TrwD, a Protein Encoded by the IncW Plasmid R388, Displays an ATP Hydrolase Activity Essential for Bacterial Conjugation

Abstract

A 1.7-kilobase pair segment from the conjugative transfer region of plasmid R388 DNA was cloned and sequenced. It contained trwD, a gene essential for plasmid R388 conjugation, for expression of the conjugative W-pilus and for sensitivity to phage PRD1. The deduced amino acid sequence of TrwD showed homology to the PulE/VirB11 superfamily of potential 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 of Escherichia coli. TrwD is probably a peripheral outer membrane protein 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 detergent-phospholipid mixed micelles. To study the importance of the nucleotide-binding site, Walker box A (GXXGXGK(T/S)), present in TrwD, the conserved lysine residue was replaced by glutamine. The mutant protein, expressed and purified under the same conditions as the wild type, did not exhibit ATPase activity. TrwD(K203Q) was not able to complement the mutation in trwD of the R388 mutant plasmid, suggesting the essentiality of the ATPase activity of the protein in the conjugative process. Furthermore, 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.

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…The weak NTPase activity of VirB11 can be enhanced by lipid binding, suggesting that, in vivo, localization of VirB11 to the bacterial membrane may stimulate NTPase activity (Krause et al, 2000b;Rivas et al, 1997). More data supporting this assumption have been reported: upon lipid binding, VirB11 is seen to undergo conformational changes (Krause et al, 2000b) and the same eVect is also observed for nucleotide binding (Savvides et al, 2003).…”

“…Along with VirB4 and the CP (VirD4), VirB11 is thought to energize the type IV secretion machinery either for complex assembly, pilus production and/or substrate secretion. The NTPase activity of several VirB11-like proteins has been determined in vitro (Christie et al, 1989;Krause et al, 2000b;Rivas et al, 1997;Sexton et al, 2004), revealing a rather weak activity similar to the one observed for chaperons like DnaK (Zylicz et al, 1983). VirB11 of A. tumefaciens has been reported to additionally possess an autophosphorylation activity (Christie et al, 1989), however, such an activity was not detected for any other member of the family of VirB11-like proteins.…”

Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification

“…The putative NTPases of type II͞IV secretion systems are soluble, found in the cytoplasm, and usually associated with the inner membrane (10)(11)(12)(13)(14)(15)(16)(17)(18). Recent electron microscopic images and cross-linking experiments show that several superfamily members homodimerize and form similar toroidal structures (15,19).…”

“…Recent electron microscopic images and cross-linking experiments show that several superfamily members homodimerize and form similar toroidal structures (15,19). Protein sequence alignments have disclosed the presence of four conserved domains in all superfamily members-two canonical nucleotide-binding motifs designated as Walker boxes A and B and two conserved regions designated as the Asp and His boxes (10,16,20). Several representative members of the type IV family of NTPases bind and hydrolyze ATP, and mutations in the Walker A motif abolish both this activity and macromolecular secretion (11,13,16,21).…”

“…Protein sequence alignments have disclosed the presence of four conserved domains in all superfamily members-two canonical nucleotide-binding motifs designated as Walker boxes A and B and two conserved regions designated as the Asp and His boxes (10,16,20). Several representative members of the type IV family of NTPases bind and hydrolyze ATP, and mutations in the Walker A motif abolish both this activity and macromolecular secretion (11,13,16,21). Similar mutations in putative NTPase genes of type II secretion systems also disrupt macromolecular secretion, and ATPase activity has been demonstrated in at least one member of this family (10,17).…”