Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus
| Autor: | Ralph Bertram, Gabriella Marincola, Christiane Goerke, Leonie Helle, Sonja Mayer, Melanie Kull, Marc-Eric Zelder, Christiane Wolz |
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| Rok vydání: | 2011 |
| Předmět: |
DNA
Bacterial Genetics Microbial Transcriptional Activation Staphylococcus aureus Operator Regions Genetic Operator (biology) Genetic Vectors Molecular Sequence Data Mutant Gene Expression lac operon Biology Microbiology chemistry.chemical_compound Plasmid TetR Promoter Regions Genetic Molecular Biology Psychological repression Regulation of gene expression Promoter Sequence Analysis DNA Tetracycline Molecular biology Repressor Proteins chemistry Plasmids |
| Zdroj: | Microbiology. 157:3314-3323 |
| ISSN: | 1465-2080 1350-0872 |
| Popis: | A set of vectors for improved tetracycline-dependent gene regulation inStaphylococcus aureusis presented. Plasmid pRAB11 was generated from pRMC2 by adding a secondtetoperator within the TetR-regulated promoter Pxyl/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 nucleasenuc1,lacZorgfp. Also, in plasmid pCG261, the pRAB11tetR–Pxyl/tetregulatory architecture permitted tight repression and a stepwise increase in transcript amounts of the target generny(putative RNase) correlated with rising ATc concentrations. Additionally, pRAB11-derived vectors harbouring semi-rationally designed Pxyl/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 TetRrevtetR-r2, which exhibits three amino acid exchanges and binds totetOin the presence of ATc, yielded an efficiently co-repressible vector within the pRAB11 system. Intriguingly,revtetRwas found to contain a fourth mutation only after propagation inS. aureus. We predict that the described vectors constitute valuable tools for staphylococcal genetics. |
| Databáze: | OpenAIRE |
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