dUTPases ubiquitously regulate cellular dUTP levels to preserve
genome integrity. Recently, several other cellular processes were
reported to be controlled by dUTPases including the horizontal
transfer of Staphylococcus aureus pathogenicity islands (SaPI).
SaPIs are mobil genetic elements that encode virulence enhancing
factors e.g. toxins. Here, phage dUTPases were proposed to
counteract the repressor protein (Stl) and promote SaPI excision
and transfer. A G protein-like mechanism was proposed which is
unexpected in light of the kinetic mechanism of dUTPase.
Here we investigate the molecular mechanism of SaPI transfer
regulation, using numerous dUTPase variants and a wide range
of in vitro methods (steady-state and transient kinetics, VIS and
fluorescence spectroscopy, EMSA, quartz crystal microbalance,
X-ray crystallography).
Our results unambiguously show that Stl inhibits the enzymatic
activity of dUTPase in the nM concentration range and
dUTP strongly inhibits the dUTPase: Stl complexation. These
results identify Stl as a highly potent dUTPase inhibitor protein
and disprove the G protein-like mechanism. Importantly, our
results clearly show that the dUTPase:dUTP complex is inaccessible
to the Stl repressor. Unlike in small GTPases, hydrolysis of
the substrate nucleoside triphosphate (dUTP in this case) is
required prior to the interaction with the partner (Stl repressor in
this case). We propose that dUTPase can efficiently interact with
Stl and induce SaPI excision only if the cellular dUTP level is low (i.e. when dUTPase resides mainly in the apo enzyme form)
while high dUTP levels would inhibit SaPI transfer. This mechanism
may serve the preservation of the integrity of the transferred
SaPI genes and links the well-known metabolic role of
dUTPases to their newly revealed regulatory function in spread
of virulence factors
