Genetic tools to enhance the study of gene function and regulation in Staphylococcus aureus

Jeffrey L. Bose, Paul D Fey, Kenneth W Bayles

Research output: Contribution to journalArticle

83 Citations (Scopus)

Abstract

The bursa aurealis transposon has been used to create transposon insertion libraries of Bacillus anthracis and Staphylococcus aureus. To provide a set of genetic tools to enhance the utility of these libraries, we generated an allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes. These tools were tested in the Nebraska Transposon Mutant Library (NTML), containing defined transposon insertions in 1,952 nonessential S. aureus genes. First, we generated a plasmid that allows researchers to replace the genes encoding green fluorescent protein (GFP) and erythromycin resistance in the transposon with a noncoding DNA fragment, leaving a markerless mutation within the chromosome. Second, we produced allelic-exchange plasmids to replace the transposon with alternate antibiotic resistance cassettes encoding tetracycline, kanamycin, and spectinomycin resistance, allowing for the simultaneous selection of multiple chromosomal mutations. Third, we generated a series of fluorescent reporter constructs that, after allelic exchange, generate transcriptional reporters encoding codon-optimized enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), DsRed.T3(DNT), and eqFP650, as well as superfolder green fluorescent protein (sGFP). Overall, combining the NTML with this allelic-exchange system provides an unparalleled resource for the study of S. aureus.

Original languageEnglish (US)
Pages (from-to)2218-2224
Number of pages7
JournalApplied and environmental microbiology
Volume79
Issue number7
DOIs
StatePublished - Apr 1 2013

Fingerprint

transposons
Staphylococcus aureus
2,6-dinitrotoluene
Green Fluorescent Proteins
Libraries
protein
gene
Plasmids
plasmid
Kanamycin Resistance
Spectinomycin
Genes
Bacillus anthracis
mutation
Mutation
genes
Erythromycin
Microbial Drug Resistance
Tetracycline
Genetic Markers

ASJC Scopus subject areas

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

Cite this

Genetic tools to enhance the study of gene function and regulation in Staphylococcus aureus. / Bose, Jeffrey L.; Fey, Paul D; Bayles, Kenneth W.

In: Applied and environmental microbiology, Vol. 79, No. 7, 01.04.2013, p. 2218-2224.

Research output: Contribution to journalArticle

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