10.17863/CAM.10063
Hooven, Thomas A
0000-0003-1959-186X
Catomeris, Andrew J
Akabas, Leor H
Randis, Tara M
0000-0003-0348-6766
Maskell, Duncan
0000-0002-5065-653X
Peters, Sarah E
Ott, Sandra
Santana-Cruz, Ivette
Tallon, Luke J
Tettelin, Hervé
Ratner, Adam J
0000-0003-1761-794X
The essential genome of Streptococcus agalactiae.
Apollo - University of Cambridge Repository (staging)
2016
Streptococcus agalactiae
Streptococcal Infections
DNA Transposable Elements
Mutagenesis, Insertional
Genomics
Signal Transduction
Gene Library
Genome, Bacterial
Genetic Vectors
Apollo - University of Cambridge Repository (staging)
Apollo - University of Cambridge Repository (staging)
2016-05-26
Article
1471-2164
1471-2164
Attribution 4.0 International
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.