Evaluation of a Tetracycline-Inducible Promoter in Staphylococcus aureus In Vitro and In Vivo and Its Application in Demonstrating the Role of sigB in Microcolony Formation

Abstract

An inducible promoter system provides a powerful tool for studying the genetic basis for virulence. A variety of inducible systems have been used in other organisms, including pXyl-xylR-inducible promoter, the pSpaclacI system, and the arabinose-inducible P BAD promoter, but each of these systems has limitations in its application to Staphylococcus aureus. In this study, we demonstrated the efficacy of a tetracycline-inducible promoter system in inducing gene expression in S. aureus in vitro and inside epithelial cells as well as in an animal model of infection. Using the xyl/tetO promoter::gfp uvr fusion carried on a shuttle plasmid, we demonstrated that dose-dependant tetracycline induction, as measured by bacterial fluorescence, occurred in each of the above environments while basal activation under noninduced conditions remained low. To ascertain how the system can be used to elucidate the genetic basis of a pathogenic phenotype, we cloned the sigB gene downstream of the inducible promoter. Induction of SigB expression led to dose-dependent attachment of the tested strain to polystyrene microtiter wells. Additionally, bacterial microcolony formation, an event preceding mature biofilm formation, also increased with tetracycline induction of SigB.

The cidA murein hydrolase regulator contributes to DNA release and biofilm development in Staphylococcus aureus

“…Many S. aureus infections are thought to involve the formation of biofilm, surface-associated communities of microbes encompassed by an extracellular matrix (1,2). Several S. aureus genes have been reported to contribute to its biofilm-forming ability, including agr (3)(4)(5), sarA (5-7), sigB (8), ica (9), rbf (10), tcaR (11), arlRS (12,13), and alsSD (14). Microarray analyses of S. aureus (15,16), Pseudomonas aeruginosa (17,18), Bacillus subtilis (19,20), and Escherichia coli (21) have also illustrated that there are major differences in gene expression between biofilm and planktonic cultures of these organisms.…”

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

“…Tet regulation vectors for S. aureus described in this study are the result of stepwise modifications along the line of the closely related vectors pALC2073 (Bateman et al, 2001) and pRMC2 (Corrigan & Foster, 2009). A strong and constitutive promoter for expression of tetR in pRMC2 was combined with a P xyl/tet version containing two tetO sites in pRAB11 yielding a very tight repression, combined with a high degree of inducibility.…”

“…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).…”

“…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). Such one-plasmid systems carry both tetR and a target gene located adjacently on one common vector.…”

Genetic characterization of the natural SigB variants found in clinical isolates of Staphylococcus aureus

“…In S. aureus, the sigB operon encodes the orthologs of RsbU, RsbV, and RsbW (Wu et al, 1996), and they are thought to have similar functions as in B. subtilis. In fact, S. aureus RsbW can bind SigB as the "anti-sigma factor" to inhibit its association with the core enzyme (Miyazaki et al, 1999), while RsbU positively regulates the SigB activity via the modulation of the RsbW activity (Gertz et al, 1999;Giachino et al, 2001;Horsburgh et al, 2002;Palma and Cheung, 2001). In addition, the expression of the sigB operon is positively auto-regulated by SigB because one of the two promoters located upstream of rsbV is recognized by the RNA polymerase holoenzyme containing SigB (Gertz et al, 1999).…”