinsProteins

RobotRobotBlueprintBlueprint PartsParts

Systems Engineering

OrganismOrganism

HumanHuman

E. coliE. coli

Systems Biology

3

Escherichia coli K-12

Genome sequenced at 1997

Revised at 2006

Re-annotation at 2006

Well-characterized Bacteria

Science (1997)

Riley M., Nuc. Acid. Res., 34, 1-9 (2006)

54%

32%

14%

4

Ultimate Goal : Comprehensive Understanding of Life

ORF

mRNA

Transcription

E. coli cellTranslation

MetabolitesChromosome

Genome DNA

Regulation

ReplicationCell-Division

TransportSecretionSignal-Transduction etc

EnzymePromoter

Unknown

Yes it is, however, life is so complicated systems.

Simple Question

Which genes are essential for the life systems?

Protein

1,484

756

446

405

310

691

5

Gene-Knockout Experimental Method

Red recombinase

Chromosome

Target Gene

Step 2. Transformation

pKD46

(ts-ori)

kanStep 1. PCR of Maker gene

E. coli

Wild type cell

(BW25113)

(30 )℃

bla

37 , LB(Km℃ +)

Chromosome

Gene-knockout cell

1, Precise design

2, Complete deletion

Datsenko K.A., Wanner B.L., (2000) Proc. Natl. Acad. Sci. 97, 6640.

6

P1

P2

H1

H2

Kanamycin resistance gene

FRTFRT FRTFRT

21 bp

geneB (target)geneAgeneA geneCgeneC

H1 H2

Met

Step 1. PCR of Kanamycin resistance (kan) gene

Designing for In-frame Deletion

FRT

geneA

geneC

FRT21 bp

34 a.a. (102 bp)MIPGIRRPAVRSSTSLGSIGTSKQLQPT+6aa

( Step 3. Elimination of kan gene)FLP recombinase

FRTFRT FRTFRT

Kanamycin resistance gene

Step 2. Transformation Red recombinase

SD of downstream geneSD of downstream gene

7

Total targeted ORFs4288

3985Keio collection

Essential

3912with annotation

73 Small ORFs (no longer annotated)

303

Single Gene-Knockout Results

C: 1655

E: 100C: 331

E: 1819

Named genes

2157y genes

1755

ORF evidenceE; experimentalC; computational

Baba T., (2006) Mol. Syst. Biol. 2006, 2, 8.

Result

• 303 genes (7% of total genes) are essential in E. coli K-12.

8

Classification of E. coli K-12 Gene Functions

6

15

77

15

170

288

237

39

11

219

151

9 133

15 448

6 187

18 447

26 1214

% of Essential or Total ORFs

Essential303

COGs (Clusters of Orthologous Groups of proteins) categoryInformation storage and processing ( 107 / 695 )

J Translation, ribosomal structure and biogenesis

K Transcription

L DNA replication, recombination and repair

Cellular processes ( 85 / 977 )

D Cell division and chromosome partitioning

O Posttranslational modification, protein turnover, chaperones

M Cell envelope biogenesis, outer membrane

N Cell motility and secretion

P Inorganic ion transport and metabolism

T Signal transduction mechanisms

Metabolism ( 90 / 1,444)

C Energy production and conversion

G Carbohydrate transport and metabolism

E Amino acid transport and metabolism

F Nucleotide transport and metabolism

H Coenzyme metabolism

I Lipid metabolism

Q Secondary metabolites biosynthesis, transport and catabolism

Poorly characterized ( 59 / 2,109)

R General function prediction only

S, U or V Function unknown

No COG assignment

2531

5 95

24

7

11

390

83

8 355

29 144

281

19 34

96

Total4288

10 10 20020

Baba T., (2006) Mol. Syst. Biol. 2006, 2, 8.

Result

2. 90 genes (6% of metabolic genes) are essential.

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Growth of Gene-Knockout Mutants

0.0 0.2 0.4 0.6 0.8 1.0 1.2LB (22 hrs) OD600

AVE-2SD

0.0

0.2

0.4

0.6

0.8

1.0

1.2

MO

PS-

0.4%

Glu

cose

(48

hrs

)

OD600 AVE+2SDAVE-2SD

AVE+2SD

COGs category

◆; Information storage and processing

■; Cellular processes

▲; Metabolism

●; Poorly characterized

●; No COG assignment

Amino Acid81 Genes

Auxotroph

Conditionally Essential(Metabolism Genes)

Nucleotide23 Genes

Baba T., (2006) Mol. Syst. Biol. 2006, 2, 8.

Result

3. 104 genes (7% of metabolic genes) are conditionally essential in glucose minimum medium.

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Metabolic Pathway of E. coli (1,163 genes / 1,119 metabolites)

Glucose

TCATCA

GlycolysisPentose phosphate

pathway(Detour pathway)

Pyruvate

Result

4. A lot of detour pathways are existing in E. coli metabolic pathway.

110.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 6 12 18 24 30 36 42 48 Time (h)

Gro

wth

(O

.D.=

600n

m)

wild

galM

glk

pgm

pgi

pfkA

pfkB

fbp

fbaB

tpiA

gapC

gpmB

pykA

pykF

ppsA

Glucose

Minimal Medium

GLC G6P FBP

ATPADP

GAP

DHAPNAD

NADH

BPG

ADPATP

3PG 2PG PEP PYR

6PG ACoA

FORM

LAC

F6P

OAA

pfkApfkB

fbaAfbaB

pgi

tpiA

gpmAgpmB

ppsA

ATPADP

gapA gapC

pgk enofbp

G1P

pgmATPADP

glk

GLC

galMpykApykF

E4PS7P

X5PE4P

R5P, S7P4 Essential Genes

(Far from Detour pathway?)Pentose phosphate

pathway(Detour pathway)

TCA

Cell Growth of Glycolysis Gene Knockouts in E. coli

Glucose

Time (h)

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Intracellular Expression of E. coli Cells

mRNA Proteins 　Metabolites

遺伝子欠失株

rpe

Ishii N., Science, 316, 593 (2007)

Metabolites

134

Proteins

68

mRNA

85

Gene-Knockouts

Glycolysis

Pentose phosphate pathway

(Detour pathway)

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Robustness of Metabolic Network

Metabolites

Proteins

Metabolic Flux (path-flow)

Forward

(Wild type)

ReverseKeeping Metabolic

Balance

Regulation of Gene Expression

Pentose phosphate pathway

(Detour pathway)

Ishii N., Science, 316, 593 (2007)

Result

5. E. coli cells regulate the gene expression level for keeping metabolic balance .

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Conclusions

1. 303 genes (7% of total genes) are essential in E. coli K-12.

2. 90 genes (6% of metabolic genes) are essential.

3. 104 genes (7% of metabolic genes) are conditionally essential in glucose minimum medium.

4. A lot of detour pathways are existing in E. coli metabolic pathway.

5. E. coli cells regulate the gene expression level for keeping metabolic balance .

Further Question

What is meaning of metabolic essential and conditional essential genes in E. coli?

Are the detour pathway lengths (steps) related for them?

→ Takehide Soh “Predicting Gene Knockout Effects by Minimal Pathway Enumeration”

Are there another type or meaning of metabolic essential genes in E. coli?

→ Takeyuki Tamura “Predicting essential genes via impact degree on metabolic networks”