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J. Biol. Chem., Vol. 280, Issue 12, 11422-11431, March 25, 2005

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Engineering a Selectable Marker for Hyperthermophiles^*

Stan J. J. Brouns, Hao Wu, Jasper Akerboom, Andrew P. Turnbull¶, Willem M. de Vos, and John van der Oost

From the Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands and ^¶Proteinstrukturfabrik c/o Berliner Elecktronenspeicherring-Gesellschaft für Synchrotronstrahlung GmbH, Albert Einstein Strasse 15, D-12489 Berlin, Germany

Limited thermostability of antibiotic resistance markers has restricted genetic research in the field of extremely thermophilic Archaea and bacteria. In this study, we used directed evolution and selection in the thermophilic bacterium Thermus thermophilus HB27 to find thermostable variants of a bleomycin-binding protein from the mesophilic bacterium Streptoalloteichus hindustanus. In a single selection round, we identified eight clones bearing five types of double mutated genes that provided T. thermophilus transformants with bleomycin resistance at 77 °C, while the wild-type gene could only do so up to 65 °C. Only six different amino acid positions were altered, three of which were glycine residues. All variant proteins were produced in Escherichia coli and analyzed biochemically for thermal stability and functionality at high temperature. A synthetic mutant resistance gene with low GC content was designed that combined four substitutions. The encoded protein showed up to 17 °C increased thermostability and unfolded at 85 °C in the absence of bleomycin, whereas in its presence the protein unfolded at 100 °C. Despite these highly thermophilic properties, this mutant was still able to function normally at mesophilic temperatures in vivo. The mutant protein was co-crystallized with bleomycin, and the structure of the binary complex was determined to a resolution of 1.5 Å. Detailed structural analysis revealed possible molecular mechanisms of thermostabilization and enhanced antibiotic binding, which included the introduction of an intersubunit hydrogen bond network, improved hydrophobic packing of surface indentations, reduction of loop flexibility, and -helix stabilization. The potential applicability of the thermostable selection marker is discussed.

Received for publication, December 3, 2004 , and in revised form, December 27, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY780486.

The atomic coordinates and structure factors (code 1XRK) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by a grant from the European Union in the framework of the SCREEN project (Contract QLK3-CT-2000-00649). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Table 1.

To whom correspondence should be addressed. Tel.: 31-317-483110; Fax: 31-317-483829; E-mail: stan.brouns{at}wur.nl.

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