The XcpR protein of Pseudomonas aeruginosa dimerizes via its N‐terminus

Abstract

SummaryExtracellular protein secretion by the main terminal branch of the general secretory pathway in Pseudomonas aeruginosa requires a secretion machinery comprising the products of at least 12 genes. One of the components of this machinery, the XcpR protein, belongs to a large family of related proteins distinguished by the presence of a highly conserved nucleotide binding domain (Walker box A). The XcpR protein is essential for the process of extracellular secretion and amino acid substitutions within the Walker A sequence result in inactive XcpR. The same mutations exert a dominant negative effect on protein secretion when expressed in wild-type bacteria. Transdominance of XcpR mutants suggests that this protein is involved in interactions with other components of the secretion machinery or that it functions as a multimer. In this study, the amino-terminal portion of the cI repressor protein of phage was used as a reporter of dimerization in Escherichia coli following fusion to full-length as well as a truncated form of XcpR. The cI-XcpR hybrid proteins were able to dimerize, as demonstrated by the immunity of bacteria expressing them to killing by phage. The full-length XcpR as well as several deletion mutants of XcpR were able to disrupt the dimerization of the chimeric cI-XcpR protein. The disruption of cI-XcpR dimers using the deletion mutants of XcpR, combined with the analysis of their dominant negative effects on protein secretion, was used to map the minimal dimerization domain of XcpR, which is located within an 85 amino acid region in its N-terminal domain. Taken together, the data presented in this paper suggest that the XcpR protein dimerizes via its N-terminus and that this dimerization is essential for extracellular protein secretion.

Molecular Analysis of the Vibrio cholerae Type II Secretion ATPase EpsE

“…We also demonstrate that EpsE displays concentration-dependent ATPase activity. Taken together with earlier findings from genetic lambda repressor and yeast-two hybrid analyses of the EpsE homologues XcpR and OutE, respectively, our size exclusion chromatography results indicate that the type II secretion ATPases can assemble into oligomers (23,34). That the functional form of these ATPases is oligomeric in nature is supported by our discovery of a small population of oligomers that displayed much higher specific activity than the monomer.…”

Self-Assembly of the Agrobacterium tumefaciens VirB11 Traffic ATPase

“…Similarly, SecA translocase functions as a dimer (12). Multimerization also was demonstrated for two VirB11-like ATPases associated with type II secretion, PulE, an essential component of the Klebsiella oxytoca pullulanase secretion machinery (24), and Pseudomonas aeruginosa XcpR, also required for type II protein secretion (34). Interestingly, PulE migrates in nonreducing SDS-polyacrylamide gels as higher-order disulfidecross-linked homo-oligomers (24).…”

Modification of Type IV Pilus-Associated Epithelial Cell Adherence and Multicellular Behavior by the PilU Protein of Neisseria gonorrhoeae

“…Given the highly related structures of the proteins, PilU might modify PilT activity by competing for a common interactive component or site on the inner membrane (24). Since the related traffic ATPases of the Helicobacter pylori type IV secretion system and conjugative plasmid systems can form hexameric ring structures (35), it is also possible that PilU has an effect by forming nonfunctional multimers with PilT. Further understanding of the functional relationships between PilT and PilU is required in order to test these hypotheses.…”