S. Rivas et al., TRWD, A PROTEIN ENCODED BY THE INCW PLASMID R388, DISPLAYS AN ATP HYDROLASE ACTIVITY ESSENTIAL FOR BACTERIAL CONJUGATION, The Journal of biological chemistry, 272(41), 1997, pp. 25583-25590
A 1.7-kilobase pair segment from the conjugative transfer region of pl
asmid R388 DNA was cloned and sequenced, It contained trwD, a gene ess
ential for plasmid R388 conjugation, for expression of the conjugative
W-pilus and for sensitivity to phage PRD1. The deduced amino acid seq
uence of TrwD showed homology to the PulE/VirB11 superfamily of potent
ial ATPases involved in various types of transport processes, A fusion
of trwD with the glutathione S-transferase (GST) was constructed, and
the resulting fusion protein was purified from overproducing bacteria
. Factor X-a hydrolysis of GST-TrwD and further purification rendered
TrwD protein with more than 95% purity, Antibodies raised against TrwD
localized it both in the soluble fraction and in the outer membrane o
f Escherichia coli. TrwD is probably a peripheral outer membrane prote
in because it could be solubilized by increasing salt concentration to
0.5 M NaCl in the lysis buffer, Both purified GST-TrwD and TrwD could
hydrolize ATP, ATPase activity increased 2-fold in the presence of de
tergent-phospholipid mixed micelles, To study the importance of the nu
cleotide-binding site, Walker box A (GXXGXGK(T/S)), present in TrwD, t
he conserved lysine residue was replaced by glutamine, The mutant prot
ein, expressed and purified under the same conditions as the wild type
, did not exhibit ATPase activity, TrwD(K203Q) was not able to complem
ent the mutation in trwD of the R388 mutant plasmid, suggesting the es
sentiality of the ATPase activity of the protein in the conjugative pr
ocess. Further more, the dominant character of this mutation suggested
that GST-TrwD(K432Q) was still able to interact either with itself or
with other component(s) of the conjugative machinery.