Monday, June 11, 2012

Development of a Tn-Seq method to study gene expression in Salmonella sp.

The EZ-Tn5™ R6Kγori/KAN-2 Transposome has been used for elucidation of bacterial gene structure and function for the last 12 years. In a recent publication by Khatiwara et al., it was used to mutagenize a strain of Salmonella enterica serotype Typhimurium to link specific gene expression information to the gene of interest. The mutagenesis work also demonstrates a new method of transposome insertion sequencing using Illumina technology instead of older Sanger sequencing techniques.

The researchers noted that the 19-base mosaic end (ME) of the transposome contains a DNA sequence similar to the recognition sequence of type II restriction enzyme BsmF I (recognition site 5'-GGGAC(N)10|/3'-CCCTG(N)14|5'), except for one nucleotide. Since BsmF I cuts the site 14 bp away from the recognition site, the researchers exploited this finding to extract 12-nucleotide sequences immediately adjacent to Tn5 insertion sites. These 12-bp transposon-junction sequences were selectively amplified from a mutant library and sequenced. The resulting profile provides information on both the identity and relative quantity of each insertion in the library. Next, the researchers tested whether any mutagenesis in the ME would negatively affect the activity of the transposome. The results suggested that a single nucleotide change in the ME would not affect the transposition reaction significantly, as determined by comparative numbers of Kan-resistant colonies after mutagenesis.

One of the MEs was mutated from A to G to create the BsmF I site. This change allowed the ligation of a "Tn-Seq" linker to the mutated ME, which provided an Illumina single-read sequencing priming site and index into the molecule. The transposome generated was electroporated into the Salmonella spp. bacterium and allowed to generate colonies as per the normal in vivo transposition protocol. Mutants were then screened for changes in activity of selected loci. Individual mutants with expression changes in the same locus were pooled, digested with BsmF I, ligated to the Tn-Seq linkers and amplified by PCR, using a short extension time to generate short amplicons for single-read Illumina sequencing with five separate indices.

Sequencing information permitted mapping of the transposition mutants and then mapping the transposon insertions that provided changes in gene expression activity. The study highlights the adaptations researchers are developing to enable deep sequencing applications for gene expression studies in bacteria.

ResearchBlogging.orgKhatiwara, A. et al. (2012). Genome Scanning for Conditionally Essential Genes in Salmonella enterica Serotype Typhimurium Applied and Environmental Microbiology, 78 (9), 3098-3107 DOI: 10.1128/AEM.06865-11

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