1887

Abstract

A set of vectors for improved tetracycline-dependent gene regulation in is presented. Plasmid pRAB11 was generated from pRMC2 by adding a second operator within the TetR-regulated promoter P. 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 , or . Also, in plasmid pCG261, the pRAB11 –P regulatory architecture permitted tight repression and a stepwise increase in transcript amounts of the target gene (putative RNase) correlated with rising ATc concentrations. Additionally, pRAB11-derived vectors harbouring semi-rationally designed P-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 , which exhibits three amino acid exchanges and binds to in the presence of ATc, yielded an efficiently co-repressible vector within the pRAB11 system. Intriguingly, was found to contain a fourth mutation only after propagation in . We predict that the described vectors constitute valuable tools for staphylococcal genetics.

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2011-12-01
2020-01-27
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