Transcript Informationsverarbeitung in Bakterien

Isolation of M. pneumoniae Mutant Strains
Myco lasma
M tants
• no natural recombination system present (genes are not
expressed)
• our attempts:
Does recombination work in spite of the missing
recombination system?
Does the expression of an antisense RNA switch off
translation?
Does the expression of M. genitalium recombination
genes (ruvAB, recU) allow recombination?
nothing works
Isolation of M. pneumoniae Mutants by
Haystack Cloning
Myco lasma
M tants
Idea: Development of an efficient screening system
for a large pool of transposon mutants
Basic assumptions:
816 kb, 688 genes → 1011 bp/gene (Himmelreich et al. 1996)
probably 180 – 215 non-essential genes (Hutchison et al. 1999)
ergo:
essential genome: ~ 600 kb
non-essential genome: ~ 200 kb
Isolation of M. pneumoniae Mutants by
Haystack Cloning
Myco lasma
M tants
How many individual random transposon insertion mutants
have to be collected to obtain a desired mutant with a
minimum probability of 99% ?
920 clones: 99% probability to find an insertion
in any non-essential gene
The tranposon mutant library: 2976 individual clones
probability is about 99.999%
Isolation of M. pneumoniae Mutants by
Myco lasma
Haystack Cloning
M tants
How do we find the needle?
We need an ordered collection of the tranposon
mutants!
• 60 pools of each 50 clones were prepared
• PCR with primers of goi and transposon to identify
positive pools
• PCR with individual clones to identify the mutant
• the system can be used for multiplex analysis
Isolation of M. pneumoniae Mutants by
Myco lasma
M tants
Haystack Cloning
pMT85
saturated transposon mutagenesis
pick 3000 transposants
grow transposant library
make pools of 50 transposants
Tn
search 60 pools by PCR for goi-Tn junction
using primers specific for the goi and the Tn
goi
identify positive pools
subscreen to identify the causative clone within a positive pool
Myco lasma
Isolation of a glpD Mutant
M tants
Identification of the pool
mini-Tn4001
SH29
glpD
0,95
0,83
0,56
SH7
control
wt
H3
G3
F3
E3
D3
C3
B3
A3
H2
G2
F2
E2
D2
C2
B2
A2
H1
G1
F1
E1
D1
Identification of positive candidates
Myco lasma
WT
glpD::Tn
M tants
glpD::Tn
[kb]
WT
Isolation of a glpD Mutant
21,3
aac-ahpD
5,1
4,3
3,5
B
MPN052
2,0
1,6
1,4
EcoRV
glpD
A
glpK
NdeI
probe B
probe A
26 bp
inverted repeat
8 bp
target duplication
SH29
→...GTGCCATGGGTTTTTACACAATTATACGGACTTTATCATCAACTTGCTTACTAAT...
glpD pos 555
Myco lasma
The Mutant Collection so far …
M tants
Mutants isolated
glpD
hprK
prpC
ldh
mpn474: surface protein
mpn372 (cytotoxin,
ADP-ribosyltransferase)
No mutants found
?essential genes?
nox
glpF
glpK
mpn239 (GntR-like
hrcA
regulator)