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Gene Essentialities of Bacterial Systems

Gene Essentialities of Bacterial Systems. 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 ”. TOMOYA BABA

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Gene Essentialities of Bacterial Systems

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  1. Gene Essentialities of Bacterial Systems 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” TOMOYA BABA Research 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

  2. E. coli Systems Engineering Blueprint Parts Robot Genes (Genome) Organism Proteins Human Systems Biology

  3. Escherichia coli K-12 Genome sequenced at 1997 Revised at 2006 Re-annotation at 2006 Well-characterized Bacteria Science (1997) 32% 14% 54% Riley M., Nuc. Acid. Res., 34, 1-9(2006)

  4. 310 Protein 756 1,484 405 446 691 Ultimate Goal : Comprehensive Understanding of Life E. coli cell Translation mRNA Transcription Transport Secretion Signal-Transduction etc ORF Promoter Enzyme Regulation Chromosome Genome DNA Metabolites Replication Cell-Division Unknown Yes it is, however, life is so complicated systems. Simple Question Which genes are essential for the life systems?

  5. kan Step 1. PCR of Maker gene Step 2. Transformation bla 37℃, LB(Km+) Gene-knockout cell 1, Precise design 2, Complete deletion Chromosome Gene-Knockout Experimental Method l Red recombinase E. coli Wild type cell (BW25113) (30℃) pKD46 (ts-ori) Target Gene Chromosome Datsenko K.A., Wanner B.L., (2000) Proc. Natl. Acad. Sci. 97, 6640.

  6. Step 2. Transformation l Red recombinase FRT FRT Kanamycin resistance gene ( Step 3.Elimination of kan gene) FRT geneC FLP recombinase 21 bp geneA FRT 34 a.a. (102 bp) MIPGIRRPAVRSSTSLGSIGTSKQLQPT+6aa Designing for In-frame Deletion Step 1. PCR of Kanamycin resistance (kan) gene H1 P1 FRT FRT Kanamycin resistance gene H2 P2 H1 H2 geneB (target) geneA geneC Met SD of downstream gene 21 bp

  7. 73 Small ORFs (no longer annotated) 3912 with annotation Named genes 2157 y genes 1755 E: 1819 C: 1655 ORF evidence E; experimental C; computational C: 331 E: 100 Single Gene-Knockout Results Essential • Result • 303 genes (7% of total genes) are essential in E. coli K-12. 303 3985 Keio collection Total targeted ORFs 4288 Baba T., (2006) Mol. Syst. Biol. 2006, 2, 8.

  8. Classification of E. coli K-12 Gene Functions COGs (Clusters of Orthologous Groups of proteins) category Information 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 Essential 303 Total 4288 77 170 15 288 237 15 19 34 • Result • 90 genes (6% of metabolic genes) are essential. 9 133 39 219 11 151 1 253 6 187 6 281 8 355 7 390 83 11 29 144 96 24 5 95 15 448 18 447 26 1214 20 10 0 10 20 % of Essential or Total ORFs Baba T., (2006) Mol. Syst. Biol. 2006, 2, 8.

  9. Growth of Gene-Knockout Mutants OD600 AVE-2SD AVE+2SD 1.2 COGs category ◆; Information storage and processing ■; Cellular processes ▲; Metabolism ●; Poorly characterized ●; No COG assignment • Result • 104 genes (7% of metabolic genes) are conditionally essential in glucose minimum medium. 1.0 0.8 Nucleotide 23 Genes MOPS-0.4% Glucose (48 hrs) AVE+2SD 0.6 Auxotroph Conditionally Essential (Metabolism Genes) 0.4 0.2 AVE-2SD Amino Acid 81 Genes 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 OD600 LB (22 hrs) Baba T., (2006) Mol. Syst. Biol. 2006, 2, 8.

  10. Metabolic Pathway of E. coli (1,163 genes / 1,119 metabolites) • Result • A lot of detour pathways are existing in E. coli metabolic pathway. Glucose Pentose phosphate pathway (Detour pathway) Glycolysis TCA Pyruvate

  11. aGLC G1P DHAP tpiA ADP ADP ADP NAD ADP pfkA pfkB pykA pykF ATP ATP pgm NADH ATP ATP galM G6P F6P FBP GAP BPG 3PG 2PG PEP PYR bGLC fbp fbaA fbaB gapAgapC gpmA gpmB eno ppsA glk pgi pgk 6PG E4P X5P OAA ACoA S7P LAC E4P R5P, S7P FORM Cell Growth of Glycolysis Gene Knockouts in E. coli Glucose 4 Essential Genes (Far from Detour pathway?) TCA Pentose phosphate pathway (Detour pathway) Glucose Minimal Medium Time (h)

  12. Intracellular Expression of E. coli Cells Glycolysis 遺伝子欠失株 rpe Metabolites 134 mRNA Proteins Metabolites Pentose phosphate pathway (Detour pathway) Proteins 68 mRNA 85 Gene-Knockouts Ishii N., Science, 316, 593 (2007)

  13. Robustness of Metabolic Network Pentose phosphate pathway (Detour pathway) Metabolites Regulation of Gene Expression Proteins Metabolic Flux (path-flow) Forward (Wild type) Reverse Keeping Metabolic Balance • Result • E. coli cells regulate the gene expression level for keeping metabolic balance . Ishii N., Science, 316, 593 (2007)

  14. Conclusions • 303 genes (7% of total genes) are essential in E. coli K-12. • 90 genes (6% of metabolic genes) are essential. • 104 genes (7% of metabolic genes) are conditionally essential in glucose minimum medium. • A lot of detour pathways are existing in E. coli metabolic pathway. • 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”

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