• 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 offtranslation?
Does the expression of M. genitalium recombinationgenes (ruvAB, recU) allow recombination?
nothing works
Isolation of M. pneumoniae Mutant Strains Myco lasma
M tantsu
p
Idea: Development of an efficient screening systemfor 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 tantsu
p
920 clones: 99% probability to find an insertionin any non-essential gene
The tranposon mutant library: 2976 individual clones
probability is about 99.999%
Isolation of M. pneumoniae Mutants by
Haystack Cloning
Myco lasma
M tantsu
p
How many individual random transposon insertion mutants have to be collected to obtain a desired mutant with a minimum probability of 99% ?
How do we find the needle?
We need an ordered collection of the tranposonmutants!
• 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
Haystack Cloning
Myco lasma
M tantsu
p
Isolation of M. pneumoniae Mutants by
Haystack Cloning
Myco lasma
M tantsu
p
goi
pMT85
Tn
saturated transposon mutagenesis
pick 3000 transposants
make pools of 50 transposants
grow transposant library
search 60 pools by PCR for goi-Tn junctionusing primers specific for the goi and the Tn
identify positive pools
subscreen to identify the causative clone within a positive pool
control
wt
H3
G3
F3
E3
D3
C3
B3
A3
H2
G2
F2
E2
D2
C2
B2
A2
H1
G1
F1
E1
D1
0,950,83
0,56
mini-Tn4001
glpD
SH7
SH29
Identification of the pool
Identification of positive candidates
Isolation of a glpD Mutant Myco lasma
M tantsu
p
SH29 →...GTGCCATGGGTTTTTACACAATTATACGGACTTTATCATCAACTTGCTTACTAAT...
26 bp inverted repeat
8 bp target duplication
glpD pos 555
Isolation of a glpD Mutant Myco lasma
M tantsu
p
MPN052 glpK
EcoRV NdeI
aac-ahpD
B
glpDA
21,3
5,14,33,5
2,0
1,61,4
[kb] WT
glpD::
Tn
probe A
WT
glpD::
Tn
probe B
The Mutant Collection so far … Myco lasma
M tantsu
p
Mutants isolated No mutants foundglpD ?essential genes?hprK noxprpC glpFldh glpKmpn474: surface protein mpn239 (GntR-like mpn372 (cytotoxin, regulator) ADP-ribosyltransferase) hrcA
