Background: Next-generation sequencing of transposon-genome junctions from a saturated bacterial mutant library (Tn-seq) is a powerful tool that permits genome-wide determination of the contribution of genes to fitness of the organism under a wide range of experimental conditions. We report development, testing, and results from a Tn-seq system for use in Streptococcus agalactiae (group B Streptococcus; GBS), an important cause of neonatal sepsis. Methods: Our method uses a Himar1 mini-transposon that inserts at genomic TA dinucleotide sites, delivered to GBS on a temperature-sensitive plasmid that is subsequently cured from the bacterial population. In order to establish the GBS essential genome, we performed Tn-seq on DNA collected from three independent mutant libraries-with at least 135,000 mutants per library-at serial 24 h time points after outgrowth in rich media. Results: After statistical analysis of transposon insertion density and distribution, we identified 13.5% of genes as essential and 1.2% as critical, with high levels of reproducibility. Essential and critical genes are enriched for fundamental cellular housekeeping functions, such as acyl-tRNA biosynthesis, nucleotide metabolism, and glycolysis. We further validated our system by comparing fitness assignments of homologous genes in GBS and a close bacterial relative, Streptococcus pyogenes, which demonstrated 93% concordance. Finally, we used our fitness assignments to identify signal transduction pathway components predicted to be essential or critical in GBS. Conclusions: We believe that our baseline fitness assignments will be a valuable tool for GBS researchers and that our system has the potential to reveal key pathogenesis gene networks and potential therapeutic/preventative targets.
CITATION STYLE
Hooven, T. A., Catomeris, A. J., Akabas, L. H., Randis, T. M., Maskell, D. J., Peters, S. E., … Ratner, A. J. (2016). The essential genome of Streptococcus agalactiae. BMC Genomics, 17(1). https://doi.org/10.1186/s12864-016-2741-z
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