Jizhong Zhou, Dorothea K. Thompson, Ying Xu

1. Microbial functional genomics Jizhong Zhou, Dorothea K. Thompson, Ying Xu Prepared by: Merced M. Gutierrez Nano Biomaterials Science Laboratory Supervisor: Prof. Sung Chul Yoon Gyeongsang National University

2. Chapter 11 The Functional Genomics of Model Organisms: Addressing Old Questions from a New Perspective Dorothea K. Thompson and Jizhong Zhou

3. Table of Contents 11.1 Introduction 11.2 Escherichia coli: A Model Eubacterium 11.2.1 E. coli genome 11.2.2 E. coli transcriptomics 11.2.3 E. coli proteomics 11.2.4 Modeling E. coli metabolism: in silico metabolomics 11.3 Bacillus subtilis: A Paradigm for Gram-Positive Bacteria 11.3.1 B. subtilis genome 11.3.2 B. subtilis transcriptomics 11.3.3 B. subtilis proteomics 11.4 Saccharomycescerevisiae: A Model for Higher Eukaryotes 11.4.1 Yeast genome 11.4.2 Yeast transcriptomics 11.4.3 Yeast proteomics 11.4.4 Yeast interactome: mapping protein–protein interaction 11.5 Comparative Genomics of Model Eukaryotic Organisms

4. Introduction • E. coli , Bacillus subtilis and S. cerevisiaeserves as a model organisms because of their reduced structural and functional complexity and their intrinsic advantages as experimental systems. • In this chapter, we focused on the impact of structural and functional genomics on elucidating the dynamics of the transcriptome, proteome, metabolome and interactome of the well-studied model organisms.

5. Model Organisms: The Genome Sequence E.coli B. subtilis S. cerevisiae Dna Microarray TRANSCRIPTOME Two-hybrid system Bioinformatics Phage display METABOLOME INTERACTOME Mass spectrometry Computer modeling Fig 11.1 Elucidation of the cellular domains of model organisms using a functional genomics approach and comprehensive technologies. PROTEOME Protein chips 2D-PAGE Mass Spectrometry

6. Escherichia coli: A MODEL EUBACTERIUM • best-characterized free-living, single-celled organism, served as a biological model for cellular processes. • 38% of the 4,288 protein-coding genes ( no attributed biological role) and 1,853 previously described (Blatter et.al, 1997) DNA replication and repair, DNA transcription, Metabolic pathways, Adaptive stress responses, Signal transduction, Genetic regulation

7. lyst operon Transcriptional units unit Transcription regulator for encoding the aromatic degradation Operon for the degradation of aromatic compounds 6 newly proposed genes Pathway for degradation of aromatic compounds valZ ileY asnW lysZ lysY lysQ Escherichia coli: GENOME Structural Analysis - Genome-Wide Parallel Functional Analysis Bioinformatic analysis- Structural and Regulatory Components of Various Biochemical pathways or Cellular Machineries

8. E. Coli Transcriptomics A. The Heat Shock Response (i) Correlation of Gene Expression & Function * alternate sigma (δ) factor rpoH (δ32) and rpoE(δE) - heat shock proteins, homeostatic mechanism exhibited by living cells when exposed to suboptimal elevated temperature (ii) Connection between Gene Expression & Physiological State * When complexed with the core RNA polymerase, the E. coli δ32 transcription factor permits the transcription machinery to initiate heat shock-regulated promoters fro both steady state and stress-activated levels of heat shocks gene expression. .

9. E. Coli Transcriptomics A. The Heat Shock Response (iii) Technological Advantages * DNA microarray technology accurately detect alternatives in bacterial transcript abundance & illustrated how functional genomics allow well-characterized cellular processes to be examined from a new & global perspective. Transformation Large scale culture Separation Mass spectrometry An integrated workflow

10. E. Coli Transcriptomics B. Transcriptome Analysis of Cellular Metabolism and Growth (i) Correlation of Gene Expression & Function * trpoperon ( trpE, trpD, trpC, trpBandtrpA) - enzymes required for the conversion of chorismate, a branch-point intermediate in the aromatic amino acid pathway to tryptophan. (ii) Importance of Transcriptional Regulation * Transcription of the trpoperon is governed by repression control via the repressor protein TrpR and an entirely different mode of regulation termed transcriptional attenuation.

11. E. Coli Transcriptomics B. Transcriptome Analysis of Cellular Metabolism and Growth (iii) Technological advantages 1. DNA microarray technology monitor global change in mRNA abundance connected with tryptophan biosynthesis, transport and regulation. trp– tryptophan biosynthesis aroH – aromatic amino acid biosynthesis mtr- tyrptophan-specific permease trpR- tryptophan repressor regulon

12. E. Coli Transcriptomics B. Transcriptome Analysis of Cellular Metabolism and Growth (iii) Technological advantages 2. DNA microarray technology provides genomic expression for cell growth particularly in the differences in rRNA and tRNA. 1. translational apparatus 2. nitrogen metabolism 3. amino acid biosynthesis 4. biosynthesis of vitamins, cofactors, prosthetic groups and carriers 5. nucleotide biosynthesis 6. fatty acid biosynthesis and degradation 7. carbon and energy metabolism 8. cellular processes and global regulators

13. E. Coli Transcriptomics B. Transcriptome Analysis of Cellular Metabolism and Growth (iii) Technological advantages 3. DNA microarray technology provides genomic expression for cell growth particularly in the differences protein synthesis and ribosome abundance. ilv GMEDA operon– isoleucine and valine synthesis leuABCDoperon – leucine synthesis trpEDCBAoperon- tryptophan synthesis

14. E. Coli Transcriptomics B. Transcriptome Analysis of Cellular Metabolism and Growth (iii) Technological advantages 4. DNA microarray technology provides genomic expression for cell growth particularly in the number of carbon and energy metabolism genes. dld– D-lactate utilization tpoS – regulation of poxB expression poxB- acetate formation aceA, aceB, gltA, lcd, & mdh- acetate utilization uspA- (universal stress protein) coupling of glucose and acetate cometabolism The step required in a microarray experiment

15. E. Coli Transcriptomics B. The NtrCregulon (i) Correlation of Gene Expression & Function * NTr(nitrogen-regulated) system & NtrC(nitrogen regulatory protein) - perform a cellular function in response to information processes about the physiological state of the cell ndNtrC transcriptional genes response to external nitrogen limitation. (ii) Connection between Gene Expression & Physiological States * NtrC protein activates transcription of sigma 54-dependent genes and Nac (nitrogen assimilation control) protein serves as an adapter to activate transcription of sigma 70-dependent genes.

16. E. Coli Transcriptomics B. The NtrCregulon (iii) Technological advantages * DNA microarray technology shows operons specified ATP-binding cassette transporters. potFGHI– putrescine dppABCDF – dipeptides oppABCDF- oligopeptides nupC- secondary ion-coupled transporter for nucleosides cycA- D-alaline/D-serine/glycine

17. E. Coli Proteomics A. Protein identification and analysis Proteome analysis concern the authencity of the ORF ( sequence annotation) and physical properties (isoelectric point (pI) and molecular mass) * vivo protein abundance , post translational modification and proteolysis B. Proteome-wide differential display of protein in various conditions 2-DE - 60% of the 223 identified loci encoded proteins 364 2-DE spots using - 18% of 2DE-spots, represented amino acid (N) terminalisoforms in a narrow pI range 4 to 7 Edman sequence analysis molecular mass 10 to 100KDa

18. E. Coli Proteomics C. Protein-protein interaction Tryptophanase (TnaA), which catalyzes both the degradation and synthesis of tryptophan. b. Galactose –binding protein (MglB), which is involved in the transport of galatose into the cell. c. Starvation- inducible protein (Dps), which froms stable complexes with DNA and thus protects DNA from oxidative damage.

19. E. Coli Proteomics C. Protein-protein interaction Figure 11.2 : Proteomic analysis of the outer membrane of E coli. • Proteins in carbonate-treated membranes of E. coli were separated by two-dimensional gel electrophoresis and then identified using mass spectrometry. (b) E. coli cells grown under conditions of iron limitation were subjected to carbonate treatment and then separated by two-dimensional gel electrophoresis.

20. Modeling E. Coli Metabolism: In silicoMetabolomics • Functional analysis by coresponses : Metabolite analysis Figure 11.3 Reconstruction of microbial metabolic networks from annotated genome sequence, biochemical data, and physiology data.

21. 77 ABC transporter – gene duplication 4.2Mb resembles E. coli (25%) in terms of size 43 temperature shock/ stress proteins 4% codes for multi functional enzymes Helix-turn-helix (HTH) family transcription factor 15of 19 LysR family 34 response regulators 5 of 12 LacI family 18 of 20 GntR family 37 sensor kinases 10 of 11 AraC family Two-component regulatory system B. subtilis : GENOME Structural Analysis – Genome Mapping Sequence analysis Physiological analysis – Adaptation of B. subtilis

22. B. subtilis Transcriptomics A. Global characterization of Heat Shocks (i) Correlation of Gene Expression & Function * alternate sigma transcription factor - δBstress regulon, HrcA, heat-inducible genes and CtsR, transcriptional regulators (ii) Connection between Gene Expression & Physiological State * transcription of many general stress genes occurs at a basal level from vegetative δA –dependent promoters, but is increased dramatically in a δB -dependent manner in response to stress and starvation.

23. B. subtilis Transcriptomics B. Two-Component Regulatory Systems (a) DegS/DegU two component system - controls exoprotease production, competence development and motility (b) ComP/ComA two component system - cell density signal activator (i) PhoP/PhoR two component system - induction of the Pho regulon in response to phosphate starvation

24. B. subtilis Transcriptomics C. DNA Microarray Analysis (a) aprE, nprE and ispA - 116 target gene DegU overproduction (b) bpr, yukl, ycdA and murD - newly DegUoperon (c.) YdbG/YdbF two component system - overproduction of YdbFchemostaxis (d.) YufL/ YufM two component system - regulating competence controlled by ComK (e.) YvrG/YvrH two component system - cell membrane related proteins

25. Stages of Sporulation in Bacillus subtilis Germination Stage 0 Stage II1 Stage II3 Stage III Stage IV Stage V Stage VI Stage VII Asymmetric septation Engulfment Prespore protoplast Cortexformation Coatformation Maturation

26. Stages of Sporulation in Bacillus subtilis How is the process regulated? Stage 0 Stage II1 Stage II3 Stage III Stage IV Stage V Stage VI Stage VII spo0 spoII spoIII spoIV spoV spoVI

27. Stages of Sporulation in Bacillus subtilis Golden age of Bacillus genetics • Identify spo genes • Clone and characterize spo genes • Determine their interdependencies Stage 0 Stage II1 Stage II3 Stage III Stage IV Stage V Stage VI Stage VII spo0 spoII spoIII spoIV spoV spoVI

28. Stages of Sporulation in Bacillus subtilis • Care in using reporter gene (lacZ) • How is regulation exerted? • Dependence of spoIIA expression on other genes Stage 0 Stage II1 Stage II3 Stage III Stage IV Stage V Stage VI Stage VII spo0 spoII spoIII spoIV spoV spoVI spoIIA

29. Global Gene Expression During Growth and Sporulation of B. subtilis Figure 11.4 Hierarchical cluster analysis of microarray-derived transcription profiles of 586 Bacillus subtilis genes whose expression levels depended on Spo0A. genes whose expression was dependent on Spo0A but not on sF; (II) genes whose expression was inhibited by Spo0A; and (III) genes whose expression was under the control of sF or some downstream transcription factor in sporulation.

30. B. subtilis Proteomics A. The Extracellular Proteome Two-dimensional map of cytosolic proteins of B. subtilis indicated that most abundant proteins perfomed mainly housekeeping functions in glycolysis, TCA cycle, amino acid biosynthesis, translation and protein quality control. B. Heat Stress Proteome during Sporulation 60 stress proteins were synthesized de novo and/or overexpressed in B. subtilis during sporulation concurrent to acquired thermotolerance in spores.

31. Saccharomycescerevisiae :A MODEL FOR HIGHER EUKARYOTES A yeast cells is about 4-7mm large, the ”eyes” at the bottom are bud scars • simple unicellular eukaryote, valuable for studying the basic mechanisms of cell life particularly in human genetic diseases. Very manipulable, capable of being easily deleted, mutated, reintroduced, overexpressed, tagged and comprehensively analyzed.

32. Yeast GENOME • The S. cerevisiae nuclear genome has 16 chromosomes • 6, 274 potential ORFs • Genetical genomes (1) a much lower gene density and largely untranscribeed DNA (2) the presence of several apparent pseudogenes and a 15-kbp redundant sequence (3) the absence of genes essential for vegetative growth

33. Yeast Transcriptomics • Genetic Basis of Metal Homeostasis (i) Correlation of Gene Expression & Function * Zap1p transcription factor - senses the status of cellular zinc levels and stimulates expression of its target genes in response to zinc limitation be examined from a new & global perspective.

34. Yeast Transcriptomics • Genetic Basis of Metal Homeostasis (ii) Connection between Gene Expression and Physiological State * copper ions 3 functional cofactors key enzymes (1) an active cytochromeoxidase complex, which enables yeast cells to grow on nonfermentable carbon sources (2) the copper-metalloenzyme superoxide dismutase, which protects the cell against the detrimental effects of reactive superoxide dismutase (3) the copper-metalloenzyme Fet3, a ferro-oxidase that is critical for fe(II) uptake

35. Yeast Transcriptomics B. Functional Genomics of Metabolic Reprogramming (i) Connection between Gene Expression and Physiological State * Genome-wide transcription patterns under aerobic and anaerobic 219 genes displayed a greater 3-fold higher transcription level, while 140 genes showed a greater than 3-fold increase in transcript level in response to anaerobosis. 2) 34 genes whose expression at the diauxin shift is dependent on a functional Cat8p, a zinc cluster-containing transcriptional activator that is essential for growth on nonfermentable carbon source.

36. Yeast Transcriptomics C. Nucleosome Remodeling Complexes in Gene Regulation (i) Connection between Gene Expression and Physiological State * Nucleosomal inhibition of gene transcription can occur at the stages of transcription factor binding, preinitiation complex formation, or transcription elongation.

37. Yeast Transcriptomics C. Nucleosome Remodeling Complexes in Gene Regulation (i) Connection between Gene Expression and Physiological State * Hierarchical cluster analysis of whole genome microarray data from a study assessing the genome-wide effects of Rvb inactivation. a. Green and red indicates a decrease or an increase in mRNA abundance b. Rvb1p and Rvb2p are essential components of a chromatin remodeling complex that regulates transcription of over 5% of yeast genes.

38. Yeast Proteomics A. The Extracellular Proteome 2,774 yeast proteins was determined by high-throughput immunolocalization of epitope-tagged gene products

39. Yeast Proteomics B. Proteome Microarray A.) Protein chip fabrication and analysis of yeast protein kinases. A protein chips were constructed by pouring PDMS over an acrylic mold, curing, and mounting the wells on a glass slides. The surface of the wells was then modified, followed by protein attachment.

40. Yeast Proteomics B. Proteome Microarray B.) Protein chip fabrication and analysis of yeast protein kinases. Kinase activities were detected using protein chips. Images of phosphorylation signals in the presence of 12 substrates are shown.

41. Yeast Interactome: Mapping Protein-Protein Interactions A. Genomic Two-hybrid Screens A.) GAL4, protein, a transcriptional activator that control expression of genes involved in galatose utilization. It contain two separable and functionally distinct domains that are both essential for activation of target gene expression: 1. N-terminal domain –responsible for specific DNA-binding activity 2. C-terminal domain–contains acidic regions that are required for activation of transcription.

42. Yeast Interactome: Mapping Protein-Protein Interactions A. Genomic Two-hybrid Screens B.) Ydr016 protein, has potential involvement in spindle pole body function. Three hypothetical proteins, Ydr016c, Ykr083c and Ylr423c, are implicated in spindle pole body function based on their interactions with proteins of known function.

43. Yeast Interactome: Mapping Protein-Protein Interactions B. Visualizing Protein-Protein Interaction Network in silicos 1.) 63% of the assembled connections occurring between proteins assigned a common functional role and 76% occuring between proteins residing in the same subcellular compartment. 2.) 21 proteins involved in membrane fusion and the 141 proteins involved in vesicular transport.

44. Yeast Interactome: Mapping Protein-Protein Interactions B. Visualizing Protein-Protein Interaction Network in silicos Protein–protein interaction map for Cdc28 and Fkh1/2 complexes involved in signaling pathways. Gray dotted arrows indicate new interactions determined by high-throughput mass spectrometric protein complex identification (HMS-PCI).

45. Yeast Interactome: Mapping Protein-Protein Interactions C. Analysis of Yeast Multiprotein Complexes by Mass Spectrometry 1.) High-throughput mass spectrometric protein complex identification (HMS-PCI) detects 3, 617 associated proteins , representing 25% of the yeast proteome. 2.) Immuno-affinity purification based on the Flag epitope tag was used to capture 100 protein kinases, 36 phosphatases and regulatory subunits, and 86 proteins functionally implicated in the cellular response to DNA damage.

46. COMPARATIVE GENOMICS OF MODEL EUKARYOTIC ORGANISM Distribution of core biological functions conserved in both yeast and worm

47. Large scale culture Transformation Separation LIMS system An integrated workflow Mass spectrometry

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