TRWD, A PROTEIN ENCODED BY THE INCW PLASMID R388, DISPLAYS AN ATP HYDROLASE ACTIVITY ESSENTIAL FOR BACTERIAL CONJUGATION

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.