dUTPase based switch controls transfer ofudvirulence genes in order to preserve integrityudof the transferred mobile genetic elements

摘要

dUTPases ubiquitously regulate cellular dUTP levels to preserveudgenome integrity. Recently, several other cellular processes wereudreported to be controlled by dUTPases including the horizontaludtransfer of Staphylococcus aureus pathogenicity islands (SaPI).udSaPIs are mobil genetic elements that encode virulence enhancingudfactors e.g. toxins. Here, phage dUTPases were proposed toudcounteract the repressor protein (Stl) and promote SaPI excisionudand transfer. A G protein-like mechanism was proposed which isudunexpected in light of the kinetic mechanism of dUTPase.udHere we investigate the molecular mechanism of SaPI transferudregulation, using numerous dUTPase variants and a wide rangeudof in vitro methods (steady-state and transient kinetics, VIS andudfluorescence spectroscopy, EMSA, quartz crystal microbalance,udX-ray crystallography).udOur results unambiguously show that Stl inhibits the enzymaticudactivity of dUTPase in the nM concentration range anduddUTP strongly inhibits the dUTPase: Stl complexation. Theseudresults identify Stl as a highly potent dUTPase inhibitor proteinudand disprove the G protein-like mechanism. Importantly, ourudresults clearly show that the dUTPase:dUTP complex is inaccessibleudto the Stl repressor. Unlike in small GTPases, hydrolysis ofudthe substrate nucleoside triphosphate (dUTP in this case) isudrequired prior to the interaction with the partner (Stl repressor inudthis case). We propose that dUTPase can efficiently interact withudStl and induce SaPI excision only if the cellular dUTP level is low (i.e. when dUTPase resides mainly in the apo enzyme form)udwhile high dUTP levels would inhibit SaPI transfer. This mechanismudmay serve the preservation of the integrity of the transferredudSaPI genes and links the well-known metabolic role ofuddUTPases to their newly revealed regulatory function in spreadudof virulence factors.