Microbiology 微生物学 Spring-Summer

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2 Microbiology 微生物学 2016 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 Technology changes quickly, but for many years we ve on Sanger s cool trick.

9 Sanger s Trick

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

11 Sanger s Trick Sanger sequencing (Dideoxy)

12 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 ddgtp ddatp ddttp ddctp Sanger sequencing (Dideoxy) 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

13 New Generation Sequencing Technology

14 454 Pyrosequencing

15 DNA Sequencing

16 DNA Sequencing

17 DNA Sequencing

18 DNA Sequencing

19 PyroSequencing

20 DNA Sequencing

21 DNA Sequencing

22 DNA Sequencing

23 DNA Sequencing

24 DNA Sequencing

25 Genomics

26 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

27 Prokaryotic Genomes

28 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

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

30 Gene Content of Syn3.0

31 Eukaryotic Organelle Genomes Large single copy region rbcl rpo genes Chloroplast Genome Inverted repeat A 2 copies of rrna genes Inverted repeat B Small single copy region

32 Eukaryotic Organelle Genomes Phe Thr 16S Val 12S D loop Pro Cytb Glu ND6 Leu Ile Met ND1 ND2 Gln Ala Asn Mitochondrial Genome ND5 ND4 Leu Ser His Trp Cys Tyr ND4L COI Ser COII Asp Lys COIII ATPase 6 ATPase 8 Arg ND3 Gly

33 Eukaryotic Genomes

34 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.

35 Chapter 12 Microbial Genomics II Genome Function and Regulation

36 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

37 Microarrays Microarrays: Transcriptome,

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

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

40 Proteomics Interactome

41 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)

42 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.

43 Comparative Genomics A genome alignment of eight Yersinia isolates

44 Comparative Genomics Evolutionary conservation of Human FOXP2 gene.

45 Chapter 12 Microbial Genomics III The Evolution of Genomes

46 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

47 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

48 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

49 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

50 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

51 13.12 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