Protocols for Tn-seq Analyses in the Group A Streptococcus.

ABSTRACT

Transposon-sequencing (Tn-seq) has revolutionized forward-genetic analyses to study genotype-phenotype associations and interrogate bacterial cell physiology. The Tn-seq approach allows the en masse monitoring of highly complex mutant libraries, leveraging massive parallel DNA sequencing as a means to characterize the composition of these mutant pools on a genome-scale with unprecedented nucleotide-level high resolution. In this chapter, we present step-by-step protocols for Tn-seq analyses in the human pathogen Streptococcus pyogenes (Group A Streptococcus or GAS) using the mariner-based Krmit transposon. We detail how to generate highly complex Krmit mutant libraries in GAS and the en masse production of Krmit insertion tags for Illumina sequencing of the transposon-genome junctions for Tn-seq analyses. Most of the protocols presented here were developed and implemented using the S. pyogenes M1T1 serotype clinical isolate 5448, but they have been successfully applied to multiple GAS serotypes as well as other pathogenic Streptococci.