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New range of vectors with a stringent 5-fluoroorotic acid-based counterselection system for generating mutants by allelic replacement in Staphylococcus aureus.

Applied and environmental microbiology (2012-03-27)
Peter Redder, Patrick Linder
ABSTRACT

We have developed a range of vectors for allelic replacements in Staphylococcus aureus to facilitate genetic work in this opportunistic pathogen. The central feature of the vector range is a selection/counterselection system that takes advantage of the 5-fluoroorotic acid (FOA) resistance and pyrimidine prototrophy caused by the loss and gain, respectively, of the pyrF and pyrE genes. This system allows for stringent counterselection of the vectors during the second homologous recombination of a classic allelic replacement. The basic vector pRLY2, which contains the pyrFE genes from Bacillus subtilis, was combined with chloramphenicol, erythromycin, and tetracycline resistance genes and four different versions of nonreplicative or conditionally replicative origins of replication. The choice between these 12 different pRLY vectors allows for high versatility and ensures that the vectors can be used in virtually any genetic background. Finally, as proof of concept, we present six deletions or modifications of components in the S. aureus degradosome as well as the operon containing the cshB DEAD box helicase.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Orotic acid, ≥98% (titration), anhydrous
Sigma-Aldrich
REDTaq® ReadyMix PCR Reaction Mix, Complete PCR reagent with standard Taq DNA Polymerase and inert dye