Format

Send to

Choose Destination
Appl Environ Microbiol. 2013 Apr;79(7):2218-24. doi: 10.1128/AEM.00136-13. Epub 2013 Jan 25.

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

Author information

1
Center for Staphylococcal Research, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

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.

PMID:
23354696
PMCID:
PMC3623228
DOI:
10.1128/AEM.00136-13
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center