New architectures for Tet-on and Tet-off regulation in Staphylococcus aureus.
Inducible expression is a valuable approach for the elucidation of gene functions. Here, we present new configurations of the tetracycline-dependent gene regulation (tet) system for Staphylococcus aureus. To provide improved and expanded modes of control, strains and plasmids were constructed for the constitutive expression of tetR or a variant allele, rev-tetR(r2). The encoded regulators respond differently to the effector anhydrotetracycline (ATc), which causes target gene expression to be induced with TetR or repressed with rev-TetR. To quantify and compare regulation mediated by episomal or chromosomal (rev-)tetR constructs, expression from a chromosomal P(xyl/tet)-gfpmut2 fusion was measured. Chromosomally encoded TetR showed tight repression and allowed high levels of dose-dependent gene expression in response to ATc. Regulatory abilities were further verified using a strain in which a native S. aureus gene (zwf) was put under tet control in its native chromosomal location. Tight repression was reflected by transcript amounts, which were barely detectable under repressed conditions and high in ATc-treated cells. In reporter gene assays, this type of control, termed Tet-on, was more efficient than Tet-off regulation, in which addition of ATc causes downregulation of a target gene. The latter was achieved and quantified by direct rev-TetR control of P(xyl/tet)-gfpmut2. Additionally, TetR was used in trans to control the expression of antisense RNA for posttranscriptional gene silencing. Induction of antisense RNA expression of the fabI gene caused pronounced growth retardation lasting several hours. These results demonstrate the efficiency of the new tet systems and their flexible use for different purposes.
Stary
Elena
E
Lehrbereich Mikrobielle Genetik, Eberhard Karls Universität Tübingen, Tübingen, Germany.
Gaupp
Rosmarie
R
Lechner
Sabrina
S
Leibig
Martina
M
Tichy
Evelyn
E
Kolb
Martina
M
Bertram
Ralph
R
D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D017930:Genes, Reporter; D005822:Genetic Vectors; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016372:RNA, Antisense / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000235:genetics* / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D013754:Tetracyclines / Q000494:pharmacology*; D015533:Transcriptional Activation; D014161:Transduction, Genetic; D014162:Transfection
D001426:Bacterial Proteins; D006460:Hemolysin Proteins; D016372:RNA, Antisense; D012097:Repressor Proteins; D013754:Tetracyclines; C034811:tetracycline resistance-encoding transposon repressor protein; C016229:4-epianhydrotetracycline; D006655:Histone Deacetylases; D013752:Tetracycline