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Transcript
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 Isolation of a glpD Mutant glpD::Tn WT glpD::Tn [kb] WT M tants 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 The Mutant Collection so far … Myco lasma 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 regulator) hrcA