title: The essential genome of Streptococcus agalactiae.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Pediatrics, Columbia University, New York, NY, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Hooven
authors.affiliation: Department of Pediatrics, Division of Pediatric Infectious Diseases, New York University School of Medicine, 550 First Avenue (MSB 223), New York, NY, 10016, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Catomeris
authors.affiliation: Department of Pediatrics, Columbia University, New York, NY, USA.
authors.forename: Leor H
authors.initials: LH
authors.lastname: Akabas
authors.affiliation: Department of Pediatrics, Division of Pediatric Infectious Diseases, New York University School of Medicine, 550 First Avenue (MSB 223), New York, NY, 10016, USA.
authors.forename: Tara M
authors.initials: TM
authors.lastname: Randis
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Maskell
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Peters
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Sandra
authors.initials: S
authors.lastname: Ott
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Ivette
authors.initials: I
authors.lastname: Santana-Cruz
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Luke J
authors.initials: LJ
authors.lastname: Tallon
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Hervé
authors.initials: H
authors.lastname: Tettelin
authors.affiliation: Department of Pediatrics, Division of Pediatric Infectious Diseases, New York University School of Medicine, 550 First Avenue (MSB 223), New York, NY, 10016, USA. Adam.Ratner@nyumc.org.|Department of Microbiology, New York University School of Medicine, New York, NY, USA. Adam.Ratner@nyumc.org.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Ratner
mesh_terms: D004251:DNA Transposable Elements; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D016254:Mutagenesis, Insertional; D015398:Signal Transduction; D013290:Streptococcal Infections / Q000382:microbiology*; D013292:Streptococcus agalactiae / Q000235:genetics* / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements