Microbiology 微生物学 Spring-Summer

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2 Microbiology 微生物学 2017 Spring-Summer

3 Relevant Information and Resources Course slides can be found at 教学工作 Course-related questions will be answered through s. Textbook: Brock Biology of Microorganisms (13 th )

4 Chapter 12 Microbial Genomics I Genomes and Genomics

5 Prokaryotic Genomes Genome size and ORFs

6 Genome sequencing Cloning region Keep copy number low sopb cat Selectable marker sopa BAC oris repe Required for replication BAC: Bacterial Artificial Chromosome, derived from F plasmid 300kbp can be inserted

7 Frederick Sanger ( ): Twice a Nobel Laureate in Chemistry

8 The Nobel Prize in Chemistry 1958 Prize motivation: "for his work on the structure of proteins, especially that of insulin" University of Cambridge

9 The Nobel Prize in Chemistry 1980 Paul Berg (1926-) 1/2 Stanfond University Walter Gilbert (1932-)1/4 Harvard University F. Sanger 1/4 Cambridge University Prize motivation: "for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant-dna" Prize motivation: "for their contributions concerning the determination of base sequences in nucleic acids"

10 Technology changes quickly, but for many years we ve on Sanger s cool trick.

11 Sanger s Trick

12 Sanger s Trick DNA chain Direction of chain growth Base Base No free 3 -OH, replication will stop at this point

13 Sanger s Trick Sanger sequencing (Dideoxy)

14 Sanger s Trick DNA strand to be sequenced Radioactive DNA primer Add DNA polymerase, mixture of all four deoxyribonucleotide triphosphates; separate into four reaction tubes A small amount of only one dideoxynucleotide triphosphate (ddgtp, ddatp, ddttp, or ddctp) added to each tube and reaction allowed to proceed Reaction products Sanger sequencing (Dideoxy) ddgtp ddatp ddttp ddctp G A T C Reaction products separated by electrophoresis on gel and identified by autoradiography 2 1 Sequence reads from bottom of gel as A G C T A A G. Sequence of unknown is 3 T C G A T T C 5 A G C T A A G

15 Improvements The dyes are spectrally distinct, and each has a different emission wavelength.

16 Improvements

17 Shot-gun - Sanger

18 New Generation Sequencing Technology

19 DNA Sequencing 454 Sequencing

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21 454 Sequencing

22 SOLiD Sequencing

23 SOLEXA Sequencing

24 454 Pyrosequencing

25 DNA Sequencing

26 DNA Sequencing

27 DNA Sequencing

28 DNA Sequencing

29 PyroSequencing

30 DNA Sequencing

31 DNA Sequencing

32 DNA Sequencing

33 DNA Sequencing

34 DNA Sequencing

35 Assembly and Annotation Occasionally assembly is NOT possible Closure can be pursued using PCR to target areas of the genome Closed vs. draft genome Functional ORF: an open reading frame that encodes a protein or an RNA Look for start/stop codons and Shine Dalgarno sequences ORFs can be compared to ORFs in other genomes

36 Total ORFs in genome Genome size Genome size (megabases)

37 Syn3.0: less than 500 genes

38 Sugar ABC transport systems Glycolysis Prokaryotic Genomes Peptide ABC transport systems PENTOSE PHOSPHATE PATHWAY Glucose Gluconate ENTNER DOUDOROFF PATHWAY Glucose-6-P 6 Phosphogluconate Glycine Acetamide Threonine Branched chain amino acids Amino acids Fructose-6-P Gly-3-P KDPG Gly-3-P + Pyruvate NH 3 + CO 2 + H 2 Polyamines Phosphate PEP DHAP Glycerol-3-P Glycerol 33 flagellar & motor genes Flagellum Aspartate Malate Pyruvate Oxaloacetate Aspartate chea/b/c/d/r/w/y H 2 and CO 2 OR Valine Acetyl-CoA Lactate Histidine 7 MCPs Zinc -ketoglutarate Aldehydes Ketoisovalerate ADP + P i Glutamate ATP Proline Glutamine Leucine PRPP Ribose-5-P Iron Chemotactic signals Cations Ribose Maltose Glycerol 3-P H + ATP synthase Glycerol uptake Uracil NH 4 + K + Fe 3+ Na + Cations

39 Prokaryotic Genomes

40 Relative percent of ORFs Prokaryotic Genomes DNA replication Translation Transcription Signal transduction Energy generation ,000 Total ORFs in genome Gene categories as a function of genome size

41 Percent of genes Prokaryotic Genomes Bacteria Archaea Carbohydrate metabolism Cell membrane Coenzyme metabolism Energy production Unknown function General prediction Functional category

42 Gene Content of Syn3.0

43 Eukaryotic Organelle Genomes rbcl Large single copy region Chloroplast Genome rpo genes Proteins for ribosome, trna, RNA polymerase, photosynthesis and autotrophy Inverted repeat A 2 copies of rrna genes Inverted repeat B RNA Editing Small single copy region

44 Eukaryotic Organelle Genomes Leu Ile Met ND1 ND2 16S Val Gln Ala Asn 12S Phe D loop Pro Thr Cytb Mitochondrial Genome Glu ND6 ND5 ND4 Leu Ser His Smaller than Chloroplast genome; Proteins for ribosome, trna, and oxidative phosphorylation Trp Cys Tyr ND4L COI Ser COII Asp Lys COIII ATPase 6 ATPase 8 Arg ND3 Gly RNA Editing

45 Eukaryotic Genomes

46 Metagenomics : Jo Handelsman Metagenomics: Total gene content of the organisms inhabiting an environment. phylogenetic analysis, functional gene analysis, and direct sequencing. Jo Handelsman (born 1959, New York, NY) is a Howard Hughes Medical Institute professor of molecular, cellular and developmental biology at Yale University. She is editor-in-chief of the academic journal DNA and Cell Biology and author of books on scientific education, most notably Scientific Teaching. On Wednesday, July 31, 2013, Dr. Handelsman was nominated by President Barack Obama to serve as Associate Director for Science at the Office of Science and Technology Policy.

47 Chapter 12 Microbial Genomics II Genome Function and Regulation

48 Microarrays Transcriptome, Functional Gene Chip Identification Gene X Gene Y Gene Z Synthesize short ss oligonucleotides complementary to genes X, Y, and Z Affix DNA to chip at known locations Gene X Gene Y Gene Z Growth condition 1 DNA chip Growth condition 2 Probe chip with labeled mrna and scan chip Gene X expressed Genes Y and Z not expressed Gene X not expressed Genes Y and Z expressed

49 Microarrays Microarrays: Transcriptome,

50 Proteomics Proteomics: 2-D gel, LC-MS M r (kda) ph

51 Proteomics Proteomics: 2-D gel, LC-MS

52 Proteomics Interactome

53 Intensity Metabolomics Flight tube Laser beam TOF detector Sample MALDI-Matrixassisted laser desorption ionization Matrix Ions Charged grid Time of flight (TOF): depends on mass/charge Mass (m/z)

54 Comparative Genomics Comparative genomics is a field of biological research in which the genomic features of different organisms are compared. Comparative genomics exploits both similarities and differences in the proteins, RNA, and regulatory regions of different organisms to infer how selection has acted upon these elements. The major principle of comparative genomics is that common features of two organisms will often be encoded within the DNA that is evolutionary conserved between them.

55 Comparative Genomics A genome alignment of eight Yersinia isolates

56 Comparative Genomics Evolutionary conservation of Human FOXP2 gene.

57 Chapter 12 Microbial Genomics III The Evolution of Genomes

58 The Evolution of Genomes Paralogs Paralogs Paralogs Homologous genes: related in sequence due to shared evolutionary ancestry Gene family. Homologs: 1) Paralogs, from gene duplication, within the same organism; 2) Orthologs, from different species; Orthologs Paralogs Orthologs A1 A2 B1 B2 Species 1 Species 2 Gene A Divergence of species Paralogs Species 2 Species 1 Gene B Gene duplication Ancestral gene in ancestral species

59 The Evolution of Genomes Paralogs Paralogs Paralogs Gene Duplications and Deletions: The major events in the evolution of genomes. Orthologs Paralogs Orthologs Other major events: Horizontal Gene Transfer and transposon: A1 A2 B1 B2 Species 1 Species 2 Divergence of species Species 2 Species 1 Paralogs Gene A Gene B Gene duplication Ancestral gene in ancestral species

60 Orthologs or paralogs?

61 The Evolution of Genomes Horizontal gene transfer (HGT): the transfer of genes between organisms in a manner other than traditional reproduction. Also termed lateral gene transfer (LGT), it contrasts with vertical transfer. Means: Transformation, Transduction, Conjugation Genes: Non-essential, mostly metabolic, antibiotic, so on

62 The Evolution of Genomes Transposon : Insertion Sequences Chromosomal rearrangement Integron genetic units characterized by their ability to capture and incorporate gene cassettes by sitespecific recombination. ln0 intl1 P atti suli ln7 aadb

63 The Evolution of Genomes Pathogenicity Islands (PAIs) : a distinct class of genomic islands acquired by microorganisms through horizontal gene transfer E. coli strain Genome (bp) K-12 4,639, ,938, ,231,428 Prophage

64 The Evolution of Genomes Core Genome: chromosome fragments present in all strains a species; Pan Genome: optional parts present in some but not all strains of the species; Integrated phage DNA Transposon Chromosomal islands Plasmids Pathogenicity island