Integrated Bioinformatics Nature of research articles Comparison of genomes – Scenario Regular expressions in Python Installing and running Blast How to find differences

E. coli: What makes it kill? Escherichia coli very small lab rats Courtesy of Kent State University Microbiology

E. coli: What makes it kill? Escherichia coli... Shiga toxin-producing a.k.a. enterohemorrhagic E. coli (EHEC). Enterotoxigenic E. coli Enteropathogenic E. coli (EPEC) Enteroaggregative E. coli (EAEC) Enteroinvasive E. coli (EIEC) Diffusely adherent E. coli (DAEC) At least six different dirarrheal "pathotypes" of E. coli !

E. coli: What makes it kill? E. coli O157:H7... The classic baddie!! Shiga toxin-producing (STEC) a.k.a. enterohemorrhagic E. coli (EHEC) First described as a pathogen in 1982 The most prevalent in foodborne outbreaks Most self-resolving diarrhea and vomiting Hemolytic urinary syndrome a serious complication Easily the most extensively studied pathogenic E. coli

E. coli: What makes it kill? E. coli O104:H4... The up-and-comer Enteroaggregative E. coli (EAEC) …but with acquired Shiga toxin genes! First described as a pathogen in 2005 (only one case!), but caused a huge outbreak in Germany in 2011 German outbreak traced back to sprouts! 3,950 people affected, with 53 deaths, and 800 cases of hemolytic urinary syndrome Good example of how "new" pathogens can suddenly emerge

Research papers Perna, N. T., G. Plunkett, 3rd, et al. (2001) Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409(6819): Hayashi, T., K. Makino, et al. Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12. DNA Res 8(1): Loman, et al. (2013). A culture-independent sequence- based metagenomics approach to the investigation of an outbreak of Shiga-toxigenic Escherichia coli O104:H4 JAMA 309(14): Hauser, et al. (2013). Phylogenetic and molecular analysis of food-borne shiga toxin-producing Escherichia coli. Appl Environ Microbiol. 79(8):

E. coli: What makes it kill? E. coli K12E. coli O157:H7 Gene finder TCTACTTATA TTCAATCCAC AGGGCTACAC AAGAGTCTGT TGAATGAACA CATACATGGT TTCTGTCTGC TCTGACCTCT GGCAGCTTTC TGGATTTCGG AACTCTAGCC TGCCCCACTC GAACCTTAGT GACTTCTGCT ATACCAAAGT CTCCGTAAAC CTCTAACATG ATGTCAGCAA TGAATAAACT TTGTTAAAGG TACAAATGAA AAGAGTTTAA AGTTAAAAAC GAATTGCAGT AAACCTGTAT GGTTACATGA ACTGCCTAAA TTATATATTT TAAGAAATTA ATTGCAATTA CCCCAGCTGT CATTAAAAAG AGGCAAATAC GACAGCACTG ACCCTCAAGA AGGCACCGGC GCTGAAATTC CGCTGAGAGC AGAGTGGTAC CCCTGCACCA GGTCTTTCCT GTGGGCACTG ATGAATGACT GAACGAACGA TTGAATGAAA TCTACTTATA TTCAATCCAC AGGGCTACAC AAGAGTCTGT TGAATGAACA CATACATGGT TTCTGTCTGC TCTGACCTCT GGCAGCTTTC TGGATTTCGG AACTCTAGCC TGCCCCACTC GAACCTTAGT GACTTCTGCT ATACCAAAGT CTCCGTAAAC CTCTAACATG ATGTCAGCAA TGAATAAACT TTGTTAAAGG TACAAATGAA AAGAGTTTAA AGTTAAAAAC GAATTGCAGT AAACCTGTAT GGTTACATGA ACTGCCTAAA TTATATATTT TAAGAAATTA ATTGCAATTA CCCCAGCTGT CATTAAAAAG AGGCAAATAC GACAGCACTG ACCCTCAAGA AGGCACCGGC GCTGAAATTC CGCTGAGAGC AGAGTGGTAC CCCTGCACCA GGTCTTTCCT GTGGGCACTG ATGAATGACT GAACGAACGA TTGAATGAAA

E. coli: What makes it kill? E. coli K12E. coli O157:H7 Gene finder TCTACTTATA TTCAATCCAC AGGGCTACAC AAGAGTCTGT TGAATGAACA CATACATGGT TTCTGTCTGC TCTGACCTCT GGCAGCTTTC TGGATTTCGG AACTCTAGCC TGCCCCACTC GAACCTTAGT GACTTCTGCT ATACCAAAGT CTCCGTAAAC CTCTAACATG ATGTCAGCAA TGAATAAACT TTGTTAAAGG TACAAATGAA AAGAGTTTAA AGTTAAAAAC GAATTGCAGT AAACCTGTAT GGTTACATGA ACTGCCTAAA TTATATATTT TAAGAAATTA ATTGCAATTA CCCCAGCTGT CATTAAAAAG AGGCAAATAC GACAGCACTG ACCCTCAAGA AGGCACCGGC GCTGAAATTC CGCTGAGAGC AGAGTGGTAC CCCTGCACCA GGTCTTTCCT GTGGGCACTG ATGAATGACT GAACGAACGA TTGAATGAAA TCTACTTATA TTCAATCCAC AGGGCTACAC AAGAGTCTGT TGAATGAACA CATACATGGT TTCTGTCTGC TCTGACCTCT GGCAGCTTTC TGGATTTCGG AACTCTAGCC TGCCCCACTC GAACCTTAGT GACTTCTGCT ATACCAAAGT CTCCGTAAAC CTCTAACATG ATGTCAGCAA TGAATAAACT TTGTTAAAGG TACAAATGAA AAGAGTTTAA AGTTAAAAAC GAATTGCAGT AAACCTGTAT GGTTACATGA ACTGCCTAAA TTATATATTT TAAGAAATTA ATTGCAATTA CCCCAGCTGT CATTAAAAAG AGGCAAATAC GACAGCACTG ACCCTCAAGA AGGCACCGGC GCTGAAATTC CGCTGAGAGC AGAGTGGTAC CCCTGCACCA GGTCTTTCCT GTGGGCACTG ATGAATGACT GAACGAACGA TTGAATGAAA

E. coli: What makes it kill? Similarity finder Killer protein Membrane protein, sodium transporter Iron responsive transcriptional regulator Calcium-dependent protein kinase Unknown protein... Killer functions Suggests therapeutic interventions How do these differences arise?

Point mutation organism 1 TTT TCT GAA TCC GTA GAC GTT organism 2 TTT TCT GAA TCA GCA GAC GTG What kind of mutations arise? How do genomic differences arise? Can these explain the difference between K12 and O157:H7??

DNA mutation has multiple causes! Errors during DNA replication base mis-incorporation polymerase slippage / repeat amplification Errors during recombination or cell division chromosome loss or rearrangement large insertions or deletions Environmental factors – mutagens: radiation – UV or ionizing radiation chemical – many mechanism of action Spontaneous events: tautomerisation depurination deamination Viral infection or transposons

Addition/deletion of DNA How to distinguish from ? Where do they come from? – GC-content? How do genomic differences arise?

Infection Phage Bacterial chromosome Phage genome Lysogenic pathway Lytic pathway Phage genome Lytic pathway = cell death! General transduction How do genomic differences arise?

Infection Phage Bacterial chromosome Phage genome Lysogenic pathway Lytic pathway Phage genome How do genomic differences arise? General transduction Lysogenic pathway = Life!

How do genomic differences arise? Infection Phage Bacterial chromosome Phage genome Lysogenic pathway Lytic pathway Phage genome General transduction Lysogenic pathway = Life!

Specialized transduction Occurs at defined sites

Specialized transduction Occurs at defined sites

The gene encoding diphtheria toxin (tox) is carried on corynephage  Lysogenic conversion by corynephage  confers toxogenicity!!  tox  –  C.d. tox  +  C.d.