No natural recombination system present (genes are not expressed) our attempts: Does recombination work in spite of the missing recombination system? Does.

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Presentation transcript:

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 Mutant Strains Myco lasma M tants u p

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 u p

920 clones: 99% probability to find an insertion in any non-essential gene The tranposon mutant library: 2976 individual clones probability is about % Isolation of M. pneumoniae Mutants by Haystack Cloning Myco lasma M tants u 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 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 Haystack Cloning Myco lasma M tants u p

Isolation of M. pneumoniae Mutants by Haystack Cloning Myco lasma M tants u 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 junction using primers specific for the goi and the Tn identify positive pools subscreen to identify the causative clone within a positive pool

controlwtH3G3F3E3D3C3B3A3H2G2F2E2D2C2B2A2H1G1F1E1D1 0,95 0,83 0,56 mini-Tn4001 glpD SH7 SH29 Identification of the pool Identification of positive candidates Isolation of a glpD Mutant Myco lasma M tants u p

SH29 →...GTGCCATGGGTTTTTACACAATTATACGGACTTTATCATCAACTTGCTTACTAAT bp inverted repeat 8 bp target duplication glpD pos 555 Isolation of a glpD Mutant Myco lasma M tants u p MPN052 glpK EcoRVNdeI aac-ahpD B glpD A 21,3 5,1 4,3 3,5 2,0 1,6 1,4 [kb] WT glpD::Tn probe A WT glpD::Tn probe B

The Mutant Collection so far … Myco lasma M tants u p Mutants isolatedNo mutants found glpD ?essential genes? hprKnox prpCglpF ldhglpK mpn474: surface proteinmpn239 (GntR-like mpn372 (cytotoxin, regulator) ADP-ribosyltransferase)hrcA