A simple plasmid-based system that allows rapid generation of tightly controlled gene expression in Staphylococcus aureus.
We have established a plasmid-based system that enables tightly controlled gene expression and the generation of GFP fusion proteins in Staphylococcus aureus simply and rapidly. This system takes advantage of an Escherichia coli-S. aureus shuttle vector that contains the replication region of the S. aureus theta-mode multiresistance plasmid pSK41, and is therefore a stable low-copy-number plasmid in the latter organism. This vector also contains a multiple cloning site downstream of the IPTG-inducible Pspac promoter for insertion of the gene of interest. Production of encoded proteins can be stringently regulated in an IPTG-dependent manner by introducing a pE194-based plasmid, pGL485, carrying a constitutively expressed lacI gene. Using GFP fusions to two essential proteins of S. aureus, FtsZ and NusA, we showed that our plasmid allowed tightly controlled gene expression and accurate localization of fusion proteins with no detrimental effect on cells at low inducer concentrations. At higher IPTG concentrations, we obtained sixfold overproduction of protein compared with wild-type levels, with FtsZ-GFP-expressing cells showing lysis and delocalized fluorescence, while NusA-GFP showed only delocalized fluorescence. These results show that our system is capable of titratable induction of gene expression for localization or overexpression studies.
The ithree Institute, University of Technology Sydney, Ultimo, New South Wales 2007, Australia.
Andrew T F
ATF
Liew
The ithree Institute, University of Technology Sydney, Ultimo, New South Wales 2007, Australia.
Torsten
T
Theis
Microbiology and Infectious Diseases, School of Medicine, University of Western Sydney, New South Wales 2751, Australia.|School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia.
Slade O
SO
Jensen
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.
Jorge
J
Garcia-Lara
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.
Simon J
SJ
Foster
School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia.
Neville
N
Firth
School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales 2308, Australia.
Peter J
PJ
Lewis
The ithree Institute, University of Technology Sydney, Ultimo, New South Wales 2007, Australia.
Elizabeth J
EJ
Harry
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D007544:Isopropyl Thiogalactoside