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BMC Genomics. 2016 May 26;17:406. doi: 10.1186/s12864-016-2741-z.

The essential genome of Streptococcus agalactiae.

Author information

1
Department of Pediatrics, Columbia University, New York, NY, USA.
2
Department of Pediatrics, Division of Pediatric Infectious Diseases, New York University School of Medicine, 550 First Avenue (MSB 223), New York, NY, 10016, USA.
3
Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.
4
Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA.
5
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.
6
Department of Microbiology, New York University School of Medicine, New York, NY, USA. Adam.Ratner@nyumc.org.

Abstract

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.

PMID:
27229469
PMCID:
PMC4881062
DOI:
10.1186/s12864-016-2741-z
[Indexed for MEDLINE]
Free PMC Article

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