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Extremophiles (2011) 15:191202 DOI 10.1007/s00792-010-0343-2
ORIGINAL PAPER
Identication and characterization of a unique, zinc-containing transport ATPase essential for natural transformation in Thermus thermophilus HB27
Ilona Rose Goran Biukovi Patrick Aderhold
Volker Mller Gerhard Grber Beate Averhoff
Received: 27 July 2010 / Accepted: 1 December 2010 / Published online: 6 January 2011 Springer 2011
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 identication 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 puried. The recombinant protein displayed NTPase activity with a preference for ATP. Gel ltration 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 Zn2? in related NTPases. Using atomic absorption spectroscopy, indeed Zn2? was detected in the enzyme, but in contrast to all known zinc-binding trafc NTPases only one zinc atom was bound to the hexamer. Deletion of the four cysteine residues led to a loss of Zn2?. Nevertheless, the mutant protein retained ATPase activity and hexameric complex formation.
Keywords DNA transporter AAA-ATPase
Metalloproteins Molecular motor T. thermophilus
Introduction
Comparative genome analyses provide evidence that horizontal gene transfer is the major driving force for bacterial adaptation to different, often extreme environments and bacterial genome evolution. Among the microorganisms thriving in extreme habitats, thermophiles and hyper-thermophiles clearly stand out in terms of interdomain DNA transfer such as 24 and 16.2% of the genes in the hyperthermophilic bacteria Thermotoga maritima and Aquifex aeolicus, respectively, are suggested to be transferred from archaeal hyperthermophiles (Aravind et al. 1998; Nelson et al. 1999). Many of the transferred genes are thermophilic traits that are essential for survival under extreme conditions.
Among the principal mechanisms...