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Microbial Genomics Topics Describe the new area of genomics Outline the rapid progress in genomic sequencing Describe the analysis of sequences - bioinformatics Show the use of genomics in the study of microbes Use the sequence of a human pathogen Escherichia coli O157:H7 to illustrate the above points Ref: Perna et al. (2001) Nature 409:529 (USA) Relevant to next lectures. Dr M. D-S, 2007 Microbial genome sequences Genbank (NCBI), Bethesda, Maryland, USA 2007: 481 - completed microbial genomes 2006: 319 2003: 112 Sizes range from 0.58 - over 9 Mb Genbank - main genomic database There is some duplication... Dr M. D-S, 2007 Genomics - the study of entire genomes of organisms assumes the entire sequence of at least one representative example has been determined includes study of all the genes and gene products and non-coding regions includes study of genome organisation and evolution Dr M. D-S, 2007 The explosion of ‘-ome’ and ‘-omics’ words Functional genomics Proteome Transcriptome Metabolome, Glycome, Lipidome e.g. a recent journal article with the title: “Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis” Dr M. D-S, 2007 Genomics What can microbial genomics tell us ? • • • • • • Full gene complement of the cell Complete description of cell metabolism How genomes are structured Virulence genes Potential drug targets Gene flow between cells (evolution) Dr M. D-S, 2007 Genome Sequencing: Two methods 1. Sanger di-deoxy sequencing (using fluorescently labelled ddNTPs) on cloned DNA templates. 2. Pyro-sequencing method on 454 machine using uncloned DNA templates Dr M. D-S, 2007 Genome Sequencing: Two methods 1. Sanger di-deoxy sequencing (using fluorescently labelled ddNTPs) on cloned DNA templates. ‘Shotgun’ strategy. Dye-terminator chemistry, ABI sequencing apparatus, commercial software for handling seq. data Dr M. D-S, 2007 Genomic sequencing methods Shear DNA & isolate fragments about 2kb chDNA Clone thousands of fragments into plasmid vector (library). Prepare DNA for sequencing Dr M. D-S, 2007 Dideoxy chain termination QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. http://www.plattsburgh.edu/acadvp/artsci/biology/bio401/DNASeq.html Dr M. D-S, 2007 Sequence: methods section Applied Biosystems Inc (ABI) latest sequencing machine, PE 3700 Capillary electrophoresis 96 capillaries at a time Robotically loaded and run (24hr) - How many bp can it do in a day?? + - each run is 2hr, get 600-1000 nt per capillary, 96 capillaries/run Dr M. D-S, 2007 Sequence: methods section Applied Biosystems Inc (ABI) latest sequencing machine, PE 3700 How many bp can it do in a day?? - each run is 2hr, about 800bp each lane, 96 lanes = 24/2 800 96 = 921,000 Or about 1 Mb /machine/day Dr M. D-S, 2007 Top of capillary tubes Sequence data - Laser scanning of the 96 capillary tubes identifies the colour and positions of the closely spaced bands of ssDNA. + TAATCATGGTC.... Dr M. D-S, 2007 Shotgun sequencing: how much do you need to do? ~ 1 Mb /machine/day Want both strands, good sequence for both, random coverage means you will need 6-8x genome size in sequence data Speed makes it efficient? Counter argument is the difficulty in linking up reads, particularly when genomes have long repeat sequences. Dr M. D-S, 2007 Genome Sequencing: Two methods In the E.coli O157:H7 genome sequence paper by Perna et al., there were 2 gaps remaining in the genome sequence! They couldn’t complete it. “Extended exact matches pose a significant assembly problem.” ?? Dr M. D-S, 2007 Repeat sequences, eg. Prophage genomes Nearly identical prophage sequences at 3 locations on genome, all > 2000 nt What sequences do you observe when inside a prophage genome? Dr M. D-S, 2007 Repeat sequences, eg. Prophage genomes Nearly identical prophage sequences at 2 locations on genome What sequences do you see going across the borders of prophages? Dr M. D-S, 2007 Repeat sequences, eg. Prophage genomes Nearly identical prophage sequences at 2 locations on genome What information do you need to place the repeats properly? Dr M. D-S, 2007 Genome Sequencing: Two methods 1. Sanger di-deoxy sequencing (using fluorescently labelled ddNTPs) on cloned DNA templates. 2. Pyro-sequencing method on 454 machine using uncloned DNA templates Dr M. D-S, 2007 The 454 machines: the next revolution QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. www.454.com The 454 machines: the next revolution 40 million bases/5.5 hr QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. www.454.com The 454 machines: the next revolution 40 million bases/5.5 hr DNA immobilised on micro-beads QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Positioned in wells of special tray (44um diameter, 1.2 million per chip) Sequencing enzymes on smaller beads. Only one DNA-bead can fit in each well QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Each bead has only one DNA fragment attached, so will give unique sequence. www.454.com The 454 machines: the next revolution QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. When a base is incorporated (by DNA polymerase), light is emitted, and the light detected under each well. www.454.com The 454 machines: the next revolution 40 million bases/5.5 hr QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. When a base is incorporated (by DNA polymerase), light is emitted, and the light detected under each well. If there are multiple bases, the light is proportional to the number. Chain lengths of 200 nt are possible. With 200,000 wells, and 200nt/well, then 40 million bases can be sequenced. www.454.com Genomics Papers filled with JARGON. Mainly genetic terms. Some terms are relatively new (eg. replichore) Use the E.coli paper example, stopping to investigate each new term or concept Emphasise the uses of this data, and the future of genomic research. Dr M. D-S, 2007 What do you know about microbial genomes ? Exercise: Think of a typical bacterial genome, like that of E.coli and Sketch the genome and the most significant features you know about it (as a whole genome, not individual genes) Jot down what you think the main selective pressures are on it Dr M. D-S, 2007 Escherichia coli genome Circular, ~ 4.6 Mb Ori and Ter, bidirectional replication Replichores about equal oriC ter Dr M. D-S, 2007 Replichore ‘balance’ ? If you move oriC relative to Ter, the growth rate of E. coli K-12 is reduced. Chromosomal inversions around the origin or termination of replication are usually symmetrical, conserving the replichore balance. Hill, C. W., and J. A. Gray. 1988. Effects of chromosomal inversion on cell fitness in Escherichia coli K-12. Genetics 119:771–778. Eisen, J. A., J. F. Heidelberg, O. White, and S. L. Salszberg. 2000. Evidence for symmetric chromosomal inversions around the replication origin in bacteria. Genome Biol. 1:0011.1–0011.9 Dr M. D-S, 2007 E.coli genome - global features Gene dosage Gene direction relative to ori Recombination/inversion rates vary around chromosome Dr M. D-S, 2007 Gene Dosage Genes near the origin of replication will almost always be in multiple copy compared to genes near the terminus So the position of a gene relative to the origin will affect its expression, and the regulatory systems would have evolved to accommodate for the gene dosage effect. So what would happen oriC if you moved genes ? ter Dr M. D-S, 2007 Gene Direction What happens when a DNA pol meets an RNA pol going in the opposite direction? RNA Polymerase DNA Polymerase Dr M. D-S, 2007 Gene Direction What happens when a DNA pol meets an RNA pol going in the opposite direction? RNA Polymerase DNA Polymerase This is better…. Dr M. D-S, 2007 Gene Direction ori A preference for genes to be on ONE strand of the replichore, so that the direction of transcription and replication are the same. This bias may have other implications. Dr M. D-S, 2007 Recombination/inversions Genomes often have large repeated sequences, eg. ribosomal RNA gene clusters (16S-23S-5S), or phage genomes. Such repeats allow large inversions of DNA segments or recombination between chromosomes Dr M. D-S, 2007 Inversion via repeated sequences Homologous recombination between rRNA genes Dr M. D-S, 2007 origin GC-skew Chi sequences terminus Dr M. D-S, 2007 Genomics: What is GC-skew ? Systematic bias in base composition of one strand as you go around the genome origin [G-C] [G+C] GC skew ter ter genome Dr M. D-S, 2007 GC-skew of genomes Dr M. D-S, 2007 GC-skew of genomic DNA Compositional bias: Leading strand enriched in G/T (keto) Lagging strand enriched in C/A (amino) WHY? Perhaps due to deamination of exposed C’s in the leading strand, producing C>T mutations. Theory only. Dr M. D-S, 2007 origin GC-skew Chi sequences terminus Dr M. D-S, 2007 E.coli O157:H7 - K12 genome comparison: Chi sequences GCTGGTGG Sequence recognised (and cut) by the RecBC enzyme Promotes homologous recombination (by RecA) Dr M. D-S, 2007 Lateral Gene Transfer (LGT) Literally, the natural transfer of genetic material between different organisms (species, genera, etc) Doesn’t say how the DNA was transferred or integrated, or where it came from. Does imply that the DNA can be identified as ‘foreign’ Since DNA doesn’t have a ‘made in X’ sticker, how can the ‘foreignness’ be identified? …. Ideas?…. Dr M. D-S, 2007 Lateral Gene Transfer (LGT) Known mechanisms of DNA transfer between bacteria: Transduction transducing bacteriophages introduce host DNA, and this recombines with the genome Transformation DNA uptake from the surroundings, and recombination. Conjugation natural transfer method, sex pilus, one-way transfer, recombination. + - Dr M. D-S, 2007 Prophage Bacteriophages that are temperate (as compared to lytic) can exist inside host cells in a stable and relatively inactive state as prophages. The host cell, with a prophage, is called a lysogen. Some prophages express virulence determinants, such as toxins ( = lysogenic conversion). eg. Shiga toxin Some prophages exist as plasmids, but most integrate into the genome. If the prophage becomes damaged…. ? Dr M. D-S, 2007 E.coli genome sequences STRAIN SIZE DATE E.coli K12 4639221 bp, Oct 13 1998 E.coli O157:H7 (USA) 5528970 bp, Jan 25, 2001 E.coli O157:H7 (Japanese) 5498450 bp, Mar 7, 2001 *about 4.1Mb in common Data from NCBI: http://www.ncbi.nlm.nih.gov:80/PMGifs/Genomes/eub.html Dr M. D-S, 2007 E.coli O157:H7 - K12 genome comparison Unexpected complex segmented relationship Share a common 4.1 Mb ‘backbone’ or common, and generally colinear sequence (only 1 inversion) Homologous sequences are interspersed with HUNDREDS of ISLANDS of INTROGRESSED DNA A B C D A B X C D A B C D A B C D Dr M. D-S, 2007 E.coli O157:H7 - K12 genome comparison The specific DNA segments for each strain were named ‘O islands’ , ie O157:H7-specific DNA segments, or ‘K islands’ Backbone of 4.1 Mb common sequence. Not identical (eg 75% of proteins differ by one aa). O-islands total 1.34 Mb (about 26% of genes !) Largest O-island is 106 gene region (not small!) Dr M. D-S, 2007 E.coli O157:H7 - K12 genome comparison Virulence genes do not seem to be concentrated in one particular ‘island’; appear to be several Often (189 cases), the backbone-island junction is WITHIN an ORF. AUG O-island UGA Protein coding ORF What does this pattern suggest? Dr M. D-S, 2007 E.coli O157:H7 - K12 genome comparison Suggests that incoming DNA recombined with the genome (somehow?) rather than inserted. AUG O-island UGA Protein coding ORF Dr M. D-S, 2007 Comparative Genome Map Dr M. D-S, 2007 Genome Map Distribution of O-islands of EDL933 specific sequence (red), ‘K-islands’ of K12 specific sequence (green) and common ‘backbone’ sequence (blue) GC-content of genes, plotted around mean GC-skew for 3rd codons Scale, in base pairs Octamer Chi sequences Dr M. D-S, 2007 Genome sequence - Figure 2 O-specific ‘islands’ K-specific ‘islands’ O157:H7 genes and their orientation Scale (10kb/tick) Dr M. D-S, 2007 Genome sequence - Figure 2 CP-933 = Cryptic Prophage. Also an O island How many kb is this phage genome? Dr M. D-S, 2007 E.coli O157:H7 genome sequence Summary of main findings: 1. Many insertions of DNA around chromosome 2. Inserted DNA is foreign (HGT or Lateral GT) 3. Several virulence gene clusters; widely spread 4. Prophage genomes prominent 5. Systematic variations base composition - coding strand, GC skew, chi seqs Dr M. D-S, 2007 E.coli O157:H7 genome sequence Summary of main findings: 6. E.coli O157:H7 undergoes relatively high rates of recombination and mutation. - where is the DNA coming from ? unknown, phage, mobile elements (eg. transposons) - what is the main method of transfer ? - is defective DNA mismatch repair important ? Dr M. D-S, 2007 E.coli O157:H7 genome sequence Summary of main findings: These large differences can be exploited: Diagnostic tools (discriminate b/n E.coli strains) New virulence gene candidates can be tested for function, and new drugs developed Effects of antibiotics on toxin synthesis examined Note in the genome sequences of many microbes, the percentage of ORFs that cannot be identified is often > 20% Dr M. D-S, 2007