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Plasmid. 2015 May;79:8-14. doi: 10.1016/j.plasmid.2015.02.002. Epub 2015 Feb 7.

An rpsL-based allelic exchange vector for Staphylococcus aureus.

Author information

1
Skirball Institute Program in Molecular Pathogenesis, Departments of Microbiology and Medicine, New York University Medical Center, New York, NY 10016, USA. Electronic address: john.chen@med.nyu.edu.
2
Skirball Institute Program in Molecular Pathogenesis, Departments of Microbiology and Medicine, New York University Medical Center, New York, NY 10016, USA.
3
Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA.
4
Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK.
5
Departments of Medicine and Microbiology, New York University School of Medicine, New York, NY 10016, USA.
6
Skirball Institute Program in Molecular Pathogenesis, Departments of Microbiology and Medicine, New York University Medical Center, New York, NY 10016, USA. Electronic address: richard.novick@med.nyu.edu.

Abstract

Staphylococcus aureus is one of the most successful bacterial pathogens, harboring a vast repertoire of virulence factors in its arsenal. As such, the genetic manipulation of S. aureus chromosomal DNA is an important tool for the study of genes involved in virulence and survival in the host. Previously reported allelic exchange vectors for S. aureus are shuttle vectors that can be propagated in Escherichia coli, so that standard genetic manipulations can be carried out. Most of the vectors currently in use carry the temperature-sensitive replicon (pE194ts) that was originally developed for use in Bacillus subtilis. Here we show that in S. aureus, the thermosensitivity of a pE194ts vector is incomplete at standard non-permissive temperatures (42 °C), and replication of the plasmid is impaired but not abolished. We report rpsL-based counterselection vectors, with an improved temperature-sensitive replicon (pT181 repC3) that is completely blocked for replication in S. aureus at non-permissive and standard growth temperature (37 °C). We also describe a set of temperature-sensitive vectors that can be cured at standard growth temperature. These vectors provide highly effective tools for rapidly generating allelic replacement mutations and curing expression plasmids, and expand the genetic tool set available for the study of S. aureus.

KEYWORDS:

Allelic exchange; Counterselection; Staphylococcus aureus; Streptomycin; Temperature-sensitive; rpsL

PMID:
25659529
PMCID:
PMC4628793
DOI:
10.1016/j.plasmid.2015.02.002
[Indexed for MEDLINE]
Free PMC Article

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