STM is a two step procedure. The first step involving the pools of transposon molecules carrying unique sequence is used to generate a library of tagged mutants. Initially in the original method (Hensel et al., 1995) a complex mixture of 2 stranded DNA tags carried on transposon mini TN5Km2 was used to sequence library of random insertional mutants. The tray consists of a unique 40 bp sequence flanked by a common arm of 20 bp at both ends. There are no presents of Hind111, KPN1, Pst1 or sal 1 restriction sites at the variable region which were used for subsequent cloning of restriction tags into suicide vector. The double stranded DNA then incorporated into putmini TM5Km2 by ligation and maintained in Ecoli as pools of transposon tagged by random signature sequences. The transposon transferred into the recipient host strain by conjugation to generate a bank of mutants that carries a random mini TN5 insertion. The strains then stored individually in a 96 well microtitter plate and used to prepare dot blots. (Hensel et al., 1995)
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The second step involving in vivo screening of the library. In the original methods the suitability of tags was determined prior to the use of the PCR amplification, labelling, and hybridization to blots of DNA probes prepared from the mutants (Hensel et al., 1995). This mutants that yield clear signal on autoradiogram were reassembled into a new 96 well pools and subjected to a selective process that involved infection of an animal. (Hensel et al., 1995).Following the selective screening the mutants recover and PCR amplification was used to prepare labelled probes representing the tags found in the preselected population input pool, and those recovered from the pool. Hybridization of the labelled probes from both input and output pools to dot blots of arrayed mutants and identification of tags that were host in the output allows the isolation of strain that were unable to survive during the infection process. The nucleotide sequence of the recovered mutants then determined by sequencing method. STM relies on the ability of the mutants to propagate in vivo as a mixed population and can identify only nonvirulent mutants that cannot be transcomplemented by other virulent strains present in some inoculum (Chiang et al., 1999). The application of STM requires that a number of parameters be considered in order to obtain reproducible identification results.
Figure shows the process of STM
Piotr Mazurkiewicz, Christoph M. Tang, Charles Boone & David W. HoldenNature Reviews Genetics 7, 929-939 (December 2006)Signature-tagged mutagenesis: barcoding mutants for genome-wide screens
There are a few crucial steps involved in the generation and screening of STM. One of the critical steps in STM depends on the high efficiency of the procedure chosen to generate random tag insertion in the genome, this is not easily achievable as mutation in the important gene can lead to lethal phenotype and it wonââ‚¬â„¢t be liable (Lehoux and Levesque., 1999). Usually the difficulty associated with STM is due to lack of a suitable in vivo transposition system. Several modifications to the standard in vivo procedure have proposed to improve the efficiency of insertional mutagenesis.
A short random genomic DNA was used to construct a library of signature tagged mutants in effort to indentify a virulence gene from streptococcus pneumonia.
The fragments inserted into a suicide plasmid vector carrying molecular tag. (Polissi et al., 1998). The transposon molecule which facilitate homologous recombination between tag carrying molecule and the host chromosome was replaced by randomly selected 400-600 bp chromosomal fragments because the in vivo transposition was shown not producing random mutation in streptococcus mutants. Another modification in STM is involving utilization of shuttle vectors where the in vivo transposition is carried out in Ecoli then the resulting plasmid are subsequently transformed into the target organism. Shuttle mutagenesis using signature tagged transposon was employed to generate a library of Neisseria meningitides random mutants which allowed in vivo screening in a bacterium where it normally impossible to use STM.
In the method developed by Hensel and co workers (1995) the specificity of DNA tags was tested by hybridisation prior to use in an animal infection model. Several modifications done to avoid the prescreening process to increase sensivity. Major criteria used for tag selection based on efficiency of amplification and labelling as well as high hybridization specificity, each tagged transposon from the selected cell was used to generate 96 separate pools of mutants. The prescreening of the mutants is no longer required as the tag identity for each pool is known. Moreover the utilization of highly specific preselected tags allowed the hybridization analysis to be carried out by plasmid or tag DNA blots rather than cloning blots, this again increase the sensitivity of the assay. (Chiang et al., 1998).
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Another modification was the screening by hybridization was replaced by PCR detection with a set of reusable tags. For this method 12 specific tag design for optimal PCR amplification were generated and used to construct 12 library of mutant, which was arrayed in 96 well micro titter plates. The mutants had same tag but presumably inserted at different location in the bacterial chromosome. One mutant was picked to form 96 pool of a 12 uniquely tagged mutants that were used for in vivo screening and identification of attenuated virulence genes.
Another problem that affects the results of STM is the duration of the infection, postinoculation time point which mutant recovers from hybridisation screening. When the incubation time is too short there will be insufficient attenuation where virulence strains donââ‚¬â„¢t have enough time outgrow the attenuated mutant. Whereas long incubation will cause some virulent strains to outgrow other virulent strains in a non specific manner.
STM has been very exciting technique and since itââ‚¬â„¢s been revolutionised the field of the bacterial pathogenesis. In the near future one can envisage by researching on this technology to wide range of animal models, infection times and routes for more challenging field of persistence and reactivation disease.