Mutagenesis of Actinomycetes Workshop 

July 11 –15 2005University of Wales Swansea

 

 

ActinoGENSIXTH FRAMEWORK PROGRAMME

SIXTH FRAMEWORK PROGRAMMEPRIORITY 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 ?

Undecyl-prodigiosin

“red”

Actinorhodin

“act”

• Streptomycetes makes two- thirds of all natural antibiotics

• Genome sequenced

• 8,667,507bp Chromosome

• G+C content of 72.1%

• contains 7825 orfs

Genome sequencing was based on a detailed genetic and physical map

                                             

John Innes Centre:Proteomics &Redirect mutagenesis

University of Surrey:microarrays

UMIST:Bioinformatics & metabolomics

University of Warwick:20 metabolite analysis

University of WalesSwansea:Systematic mutagenesis

Functional genomics of Streptomyces coelicolor

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

Tn5062 [AprR oriT]

+Cosmid

Target Site

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

Ref: Bishop et al 2004 Genome Research 14: 893-900

transposase

In vitro transposition

(1) Mutant cosmid isolation

+cosmid Tn5062

+

Transform E.coli [AprRKanRAmpR ]

Isolate cosmid DNA

Sequence

In vitro transposon mutagenesis

Organisation of Tn5062

MEstop RBS gfp apramycinR MEoriTT4T4

EZR1 sequencing primer

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

http://streptomyces.org.uk/S.coelicolor/index.html

• location and description of each insertion provided at:

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

Systematic mutagenesis of Streptomyces coelicolor A3(2)

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

MEstop RBS gfp apramycinR MEoriTT4T4

Tn5062

Tn5066

Tn5069

Tn5070

MEstop RBS gfp hygromycinR MEoriTT4T4 Thyg

MEstop RBS luxABluxAB hygromycinR MEoriTT4Thyg

ME Tmmr tetR hygromycinR MEoriTT4Thygtcp

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

Select for marker replacement

[AprRKanS]

usually 1-10% of exconjugants if gene/operon is non-essential

Transfer by conjugation from

E.coli ET12567(pUZ8002) into

S. coelicolor

Transfer of mutated cosmid to Streptomyces

X X

AprKm

Apr

SCO5751

1 2 3 4 x 5 6 7

Bam

HI

Sph I osaB

response regulator

osaA

hybrid histidine kinase

SCO5750

1 kb

6279200 bp 6290053 bp

Insertional mutagenesis of cosmid SC7C7

osaB complementing DNA

Mutant phenotypes

R2YE (containing 10.3% sucrose)

A: wild type

B: osaB mutant [insertion x, Tn5493]

1) S. lividans

A B

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]

2) S. coelicolorR2YE MS + 250mM KCl MS

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

osaAB, genes involved in osmoadapation

• 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

1 2 3 4 x 5 6 7

osaB

response regulator

osaA

hybrid histidine kinase

Transcription

pro

mo

ter

Truncatedprotein

Translation Translation

eGFP

Monitoring of gene expression

Expression analysis of mutated gene

Chromo-

some

ME stop RBS gfp apramycin

resistance gene

MEoriTT4T4

Tn5062

osaB is induced by hyperosmolarity

+ sucrose

- sucrose

osaB has its own promoter

Timecourse of osaB expression: mRNA isolated from R2YE-grown cultures

12 – 72h t g c a

6285056 chromosome position……

cttctggtctcccgccgcgcttccgctacgagcacagtgacatcacggtgacagggtgtg

gcgacaggcggggtgcggctacgatgaccggcacaaggacgggcggcgcaagggagtcgt

cccccggggcggcacccgccggtgccgtgccaagtcctgtggacaggggaggccccacgc

cggggcgaggagggcgggccatggtgcagaaggccaagatcctcctggtcgatgaccggc

cggagaatctgcttgcgctggaggcgatcctctcggcgctcgatcagacgctggtgcggg

-35 -10

Translation start

Transcription start

0

0.5

1

1.5

2

2.5

20h 36h 46h 71h 84h

Time (h)

Und

ecyl

prod

igio

sin...

A53

0 m

l-1 A

450-1

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

20h 36h 46h 71h 84h

Time (h)

Tot

al b

lue

pigm

ents..

.

A64

0 m

l-1 A

450-1

osaB mutant (+S)

wild-type (+S)

osaB mutant (-S)

wild-type (-S)

Overproduction of ACT and RED in an osaB mutant

wild-type osaB mutant Complemented

strain

Overproduction of ACT and RED in an osaB mutant

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

(2) In vivo transposon mutagenesis

Kay Fowler

AimGenerate a library of transposon induced, tagged mutants for gene function studies

Tn4560 (8 kb)Derived from Tn 4556 of Streptomyces fradiae (Chung 1987)

Viomycin phosphotransferase gene for selection in Streptomyces

38 bp IRsRecombinase ?

38 bp IRs~Tn3

vph

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 plate2. Overlay with antibiotics:

Nalidixic acid or carbenicillin to kill E. coliViomycin to select Streptomyces::Tn

>1000 colonies containindependent Tn insertions

Conjugation plate2d after overlay

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

End primer

PCR Product

1. Digest DNA using EagI (C’GGCCG)

2. Ligate non-phosphorylated End primer/Adaptor

3. PCR

Genome

Tn primerNo ligation

Ligation

Tn primerEnd primer

3’ nested

Transposon Adaptor

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 GustTübingen

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