Informationsverarbeitung in Bakterien

1
Isolation of M. pneumoniae Mutant Strains

• 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
2
Isolation of M. pneumoniae Mutants by Haystack
Cloning
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
3
Isolation of M. pneumoniae Mutants by Haystack
Cloning
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
4
Isolation of M. pneumoniae Mutants by Haystack
Cloning
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

5
Isolation of M. pneumoniae Mutants by Haystack
Cloning
saturated transposon mutagenesis
pMT85
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
6
Isolation of a glpD Mutant
Identification of the pool
Identification of positive candidates
7
Isolation of a glpD Mutant
glpDTn
glpDTn
WT
WT
kb
21,3
aac-ahpD
5,1
4,3
B
3,5
glpD
MPN052
glpK
A
2,0
1,6
EcoRV
NdeI
1,4
probe A
probe B
SH29 ?...GTGCCATGGGTTTTTACACAATTATACGGACTTTATCA
TCAACTTGCTTACTAAT...
glpD pos 555
8
The Mutant Collection so far
Mutants isolated No mutants found glpD
?essential genes? hprK nox prpC gl
pF ldh glpK mpn474 surface
protein mpn239 (GntR-like mpn372 (cytotoxin,
regulator) ADP-ribosyltransferase) hrcA