Bioinformatics Datamining Bacterial Genome Sequences

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BioinformaticsData-mining Bacterial Genome
Sequences

• Professor Mark Pallen
• University of Birmingham

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BioinformaticsDefinitions

• Fusion of Biology, Computer science, Mathematics
• Broad Meaning
• Any computationally intensive research with
  biological relevance
• Narrow meaning
• Computer-based analysis and archiving of
  macromolecular sequence data

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The Scope of Bioinformatics
Algorithm Development

• Domain Hunting
• Detailed Analysis by Expert

• Interface Design
• Graphics
• Web

Large-scale (semi-) Automated Analysis
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Scale

• Large-scale
• (semi)-automated analysis of genome sequence
• Interface with functional genomics
• Medium-scale
• Domain hunting
• Small-scale
• Analysis of individual sequence by power-user

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BioinformaticsEnabling Technologies

• Maths
• Algorithm Development
• Computer science
• Ever-increasing list of free software
• Bioinformatics programs
• Operating system LINUX
• Scripting languages glue programs together
• PERL
• Growth of Internet
• Distance is dead!, Distributed Resources
• User-friendliness of Web
• Just Cut and Paste!

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BioinformaticsChallenges

• DNA Protein Sequences
• Exponential increase
• Genome Sequencing
• Need for annotation
• Molecular stamp collecting?
• Role in drug discovery
• Big business

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BioinformaticsChallenges

• Interplay between the wet and the dry
• Bioinformatics Predictions
• range from the very general to the very specific
• range from highly speculative to the almost
  certain
• But in the end they are still only predictions
• Need for experimental confirmation

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Challenges The Data Flood

• 52 bacterial genomes completed and published
• 100,000 genes
• 228 genomes ongoing
• 450,000 more genes when finished

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The Post-Genomic Iceberg
Discovered Biology
The Undiscovered Genotype Most genes are of
unknown function Undiscovered genomic diversity
Undiscovered Biology
The Undiscovered Phenotype Most bacterial
physiology inapparent in the lab Undiscovered
regulators and regulons
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Bioinformatics Approaches

• Multiple levels of analysis
• Gene Finding
• Protein function prediction
• Power of homology
• Pitfalls of homology
• Comparative genomics
• Metabolism reconstruction
• Interface with functional genomics

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What is a Sequence?

• DNA Sequence, double stranded, antiparallel
• Conventionally written 5 to 3
• 5-ATGAGTACCG CTAAATTAGT TAAATCAAAA-3
• 3-TACTCATGGC GATTTAATCA ATTTAGTTTT-5
• RNA sequence, single stranded, U instead of T
• 5-AUGAGUACCG CUAAAUUAGU UAAAUCAAAA-3
• Protein sequence
• conventionally written N-terminal to C-terminal
• 3-letter code Met Ser Thr Ala Lys Leu
• 1-letter code MSTAKLVKSKATN
• Sequences usually written in a monospaced font
  like Courier
• Times Courier
• AGCGGGCGG AGCGGGCGG
• ATCGTTCTG ATCGTTCTG

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First get your sequence!

• Most sequencing is now...
• Performed on DNA (rather than RNA or protein)
• Performed using the Sanger didexy method
• Exceptions...
• rRNA is sometimes sequenced directly
• N-terminal and mass spectrometry sequencing of
  proteins
• Template for sequencing can be
• DNA cloned in a plasmid (e.g. pUC19)
• DNA cloned in a single-stranded phage (e.g. M13)
• PCR products

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First get your sequence!

• Automated Sequencing
• Fluorescent dyes used
• Extract sequence from chromatogram
• Must extract only the error-free region

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Where to analyse?
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Assembling a contig
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Analysis of nucleotide sequence data

• Sequence Composition
• bacteria vary greatly in chromosomal GC
  content each genus has characteristic GC
• useful in
• checking you cloned what you think you have
• identifying foreign DNA in a genome
• Predicted Restriction maps
• helpful in the lab

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Analysis of nucleotide sequence data

• Search for Other Sequence Features
• Promoters
• Ribosome-binding Sites
• Repeats
• Inverted Repeats (e.g. terminators)
• Consensus Sequences for regulator binding sites

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Searching for coding regions

• Any given DNA sequence can be translated in 6
  different reading frames, 3 on each strand

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ORF maps
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The Problem of Frameshift Errors
Actual sequence
10 20 30 40
50 60 70

ATGAGTACCGCTAAATTAGTTAAATCAAAAGCGACCAATCTGCTTTAT
ACCCGCAACGATGTCTCCGACAGCGAGAAA M S T A K L
V K S K A T N L L Y T R N D V S D
S E K V P L N L N Q K R P I
C F I P A T M S P T A R K E Y
R I S I K S D Q S A L Y P Q R
C L R Q R E K
10 20 30 40
50 60 70

ATGAGTACCGCTAAATTAGTTAAATCAAAAAGCGACCAATCTGCTTTA
TACCCGCAACGATGTCTCCGACAGCGAGAA M S T A K L
V K S K S D Q S A L Y P Q R C L R
Q R E V P L N L N Q K A T N
L L Y T R N D V S D S E K E Y
R I S I K K R P I C F I P A T
M S P T A R K
Frameshifted sequence after single base error
Markov Models (GLIMMER) now commonly used to
predict coding regions
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Analysis of Protein Sequence Data
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Analysis of Protein Sequence Data Signal peptides
SignalP uses neural networks
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Homology

• Similarity that arises because of descent from a
  common ancestor
• The formation of different languages and of
  distinct species, and the proofs that both have
  been developed through a gradual process, are
  curiously parallel We find in distinct languages
  striking homologies due to community of descent,
  and analogies due to a similar process of
  formation Languages, like organic beings, can be
  classed in groups under groups and they can be
  classed either naturally according to descent, or
  artificially by other characters The survival or
  preservation of certain favoured words in the
  struggle for existence is natural selection.
• Charles Darwin, 1871 THE DESCENT OF MAN, Chapter
  3

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Homology
the cat sat on the mat die Katze sass auf
der Matte
vgeGBant88-2 ITLITCVSVKDNSKRYVVAG vgeGEfae9-1
78 LTLITCDQATKTTGRIIVIA vgeGSpne1-403
MTLITCDPIPTFNKRLLVNF sortase_staur
LTLITCDDYNEKTGVWEKRK
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Homology
Sequence homology is not just sequence similarity!
Sequences 1, 1A, 1B and 2 are all homologous to
one another Another sequence 2 is similar to
sequences 1, 1A, 1B 2, but not homologous to
them as it does not share a common ancestor with
them Another sequence 1 is neither homologous
nor similar to sequences 1, 1A, 1B 2
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Types of Homology
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Sequence Databases

• All sequences when published are deposited in
  Sequence Databases
• Nucleic Acid Sequence Databases
• EMBL, Heidelberg, http//www.embl-heidelberg.de/
• GenBank, in the NCBI, USA, http//www.ncbi.nlm.nih
  .gov/
• Protein Sequence Databases
• GenPept and TREMBL
• Curated database SwissProt, Geneva,
  http//www.expasy.ch/sprot/
• Numerous others, reviewed every year in NAR
• Problem of sequence formats
• Simplest format is FASTA
• gtsequence name
• AATGATGCGTGATGATGATGATGACTGACTGATGATGAT

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Homology Searches

• The aim of homology searches is to identify
  sequences within these databases that are
  homologous to your sequence.
• This involves comparing your sequence with all
  the database sequences, looking for stretches of
  sequence that appear to be similar, then scoring
  the matches and ranking them. Usually a measure
  of the significance of the match will be given.

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Homology Searches Translate first!
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Homology Searches with BLAST

• BLASTN
• Nucleotide query vs nucleotide database
• BLASTP
• protein query vs protein database
• BLASTX
• automatic 6-frame translation of nucleotide query
  vs protein database
• TBLASTN
• protein query vs automatic 6-frame translation of
  nucleotide database
• TBLASTX
• automatic 6-frame translation of nucleotide query
  vs automatic 6-frame translation of nucleotide
  database

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Typical Blast Output
Sum
Reading High Probability Sequences
producing High-scoring Segment Pairs
Frame Score P(N) N embX69337ECDPS
E.coli dps gene for binding protein 2 834
6.4e-109 1 gbU04242ECU04242 Escherichia
coli core starvation p... 3 828 2.7e-106
1 embX14180ECGLNHPQ Escherichia coli glutamine
permeas... 3 443 2.8e-53
1 gbU18769HDU18769 Haemophilus ducreyi fine
tangled p... 1 150 4.0e-18 2
dbjD01016ANALTI46 Anabaena variabilis lti46
gene. gte... 2 129 4.8e-12 2
gbM84990P26BPO Plasmid pOP2621 ORF1 gene,
5' end... -2 131 6.7e-09
1 gbU16121HPU16121 Helicobacter pylori
neutrophil act... 1 112 1.8e-06
1 gbM32401TRPTYF1 T.pallidum pallidum
antigen TyF1 g... 3 101 5.6e-06
2 embX71436RPNTRB R.phaseoli ntrB gene 1
67 0.76
2 gbL35598DRODGC1A Drosophila melanogaster
receptor g... 1 48 0.97 3
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Typical Blast Output
gbU18769HDU18769 Haemophilus ducreyi fine
tangled pili major pilin subunit gene Length
780 Plus Strand HSPs Score 150 (68.0 bits),
Expect 4.0e-18, Sum P(2) 4.0e-18 Identities
36/89 (40), Positives 46/89 (51), Frame
1 Query 30 ELLNRQVIQFIDLSLITKQAHWNMRGANFIAVH
EMLDGFRTALIDHLDTMAERAVQLGGV 89 E L
LLI K AHWN G FIAVHEMLD D D AER
LG Sbjct 253 EALQMRLQGLNELALILKHAHWNVVGPQFIA
VHEMLDSQVDEVRDFIDEIAERMATLGVA 432 Query 90
ALGTTQVINSKTPLKSYPLDIHNVQDHLK 118
G YPL QDHLK Sbjct 433
PNGLSGNLVETRQSPEYPLGRATAQDHLK 519
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Sequence alignments
Dps_trepo .........N MCTDGKKYHS TATSAAVGAS
APGVPDARAI AAICEQLRRH Dps_helico ..........
.......... .......... .......... MKTFEILKHL
Dps_anab .......... .......... .......MPR
INIGLTDEQR QGVINLLNQD MrgA ..........
.......... .......... MKTENAKTNQ TLVENSLNTQ
Dps_haemo MRSKTITFPV LKLTGQSQAL TNDMHKNADH
TVPGLTVATG HLIAEALQMR Dps_ecoli ..........
....MSTAKL VKSKATNLLY TRNDVSDSEK KATVELLNRQ
Dps_strep ........MT SQPHLHQHAA EIQEFGTVTQ
LPIALSHDAR QYSCQRLNRV Dps_trepo VADLGVLYIK
LHNYHWHIYG IEFKQVHELL EEYYVSVTEA FDTIAERLLQ
Dps_helico QADAIVLFMK VHNFHWNVKG TDFFNVHKAT
EEIYEEFADM FDDLAERLVQ Dps_anab LADSYLLLVK
TKKYHWDVVG PQFRSLHQLW EEHYEKLTEN IDAIAERVRT
MrgA LSNWFLLYSK LHRFHWYVKG PHFFTLHEKF
EELYD..... .HAAETWIPS Dps_haemo LQGLNELALI
LKHAHWNVVG PQFIAVHEML DSQVDEVRDF IDEIAERMAT
Dps_ecoli VIQFIDLSLI TKQAHWNMRG ANFIAVHEML
DGFRTALIDH LDTMAERAVQ Dps_strep LADTQFLYAL
YKKCHWGMRG PTAYQLHLLF DKHAQEQLEL VDALAERVQT
ClustalW most commonly used program Note problems
of indels and ragged ends Need for manual
refinement Multiple alignments useful for
identifying active sites and distant homology
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Into the twilight zoneThe search for distant
homologies
Signal Peptide
A
Proteins consist of domains
B
Signal Peptide
Transitivity of Homology
Coiled coil domain
C
Distant Homology
D
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Domain Hunting
Homology Search
Add to Alignment
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PSI-BLAST Position-Specific Iterated BLAST

• combines statistically significant alignments
  produced by BLAST into a position-specific score
  matrix
• searches the database using this matrix
• allows multiple iterations of this process
• runs at approximately the same speed per
  iteration as gapped BLAST
• is much more sensitive to weak but biologically
  relevant sequence similarities

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Role of Sequence Analysisin the Pre-Genomic Era
Confirm

• Sequence Analysis
• Homology
• Structural Features

Identify Clone Gene
Obtain Sequence
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Role of Sequence Analysisin the Post-Genomic Era
Obtain Sequence from Genome Project
Formulate Hypothesis

• Sequence Analysis
• Homology
• Structural Features
• Genomic Context

Novel Lab-based Experimental Programme Amplify
Clone Express Gene Create mutant etc.
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Case studies

• ViruloGenome has resulted in four publications on
  
• Sortase
• ARTs
• Tricorn proteases
• The ESAT-6 superfamily
• and has been cited in one other paper on
  Bestrophin

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Sortase and its targets

• Many proteins in Staphylococcus aureus sorted on
  to bacterial surface by an enzyme sortase
• Many of these sortase-sorted proteins involved
  in causing disease (wound infection, septicaemia
  etc)
• But unclear how common this mechanism of sorting
  proteins on to bacterial surfaces is in other
  similar Gram-positive bacteria
• Novel sortase-sorted proteins in S. aureus and
  other bacteria could also be involved in
  diseaseroute to novel vaccine, drug and
  epidemiological targets

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Searching new genomes

• Multiple sortase-like proteins in almost all
  Gram-positives
• examples include bacterium causing pneumonia,
  diarrhoea, botulism, anthrax, diphtheria
• all that have one have at least two
• up to six in C. diphtheriae
• genes clustered with those for sortase-sorted
  proteins

• Sortase in a sludge-dwelling archaean,
  Methanobacterium!
• Sortase in a Gram-negative bacterium, Shewanella,
  with a sortase-sorted protein!
• New sortase-sorted proteins also found in many
  different kinds of bacteria
• Eight new proteins in S. aureus
• Four new proteins in S. epidermidis

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vgeGSpyM35-21 202 ----EVTLVTCT-DIEATERIIVKG vge
GSmut53-76 199 ----EVTLVTCT-DAGATARTIVHG vgeGE
fae12-38 194 ----MITLITCG-DLQATTRIAVQG vgeGSpne
1-402 180 ----YATLLTCTPYMINSHRLLVRG vgeGSpne1-4
00 212 ----YVTLLTCTPYMINTHRLLVRG vgeGCdip26-60
233 ----LVTLVTCTPLGINTHRILVTA 3036999_acnae
193 ----LLTLVTCTPLGINTHRILLTG vgeGCdip28-52
224 ----YITLITCTPYGINTHRLMVRG vgeGCdip28-55
243 ----QVTLITCTPYGINTHRLIITA 6580649_stcoe
191 ----ELRVITCG------GGFTKQN 6759575_stcoe
180 ----ELRVITCG------GPYSRST 7160127_stcoe
173 ----EVRLITCA------GDYDHKV vgeGCdip22-146
216 ----QITLITCTPYAVNSHRLLVRA vgeGCdip26-61
245 ----LITLVTCTPYGINTHRLLVTA vgeGEfae24-45
209 ----LVTLLTCTPYMINSHRLLVRG vgeGSpyo1-123
176 ----YATLVTCTPYGVNTKRLLVRG vgeGSequ392-28
190 ----LVTLVTCTPYGVNTHRLLVRG 7211004_stcoe
177 ----ELRLITCG------GPRDGQE 6689174_stcoe
207 ---SELRLITCG------GTFDQTT ywpE_bacsu
73 -----ITLITCDKAVKTEGRLVVKG vgeGBant88-2 205
-----ITLITCVSVKDNSKRYVVAG vgeGEfae9-178 220
-----LTLITCDQATKTTGRIIVIA vgeGSpne1-403 200
----IMTLITCDPIPTFNKRLLVNF sortase_staur 178
----QLTLITCDDYNEKTGVWEKRK YHCS_BACSU 171
----ELILTTCYP-------FSYVG 5102801_stcoe 210
KAGHYITLTTCTPVYTSRYRYVVWG 2622963_methe 170
---ARLMLITCYPPGQKKAAWITHC
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Implications

• In disease-causing bacteria
• Sortase-sorted proteins could provide novel
  vaccine targets, could help track spread of
  infections
• Sortase-like proteins could prove new drug
  targets
• Lots of new scientific questions
• In harmless bacteria
• overlooked biology of model organisms
• B. subtilis, S. coelicolor, M. thermoautotrophicum

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ADP-Ribosylation

• protein modification
• transfer of ADP-ribosyl group to a protein by an
  ADP-ribosyltransferase (ART)

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ADP-Ribosyltransferases

• Many important bacterial exotoxins ADP-ribosylate
  host cell proteins
• diphtheria toxin, P. aeruginosa exotoxins A S
• clostridial toxins
• EDIN from Staphylococcus aureus
• Mtx from Bacillus sphaericus
• VIP2 from Bacillus cereus
• AB5 toxins cholera toxin, LT-I, LT-II from E.
  coli, pertussis toxin

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Search for novel ARTs

• Found nearly two dozen novel ARTs
• similarities patchy, but convincing
• Many could just be house-keeping proteins
• but at least two appear to be new toxins

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NARG_HUMAN 174 CHQVFRGVH 16 GFASASLKHVAAQ 24
PGEEEVLIPP 82 9945866 317 VVKTFRGTQ 17
GYLSTSRDPGVAR 25 GDEQEILYDK 67 ALT_BPT2 476
GITVYRAQS 18 NFVSTSLTPIIFG 69 ATEAEMLFPP 103
ART1_PSESM 182 SGQLHRGIK 21 AFMSTSTRMDVTE 24
PYEDEALIPP 29 ART3_PSESM 193 SSQLHRGIK 21
AFMSTSTHMQVSE 24 PYEDEALISP 29 ART4_PSESM 159
RGTVYRGIR 21 AFMSTTRIKDSAQ 28 PSEEEIMLPM
17 ART5_PSESM 70 KETLYRGIN 21 TFVSATPDLSTVN 30
SAEEEGIFAP 33f ART2_PSESM 152 TTCLYRPIN 60
AFMSTSTDSVIAN 24 AYEKEAIIPP 30f ART1_ENTFA
389 DIITYRGVS 14 EFKSTSINKKVAE 34 KKESEFLLNR
21 ART2_ENTFA 337 SDDLVRFAN 11 EYISTTKDIYSEQ 18
NSEKEVLFNR 25 ART_STREPY 152 LTSYYRNHQ 18
SFMSTTALKNGAM 26 PSEVELLFPR 28 EDIN_STAAU 150
DENLVRKLN 11 GYSSTQLVSGAAV 28 YGQQEVLLPR
40 ART_MYCAV 90 EGAVVRGTN 18 AFTSTSTDHTVAQ 26
PDEKEILFPA 16 6730537 343 NITVYRWCG 30
GYMSTSLSSERLA 28 ASEKEILLDK 42 ART_CLOAB 275
DMKVYRGTD 29 GFMSTALVKESSF 25 PDEAELLLNH
27 10176155 382 GIMVYRNVG 22 GYMSTSVLREGAF 26
FKDEEYEFLI 27 10336588 152 PISVYRIVS 18
GFISSGTDKTQML 31 GRTLEALFPP 42 VRP2_SALTY 466
HRVVYRGLK 23 AFMSTSPDKAWIN 23 KGEAEMLFPP
61 TOX1_BORPE 3 PATVYRYDS 36 AFVSTSSSRRYTE 63
TYQSEYLAHR 101 YD72_MYCPN 5 VRFVYRVDL 32
YFISTSETPTAAI 59 AYQREWFTDG 454 TOXA_SALTI 1
VDFVYRVDS 37 YIATTSSVNQTYA 51 RLQREYVSTL
104 TOX2_SALTI 1 VDFVYRVDS 37 YIATISDINEAYN 51
RLQSEYVSVN 52f 5902922 65 RQRLVRWDR 39
IFVSTTKTQRNKK 38 PNQMEVAFPG 680 7105990 76
CGTLYRSDS 31 PYVSTSYDHDLYK 54 QTKTEIMSDC
11 143205 92 EHRLLRWDR 38 IFVSTTRA-RYNN 41
PNEDEITFPG 668 CHTA_VIBCH 1 DDKLYRADS 47
GYVSTSISLRSAH 37 PDEQEVSALG 75 ELAH_ECOLI 2
GDKLYRADS 47 GYVSTSLSLRSAH 37 PYEQEVSALG
119 ART2_BURCE 1f --VLYRPSC 41 GYLGAFRKLDTAT 35
PEDGEVAALG 415 ART1_BURCE 380 EGALYRPSA 41
GYLGTYRSAFTAQ 32 PAQGEVAAMG 401 ART4_BURCE 438
PFALFRPSQ 41 GYVGTFTGRAEAQ 36 PRDGEVAAMG
404 ART3_BURCE 609 RGLLFRPST 41 GYLGTFQYGRTAL 34
PHIHEQAAMG 402 ART5_BURCE 772 PWVLFRPST 41
GYVGAFRLPSTAL 34 PDNQEFAAVG 405
R S E
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ESAT-6

• Small secreted protein from M. tuberculosis of
  key importance as virulence factor and T-cell
  antigen
• Missing in BCG, defined mutant attenuated
• Useful in immunodiagnosis
• Multi-protein family in M. tuberculosis
• Hard to investigate in difficult-to-handle
  mycobacteria
• But said to have no homologues outside
  mycobacteria and their close relatives...

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The ESAT-6/WXG100 superfamily
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VGE PSI-BLASTSearch for novel ESAT-6 homologues
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Bestrophin

• Best macular dystrophy or vitelliform macular
  dystrophy type-2
• human genetic disorder
• visual impairment egg-like lesions in the macula
• caused by mutations in a gene for bestrophin,
  function unknown
• VGE-PSI-BLAST searches by Goodstadt Ponting
  found homologues of bestrophin in several species
  of bacteria, including the model laboratory
  organism, Escherichia coli (protein b1520)

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From Sequence to Consequence

• Preliminary lab-based studies underway by Lihong
  Zhang on sortase substrates in
• C. diphtheriae
• B. anthracis
• C. difficile
• S. pneumoniae
• S. putrefasciens

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From Sequence to Consequence

• Preliminary lab-based studies planned on
• ARTs from S. typhi and S. pyogenes
• Martin Antonio, Claire Spreadbury with Steve
  Dove
• ESAT-6/YukA homologues in S. aureus and B.
  subtilis
• Jen Gifford-Garner, Lihong Zhang, Kate Spanchak
  mutants from Anil Wipat, bacterial 2H from Lars
  Westblade
• Bestrophin homologue in E. coli
• Lihong Zhang mutagenesis with Jeff Cole

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Conclusions
Only connect!
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Acknowledgements

• Dosh
• BBSRC,
• QUB, Brum
• Meatware
• Alex Lam
• Nick Loman
• Arshad Kahn
• Lihong Zhang
• Sequences
• provided by Sanger, TIGR, et al. , funded by
  Wellcome et al.