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

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 non-essential S. aureus genes. First, we generated a plasmid that allows researchers to replace the genes encoding GFP and erythromycin resistance in the transposon with a non-coding DNA fragment, leaving a marker-less 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, following allelic exchange, generate transcriptional reporters encoding codon-optimized ECFP, EYFP, DsRed.T3(DNT), and eqFP650, as well as sGFP. Overall, combining the NTML with this allelic exchange system provides an unparalleled resource for the study of S. aureus.