DUAL TRAFFICKING PATHWAYS OF CONNEXINS TO GAP JUNCTIONS

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  Mutagenesis of Actinomycetes Workshop   July 11
15 2005 University of Wales Swansea     ActinoGEN
SIXTH FRAMEWORK PROGRAMME SIXTH FRAMEWORK
PROGRAMME PRIORITY 1 LIFE SCIENCES, GENOMICS AND
BIOTECHNOLOGY FOR HEALTH
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Actinomycetes are an important resource for new
antibiotics

• techniques to manipulate actinomycete genes
  are vital to exploiting this resource
• precursor biosynthesis
• regulatory networks
• antibiotic biosynthetic genes
• exemplified in S. coelicolor

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Genome sequencing was based on a detailed genetic
and physical map
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Functional genomics of Streptomyces coelicolor
UMIST Bioinformatics metabolomics
John Innes Centre Proteomics Redirect
mutagenesis
University of Warwick 20 metabolite analysis
University of Wales Swansea Systematic
mutagenesis
University of Surrey microarrays
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Mutagenesis

• Three techniques that exploit the genome
  sequence
• In vitro transposon mutagenesis systematic
• (2) In vivo transposon mutagenesis identify
  genes of related function
• (3) PCR targetting (Redirect) functional
  analysis of a set of genes

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(1) In vitro shuttle transposition

• Transposition is (fairly) random
• Target site is duplicated and Insertion Sequence
  integrated

Tn5062 AprR oriT

Cosmid
Target Site
Ref Bishop et al 2004 Genome Research 14
893-900
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In vitro transposon mutagenesis
(1) Mutant cosmid isolation
cosmid
transposase
Tn5062


In vitro transposition
Transform E.coli AprRKanRAmpR
Isolate cosmid DNA
Sequence
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Organisation of Tn5062
EZR1 sequencing primer
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Analysis of Tn5062 insertions

• sequence files are directly processed using
  Transposon Express software
• finds boundary of Tn5062 sequence
• compares succeeding sequence with cosmid or
  genome sequence
• reports coordinates of insertion and identity
  of disrupted gene

Ref Herron et al 2004 Nucleic Acids Res 32 e113
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Transposon Express
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• location and description of each insertion
  provided at

http//streptomyces.org.uk/S.coelicolor/index.html
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Systematic mutagenesis of Streptomyces coelicolor
A3(2)

• Progress to date
• 105 of 319 cosmids fully processed
• 11493 independent insertions
• 10459 insertions in 2520 orfs (of 7825 in
  total)
• 4.2 insertions per orf

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Advantages of systematic in vitro transposon
mutagenesis

• High throughput
• Conjugation and the recovery of gene replacement
  clones are efficient, so that many replicate
  clones are obtained for phenotypic testing

• With one insertion per 280 bp, phenotypic
  analysis of several independent insertions in a
  given gene obviates the need for linkage analysis

• Mutations can be moved into different genetic
  backgrounds, facilitating analysis of gene
  interactions

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Advantages of systematic in vitro transposon
mutagenesis

• Mutations can be stored and shipped as
• cosmid DNA
• E coli containing cosmids
• Streptomyces mutants

• A Tn5062 insertion can be manipulated to
• change resistance marker (eg switch AprR to HygR
  )
• leave an in-frame deletion
• induce transcription of downstream genes

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Tn5062 Tn5066 Tn5069 Tn5070

• exchange cassettes can be excised as PvuII
  fragments and used to
• replace an existing Tn5062 insertion by Red
  recombination in E.coli
• for de novo in vitro transposon mutagenesis

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Transfer of mutated cosmid to Streptomyces
Transfer by conjugation from E.coli
ET12567(pUZ8002) into S. coelicolor
Select for marker replacement AprRKanS usually
1-10 of exconjugants if gene/operon is
non-essential
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Insertional mutagenesis of cosmid SC7C7
6279200 bp 6290053 bp
1 2 3 4
x 5 6
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SCO5750
osaB response regulator
osaA hybrid histidine kinase
Sph I
Bam HI
SCO5751
1 kb
osaB complementing DNA
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Mutant phenotypes
1) S. lividans
R2YE (containing 10.3 sucrose) A wild type B
osaB mutant insertion x, Tn5493
A
B
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osaAB, genes involved in osmoadapation

• osaB encodes a response regulator (insertion 5)
  that is essential for osmoadaptation during the
  transition between vegetative and reproductive
  growth

• osaA mutants (1-4) all exhibit delayed aerial
  hyphal formation in the presence of osmolyte a
  second orphan HHK (SCO7327) may also be involved
  in osmoadaptation

• SCO5750 mutants (6) are unaffected by osmolyte
  insertions 1-5 are non-polar with respect to
  SCO5750

• osaB complementation, with a fragment initially
  cloned linked to AprR of insertion 7, indicates
  osaA and osaB are independently transcribed

• insertions 1 and 5 have been successfully
  introduced into S. lividans similar phenotypes
  as for the S. coelicolor osaAB mutants were
  obtained

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osaB is induced by hyperosmolarity
sucrose
- sucrose
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osaB has its own promoter
t g c a
12 72h
Timecourse of osaB expression mRNA isolated from
R2YE-grown cultures
6285056 chromosome position cttctggtctcccgccgcg
cttccgctacgagcacagtgacatcacggtgacagggtgtg gcgacagg
cggggtgcggctacgatgaccggcacaaggacgggcggcgcaagggagtc
gt cccccggggcggcacccgccggtgccgtgccaagtcctgtggacagg
ggaggccccacgc cggggcgaggagggcgggccatggtgcagaaggcca
agatcctcctggtcgatgaccggc cggagaatctgcttgcgctggaggc
gatcctctcggcgctcgatcagacgctggtgcggg
Transcription start
Translation start
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Overproduction of ACT and RED in an osaB mutant

• osaB mutant (S)
• ? wild-type (S)
• ? osaB mutant (-S)
• ? wild-type (-S)

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Overproduction of ACT and RED in an osaB mutant
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• Osmoadaptation conclusions
• the response regulator encoded by osaB is
  essential for developmental osmoadaptation
• osaB impacts on antibiotic production in
  conditions of hyperosmolarity
• unlike most sensory kinase-response regulator
  gene pairs, osaB is independently transcribed
• the sensory kinase encoded by osaA is required
  for osmoadaptation, but not essential another
  kinase may also interact with OsaB

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(2) In vivo transposon mutagenesis
Aim Generate a library of transposon induced,
tagged mutants for gene function studies
Kay Fowler
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Tn4560 (8 kb) Derived from Tn 4556 of
Streptomyces fradiae (Chung 1987) Viomycin
phosphotransferase gene for selection in
Streptomyces
vph
Recombinase ?
Tn3
38 bp IRs
38 bp IRs
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Tn4560 delivery plasmid pKAY1

• based on temperature-sensitive plasmid
  pUC1169 (derivative of pIJ101 containing Tn4560)
• pOJ260 (contains E. coli ori and oriT) was
  cloned at the unique BamHI site
• encodes a truncated Rep protein due to
  mutation at the unique BstBI site

  -GCCCCGTTCGCGAACTCCTCGGACGGATCGGGGACCTGA -AlaPro
PheAlaAsnSerSerAspGlySerGlyThr
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Transposon delivery on pKAY1 introduced into
Streptomyces by conjugation from E. coli
1. Mix Streptomyces and E. coli on agar
plate2. Overlay with antibiotics Nalidixic
acid or carbenicillin to kill E. coli Viomycin
to select StreptomycesTn
Conjugation plate 2d after overlay
gt1000 colonies contain independent Tn insertions
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In vivo transposon mutagenesis

• wash off microcolonies
• plate on SFM viomycin
• harvest spores Tn library
• plate library using conditions to detect a
  specific phentype
• isolate DNA from mutant
• Ligation-mediated PCR

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Ligation-mediated PCR for target sequence
amplification
1. Digest DNA using EagI (CGGCCG) 2. Ligate
non-phosphorylated End primer/Adaptor 3. PCR
End primer
Genome
Transposon
Adaptor
Tn primer
No ligation
Tn primer
End primer
PCR Product
3 nested
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Target sequence identification

• Use TA cloning to clone PCR products
• Sequence inserts
• Blast sequence against genome to identify target
  gene

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(3) PCR targetting (Redirect)
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Acknowledgements Swansea Amy Bishop
osaAB Sue Fielding sequencing Paul
Herron in vitro transposition Gareth Hughes
Transposon Express Ricardo del Sol exchange
cassettes Norwich Govind Chandra ScoDB Tobias
Kieser in vivo transposon mutagenesis Kay
Fowler in vivo transposon mutagenesis