Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control

Abstract

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.

Cross-species inhibition of dUTPase via the Staphylococcal Stl protein perturbs dNTP pool and colony formation in Mycobacterium

“…This K i value is on the same low range as found in the case of the Stl-induced inhibition experiments of the S. aureus phage Φ11 dUTPase [33], suggesting that the complexation of Stl with the two dUTPases are similarly strong. However, the maximal inhibition is about 80 %, which value is somewhat lower as compared to the Φ11 dUTPase (100 %) ( Figure 1B, Table I.…”

“…containing Stl-GST fusion protein [33] were propagated in 500 ml LB to an OD 600 of 0. After that, 80 Cleavage Unit thrombin was added to perform on-column cleavage for the removal of glutathion-S-transferase tag.…”

“…The Gm-Int HindIII cassette from the pUC-Gm-Int plasmid [38] was introduced into the resulting construct to yield the integrating vectorpGem-int-empty (used as control). The Stl coding region with AU1-tag was PCR-amplified from pGEX-4T-1 expression vector (generated in [33]) and cloned after the promoter (ending in NheI restriction site) in the vector pGemint-empty with NheI restriction sites to get pGem-int-Stl. Successful cloning was verified with sequencing of the appropriate region of the plasmid.…”

“…dUTPase [33]. To investigate whether Stl may also bind to mycobacterial dUTPase (mtDUT), we carried out native gel electrophoresis experiments.…”

“…Although the presence of potential dUTPase inhibitor proteins in Bacillus subtilis and Drosophila melanogaster was suggested in the early literature [30,31], till now none of these suggestions could be confirmed at the molecular level. However, it was recently shown that a pathogenicity island repressor protein in Staphylococcus aureus, Stl SaPIbov1 (Stl), is capable of interacting with the helper phage dUTPase and that the Stl repressor activity is inhibited in the complex [32,33]. Detailed quantitative characterization of this interaction also revealed that the Stl repressor is a highly potent protein inhibitor of the Staphylococcus aureus phage Φ11 dUTPase, as well [33].…”