title: Identification and characterization of a unique, zinc-containing transport ATPase essential for natural transformation in Thermus thermophilus HB27.
abstract: Thermus thermophilus is a model strain to unravel the molecular basis of horizontal gene transfer in hot environments. Previous genetic studies led to the identification of a macromolecular transport machinery mediating DNA uptake in an energy-dependent manner. Here, we have addressed how the transporter is energized. Inspection of the genome sequence revealed four putative transport (AAA) ATPases but only the deletion of one, PilF, led to a transformation defect. PilF is similar to transport ATPases of type IV and type II secretions systems but has a unique N-terminal sequence that carries a triplicated GSPII domain. To characterize PilF biochemically it was produced in Escherichia coli and purified. The recombinant protein displayed NTPase activity with a preference for ATP. Gel filtration analyses combined with dynamic light scattering demonstrated that PilF is monodispersed in solution and forms a complex of 590 ± 30 kDa, indicating a homooligomer of six subunits. It contains a tetracysteine motif, previously shown to bind Zn(2+) in related NTPases. Using atomic absorption spectroscopy, indeed Zn(2+) was detected in the enzyme, but in contrast to all known zinc-binding traffic NTPases only one zinc atom was bound to the hexamer. Deletion of the four cysteine residues led to a loss of Zn(2+). Nevertheless, the mutant protein retained ATPase activity and hexameric complex formation.
authors.affiliation: Molecular Microbiology and Bioenergetics, Institute of Molecular Biosciences, Goethe University, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany.
authors.forename: Ilona
authors.initials: I
authors.lastname: Rose
authors.forename: Goran
authors.initials: G
authors.lastname: Biuković
authors.forename: Patrick
authors.initials: P
authors.lastname: Aderhold
authors.forename: Volker
authors.initials: V
authors.lastname: Müller
authors.forename: Gerhard
authors.initials: G
authors.lastname: Grüber
authors.forename: Beate
authors.initials: B
authors.lastname: Averhoff
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004269:DNA, Bacterial / Q000235:genetics; D036781:Fimbriae Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D020409:Molecular Motor Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013054:Spectrophotometry, Atomic / Q000379:methods; D016963:Thermus thermophilus / Q000235:genetics*; D015032:Zinc / Q000737:chemistry
chemical_list: D004269:DNA, Bacterial; D020409:Molecular Motor Proteins; C102919:PilF protein, Pseudomonas; D036781:Fimbriae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D015032:Zinc