title: A new platform for ultra-high density Staphylococcus aureus transposon libraries.
abstract: BACKGROUND
  Staphylococcus aureus readily develops resistance to antibiotics and achieving effective therapies to overcome resistance requires in-depth understanding of S. aureus biology. High throughput, parallel-sequencing methods for analyzing transposon mutant libraries have the potential to revolutionize studies of S. aureus, but the genetic tools to take advantage of the power of next generation sequencing have not been fully developed.
  
  
  RESULTS
  Here we report a phage-based transposition system to make ultra-high density transposon libraries for genome-wide analysis of mutant fitness in any Φ11-transducible S. aureus strain. The high efficiency of the delivery system has made it possible to multiplex transposon cassettes containing different regulatory elements in order to make libraries in which genes are over- or under-expressed as well as deleted. By incorporating transposon-specific barcodes into the cassettes, we can evaluate how null mutations and changes in gene expression levels affect fitness in a single sequencing data set. Demonstrating the power of the system, we have prepared a library containing more than 690,000 unique insertions. Because one unique feature of the phage-based approach is that temperature-sensitive mutants are retained, we have carried out a genome-wide study of S. aureus genes involved in withstanding temperature stress. We find that many genes previously identified as essential are temperature sensitive and also identify a number of genes that, when disrupted, confer a growth advantage at elevated temperatures.
  
  
  CONCLUSIONS
  The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA. marinasantiago@fas.harvard.edu.
authors.forename: Marina
authors.initials: M
authors.lastname: Santiago
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA. lmatano@g.harvard.edu.
authors.forename: Leigh M
authors.initials: LM
authors.lastname: Matano
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA. smoussa@hms.harvard.edu.
authors.forename: Samir H
authors.initials: SH
authors.lastname: Moussa
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA. michael_gilmore@meei.harvard.edu.|Department of Ophthalmology, Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, MA, 02114, USA. michael_gilmore@meei.harvard.edu.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Gilmore
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA. suzanne_walker@hms.harvard.edu.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Walker
authors.affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA. txm50@psu.edu.|Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, 16802, USA. txm50@psu.edu.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Meredith
mesh_terms: D001435:Bacteriophages / Q000235:genetics*; D004251:DNA Transposable Elements; D015870:Gene Expression; D015723:Gene Library; D020043:Genes, Essential; D005822:Genetic Vectors / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D009154:Mutation; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D013696:Temperature
chemical_list: D004251:DNA Transposable Elements