ATPase Activity and Multimer Formation of PilQ Protein Are Required for Thin Pilus Biogenesis in Plasmid R64

Sakai, Daisuke; Horiuchi, Takayuki; Komano, Teruya

doi:10.1074/jbc.m010652200

Cited by 49 publications

(53 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Rates of ATP hydrolysis were calculated to be 5.6 and 1.5 nmol min Ϫ1 mg Ϫ1 for EpsE and EpsE(K270A), respectively. The rate of ATP hydrolysis observed for EpsE is similar to rates observed for other type II/type IV secretion family members, including PilQ, PilT, DotB, and TrwD (9,24,26,32).…”

Section: Resultssupporting

confidence: 69%

Molecular Analysis of the Vibrio cholerae Type II Secretion ATPase EpsE

2005

View full text Add to dashboard Cite

The type II secretion system is a macromolecular assembly that facilitates the extracellular translocation of folded proteins in gram-negative bacteria. EpsE, a member of this secretion system in Vibrio cholerae, contains a nucleotide-binding motif composed of Walker A and B boxes that are thought to participate in binding and hydrolysis of ATP and displays structural homology to other transport ATPases. Here we demonstrate that purified EpsE is an Mg 2؉ -dependent ATPase and define optimal conditions for the hydrolysis reaction. EpsE displays concentration-dependent activity, which may suggest that the active form is oligomeric. Size exclusion chromatography showed that the majority of purified EpsE is monomeric; however, detailed analyses of specific activities obtained following gel filtration revealed the presence of a small population of active oligomers. We further report that EpsE binds zinc through a tetracysteine motif near its carboxyl terminus, yet metal displacement assays suggest that zinc is not required for catalysis. Previous studies describing interactions between EpsE and other components of the type II secretion pathway together with these data further support the hypothesis that EpsE functions to couple energy to the type II apparatus, thus enabling secretion.Vibrio cholerae infection of the small intestine results in severe diarrheal disease, with the main virulence factor, cholera toxin, causing many of the disease symptoms. Extracellular secretion of cholera toxin occurs via two distinct steps: inner membrane translocation of the individual toxin subunits via a Sec-dependent mechanism and outer membrane translocation of the assembled toxin complex by the type II secretion pathway. The type II pathway is conserved among gram-negative bacteria, including many pathogens, and secretes a variety of virulence factors and degradative enzymes. The type II pathway components in V. cholerae are encoded by 12 eps (extracellular protein secretion) genes, organized in a single operon, and the vcpD/pilD gene (7,17,29).The components of the Eps transport system are found in association with both inner and outer membranes and assemble into a multiprotein apparatus that most likely spans the entire cell envelope (for a review, see reference 27). A fully functional transport system requires the presence of EpsE, which is a cytoplasmic protein when it is expressed in the absence of other Eps proteins but is associated with the cytoplasmic membrane in the presence of the inner membrane proteins EpsL and EpsM (28).EpsE is a member of a larger family of secretion nucleoside triphosphatases (NTPases; the type II/type IV secretion family), which are thought to couple nucleoside triphosphate (NTP) hydrolysis to bacterial protein secretion (19). Members of this secretion NTPase family contain the following conserved motifs; Walker A and B boxes, a histidine box, and an aspartate box (20,24,25,35). Several members of the type IV secretion ATPase family, such as HP0525 from Helicobacter pylori, TrbB from the conjugat...

show abstract

Section: Resultssupporting

confidence: 69%

Molecular Analysis of the Vibrio cholerae Type II Secretion ATPase EpsE

2005

View full text Add to dashboard Cite

show abstract

“…This proximity makes it possible for His Box 2 to act as a phosphate sensor, as has been proposed for other ATPases such as RecA (Story & Steitz, 1992), with rotation of the side chain occurring upon hydrolysis. Mutation of the corresponding residue in plasmid R64 PilQ (a PilB homologue) reduced ATPase activity and DNA transfer efficiency; the other His residue was not mutated in that study (Sakai et al, 2001). Of the three T4P motor proteins (PilB, PilT and PilU) in P. aeruginosa that are required for twitching motility, the function of PilU remains the most enigmatic.…”

Section: Discussionmentioning

confidence: 82%

“…Shown are the average twitching zone areas (12 zones each, four technical replicates from three independent plates) for unmodified and mutant proteins. and DotB of Legionella pneumophila (T4S) have been shown to be dominant-negative, whereas VirB11 (T4S) WA mutants have not (Bhattacharjee et al, 2001;Sakai et al, 2001;Stephens et al, 1995). Turner et al (1993) have shown that a WA mutant of XcpR (P. aeruginosa T2S ATPase) causes dominant-negative effects on secretion in the wild-type, but that the identical mutation in PilB does not affect the presence of external T4P in a wild-type strain, as assessed by electron microscopy.…”

Section: Discussionmentioning

confidence: 99%

“…1b) is required in all three twitching ATPases for normal twitching motility, although PilU function is most severely affected by its mutation. Site-directed mutagenesis of the corresponding Asp residue in the T2S protein PulE results in reduced secretion, while mutation of the Asp residue in R64 PilQ reduces both ATPase activity and mating efficiency in liquid (Sakai et al, 2001;Possot et al, 1992). Examination of the EpsE structure shows that the D293 residue forms a salt bridge to R336, stabilizing its guanidinium moiety.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU

et al. 2008

View full text Add to dashboard Cite

“…The C-terminal segments of the pilV genes are under the control of multiple DNA inversion of shufflon and determine the recipient specificity in liquid matings (10). The pilL and pilN gene products are outer membrane lipoproteins, and the pilQ gene product is a cytoplasmic ATPase (28,29). The remaining pil genes are likely to encode structural proteins that function in the establishment of the pilin transport apparatus and thin-pilus basal body.…”

mentioning

confidence: 99%

Genes Required for Plasmid R64 Thin-Pilus Biogenesis: Identification and Localization of Products of thepilK,pilM,pilO,pilP,pilR, andpilTGenes

Sakai

Komano

2002

J Bacteriol

Self Cite

View full text Add to dashboard Cite

We have previously shown that the pilL, pilN, pilQ, pilS, pilU, and pilV genes of plasmid R64 encode outer membrane lipoprotein, secretin, cytoplasmic ATPase, major pilin, prepilin peptidase, and minor pilin, respectively, which are required for thin-pilus formation. In this work, we characterized the products of the remaining essential genes, pilK, pilM, pilO, pilP, pilR, and pilT, with regard to their localization and processing. Overexpression systems containing pilM, pilO, and pilP genes fused with N-terminal glutathione S-transferase (GST) or a His tag were constructed. Overproduced proteins were purified and used to raise specific antibodies. Localization of PilM, PilO, and PilP proteins was performed by Western blot analysis with anti-GST-PilM, anti-PilO, and anti-PilP antibodies, respectively. The pilK, pilR, and pilT products were produced with a C-terminal His tag and then detected by anti-His tag antibody. Subcellular fractionation experiments with Escherichia coli cells producing R64 thin pili revealed that PilK, PilM, and PilR are inner membrane proteins, and PilP and PilT are periplasmic proteins. PilO protein was localized to the outer membrane in the presence of other Pil proteins, whereas it was localized to the cytoplasm in the absence of these proteins. Furthermore, the cleavage site of PilP protein was determined by N-terminal amino acid sequencing of purified mature PilP protein. We predict that PilK, PilM, PilO, PilP, and PilT proteins function as the components of the pilin transport apparatus and thin-pilus basal body.

show abstract

ATPase Activity and Multimer Formation of PilQ Protein Are Required for Thin Pilus Biogenesis in Plasmid R64

Cited by 49 publications

References 39 publications

Molecular Analysis of the Vibrio cholerae Type II Secretion ATPase EpsE

Molecular Analysis of the Vibrio cholerae Type II Secretion ATPase EpsE

Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU

Genes Required for Plasmid R64 Thin-Pilus Biogenesis: Identification and Localization of Products of thepilK,pilM,pilO,pilP,pilR, andpilTGenes

Contact Info

Product

Resources

About