title: Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus.
abstract: A set of vectors for improved tetracycline-dependent gene regulation in Staphylococcus aureus is presented. Plasmid pRAB11 was generated from pRMC2 by adding a second tet operator within the TetR-regulated promoter P(xyl/tet). Pronounced repression was observed in the absence of anhydrotetracycline (ATc) combined with high induction in the presence of the drug, as demonstrated for pRAB11 bearing staphylococcal nuclease nuc1, lacZ or gfp. Also, in plasmid pCG261, the pRAB11 tetR-P(xyl/tet) regulatory architecture permitted tight repression and a stepwise increase in transcript amounts of the target gene rny (putative RNase) correlated with rising ATc concentrations. Additionally, pRAB11-derived vectors harbouring semi-rationally designed P(xyl/tet)-like fragments, mutated at up to six defined positions, were constructed. Sixteen mutant sequences with single to quadruple exchanges were analysed for transcriptional strength and ATc-dependent inducibility. A set of promoters with gradually decreased activities and improved repression is presented. Finally, the implementation of reverse TetR revtetR-r2, which exhibits three amino acid exchanges and binds to tetO in the presence of ATc, yielded an efficiently co-repressible vector within the pRAB11 system. Intriguingly, revtetR was found to contain a fourth mutation only after propagation in S. aureus. We predict that the described vectors constitute valuable tools for staphylococcal genetics.
authors.affiliation: Lehrbereich Mikrobielle Genetik, Waldhäuser Str. 70/8, Eberhard Karls Universität Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, 72076 Tübingen, Germany.
authors.forename: Leonie
authors.initials: L
authors.lastname: Helle
authors.affiliation: Lehrbereich Mikrobielle Genetik, Waldhäuser Str. 70/8, Eberhard Karls Universität Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, 72076 Tübingen, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Kull
authors.affiliation: Lehrbereich Mikrobielle Genetik, Waldhäuser Str. 70/8, Eberhard Karls Universität Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, 72076 Tübingen, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Mayer
authors.affiliation: Institut für Med. Mikrobiologie und Hygiene, Universitätsklinikum Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, 72076 Tübingen, Germany.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Marincola
authors.affiliation: Lehrbereich Mikrobielle Genetik, Waldhäuser Str. 70/8, Eberhard Karls Universität Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, 72076 Tübingen, Germany.
authors.forename: Marc-Eric
authors.initials: ME
authors.lastname: Zelder
authors.affiliation: Institut für Med. Mikrobiologie und Hygiene, Universitätsklinikum Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, 72076 Tübingen, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Goerke
authors.affiliation: Institut für Med. Mikrobiologie und Hygiene, Universitätsklinikum Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, 72076 Tübingen, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Wolz
authors.affiliation: Lehrbereich Mikrobielle Genetik, Waldhäuser Str. 70/8, Eberhard Karls Universität Tübingen, Interfakultäres Institut für Mikrobiologie und Infektionsmedizin, 72076 Tübingen, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Bertram
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013211:Staphylococcus aureus / Q000235:genetics*; D013752:Tetracycline / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D004269:DNA, Bacterial; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline