Transcript DUAL TRAFFICKING PATHWAYS OF CONNEXINS TO GAP …

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
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
Why Streptomyces coelicolor ?
• Streptomycetes makes two-thirds
of all natural antibiotics
• Genome sequenced
• 8,667,507bp Chromosome
• G+C content of 72.1%
Actinorhodin
“act”
Undecylprodigiosin
“red”
• contains 7825 orfs
Genome sequencing was based on a detailed
genetic and physical map
Functional genomics of Streptomyces coelicolor
UMIST:
Bioinformatics
& metabolomics
University of
Warwick:
20 metabolite
analysis
University of Wales
Swansea:
Systematic mutagenesis
John Innes
Centre:
Proteomics &
Redirect
mutagenesis
University of
Surrey:
microarrays
Mutagenesis
Three techniques that exploit the genome sequence:
(1) 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
(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
In vitro transposon mutagenesis
(1) Mutant cosmid isolation
cosmid
Tn5062
+
transposase
+
In vitro
transposition
Transform E.coli
[AprRKanRAmpR ]
Isolate cosmid DNA
Sequence
Organisation of Tn5062
EZR1 sequencing primer
MEstop RBS
gfp
T4 apramycinR T4 oriT
ME
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
Transposon Express
•
location and description of each insertion provided at:
http://streptomyces.org.uk/S.coelicolor/index.html
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
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
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
Tn5062
Tn5066
Tn5069
Tn5070
MEstop RBS
gfp
MEstop RBS
gfp
T4 apramycinR T4 oriT
T4 Thyg hygromycinRT4 oriT
ME
ME
MEstop RBS luxAB Thyg hygromycinRT4 oriT
ME
ME tcp Tmmr
ME
tetR
Thyg hygromycinRT4 oriT
exchange cassettes can be excised as PvuII fragments and used to:
(1)
replace an existing Tn5062 insertion by Red recombination in E.coli
(2)
for de novo in vitro transposon mutagenesis
Transfer of mutated cosmid to
Streptomyces
Transfer by conjugation from
E.coli ET12567(pUZ8002) into
S. coelicolor
X
X
Select for marker replacement
[AprRKanS]
Apr
usually 1-10% of exconjugants if
gene/operon is non-essential
Km
Apr
Insertional mutagenesis of cosmid SC7C7
6279200 bp
6290053 bp
3
hybrid histidine kinase
1 kb
x 5
osaB
6
Sph I
osaA
4
Bam HI
1 2
7
SCO5750
SCO5751
response regulator
osaB complementing DNA
Mutant phenotypes
1) S. lividans
A
B
R2YE (containing 10.3% sucrose)
A: wild type
B: osaB mutant [insertion x, Tn5493]
2) S. coelicolor
R2YE
MS + 250mM KCl
MS
A:wild type B:osaA (HK) mutant [insertion #1];
C:osaB (RR) mutant+vector; D:osaB (RR) mutant
(complemented); E:osaB (RR) mutant [insertion #5]
osaAB, genes involved in osmoadapation
1 2
3
4
x 5
6
osaA
osaB
hybrid histidine
kinase
response regulator
7
•
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
Expression analysis of mutated gene
Truncated
protein
eGFP
promoter
Translation
Chromo-
Translation
Monitoring of gene expression
Transcription
ME
stop RBS
gfp
T4
some
apramycin
resistance gene
Tn5062
T4
oriT
ME
osaB is induced by
hyperosmolarity
+ sucrose
- sucrose
osaB has its own promoter
t g c a
12 – 72h
Timecourse of osaB expression: mRNA
isolated from R2YE-grown cultures
6285056 chromosome position……
cttctggtctcccgccgcgcttccgctacgagcacagtgacatcacggtgacagggtgtg
Transcription start
-35
-10
gcgacaggcggggtgcggctacgatgaccggcacaaggacgggcggcgcaagggagtcgt
cccccggggcggcacccgccggtgccgtgccaagtcctgtggacaggggaggccccacgc
Translation start
cggggcgaggagggcgggccatggtgcagaaggccaagatcctcctggtcgatgaccggc
cggagaatctgcttgcgctggaggcgatcctctcggcgctcgatcagacgctggtgcggg
Overproduction of ACT and RED in an osaB mutant
0.08
-1
0.06
A640 ml A450
0.05
-1
0.04
0.03
0.02
osaB mutant (+S)
0.01
0
20h
36h
46h
71h
84h
 wild-type
Time (h)
2
-1
 wild-type
1.5
-1
A530 ml A450
(+S)
 osaB mutant (-S)
2.5
Undecylprodigiosin...
Total blue pigments...
0.07
1
0.5
0
20h
36h
46h
Time (h)
71h
84h
(-S)
Overproduction of ACT and RED in an osaB mutant
wild-type
osaB mutant
Complemented
strain
Osmoadaptation – conclusions
•
the response regulator encoded by osaB is
essential for developmental osmoadaptation
•
osaB impacts on antibiotic production in
•
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
conditions of hyperosmolarity
(2) In vivo transposon mutagenesis
Aim
Generate a library
of transposon
induced, tagged
mutants for gene
function studies
Kay Fowler
Tn4560 (8 kb)
Derived from Tn 4556 of Streptomyces fradiae (Chung 1987)
Viomycin phosphotransferase gene for selection in Streptomyces
vph
Recombinase ?
38 bp IRs
~Tn3
38 bp IRs
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
-AlaProPheAlaAsnSerSerAspGlySerGlyThr***
Transposon delivery on pKAY1 introduced into
Streptomyces by conjugation from E. coli
1. Mix Streptomyces and E. coli on agar plate
2. Overlay with antibiotics:
Nalidixic acid or carbenicillin to kill E. coli
Viomycin to select Streptomyces::Tn
Conjugation plate
2d after overlay
>1000 colonies contain
independent Tn insertions
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
Ligation-mediated PCR for target
sequence amplification
1. Digest DNA using EagI (C’GGCCG)
2. Ligate non-phosphorylated End primer/Adaptor
3. PCR
Ligation
End primer
Genome
Adaptor
No ligation
End primer
PCR Product
Transposon
Tn primer
Tn primer
3’ nested
Target sequence
identification
• Use TA cloning to clone PCR products
• Sequence inserts
• Blast sequence against genome to identify target gene
(3) PCR targetting (Redirect)
Bertolt Gust
Tübingen
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