New Architectures for Tet-On and Tet-Off Regulation in Staphylococcus aureus

Abstract

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.

Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus

“…In fact, the activity of P xyl/tet in the TetR unbound state exceeds that of native S. aureus promoters of low and intermediate strength, such as P folA , P zwf or P fabI and rather reflects the transcriptional level of the highly active rpsF promoter (Stary et al, 2010;Xu et al, 2010). Although tet regulation enables inducer dose-dependent responses of a target gene (reviewed by Bertram & Hillen, 2008), the determination and maintenance of a defined inducer concentration resulting in stable and reproducible intermediate expression is cumbersome.…”

“…tet-OFF responses in S. aureus were initially achieved by TetRdependent inducible expression of a target gene's antisense RNA fragment (Ji et al, 1999), resulting in post-transcriptional downregulation. More recently, however, a reverse variant of TetR which binds to tetO only in the presence of ATc has been applied for use in S. aureus (Stary et al, 2010). Enabling tet-OFF regulation acting on the level of transcription, this regulator termed revTetR-r2 exhibits a total of three amino acid exchanges (E15A, L17G and L25V) compared with wild type TetR (Scholz et al, 2004).…”

“…Enabling tet-OFF regulation acting on the level of transcription, this regulator termed revTetR-r2 exhibits a total of three amino acid exchanges (E15A, L17G and L25V) compared with wild type TetR (Scholz et al, 2004). A remarkable diversity of tet regulation architectures has been described for S. aureus, whereby TetR controls target genes encoded in cis or in trans (Bateman et al, 2001;Gründling & Schneewind, 2007;Stary et al, 2010). A convenient and popular system for complementation studies in staphylococci is represented by pALC2073 and vectors derived therefrom (Bateman et al, 2001).…”

“…An 868 bp KpnI/PstI sequence containing tetR, its promoter and the divergent two-tetO P xyl/tet version was then ligated with the 5.6 kb KpnI/PstI vector backbone of pRMC2 to obtain pRAB11. Gene gfpmut2 (Cormack et al, 1996) was amplified from plasmid pRAB4 (Stary et al, 2010) using primers pRAB4-gfpmut2-fw and -rev, and a 3.3 kb translational spoVG-lacZ fusion was obtained using primers pRAB11-lacZ-fw and -rev and pWH102 as a template (Kamionka et al, 2005). Each of the fragments was inserted into pRAB11 and pRMC2 via Bgl II and EcoRI to yield pRAB11-lacZ, pRAB11-gfp and the correspondingly designated pRMC2 derivatives.…”

Are We There Yet? A Story About Cardiac Stem Cells

“…Preferably, such a system should be based on a "tet-off" design, in which the system is activated until doxycycline (DOX) is added (Gossen & Bujard, 1992;Urlinger et al, 2000). The opposite system is called "tet-on" (rtTA is used), which is claimed to allow a tight control (Stary et al, 2010). However, when tet-on system is used, DOX must be administered in the drinking water of mice, which can be rather expensive.…”