For reverse genetic approaches inactivation or selective modification of genes are required to elucidate their putative function. Sulfolobus acidocaldarius is a thermoacidophilic Crenarchaeon which grows optimally at 76°C and pH 3. As many antibiotics do not withstand these conditions the development of a genetic system in this organism is dependent on auxotrophies. Therefore we constructed a pyrE deletion mutant of S. acidocaldarius wild type strain DSM639 missing 322 bp called MW001. Using this strain as the starting point, we describe here different methods using single as well as double crossover events to obtain markerless deletion mutants, tag genes genomically and ectopically integrate foreign DNA into MW001. These methods enable us to construct single, double, and triple deletions strains that can still be complemented with the pRN1 based expression vector. Taken together we have developed a versatile and robust genetic tool box for the crenarchaeote S. acidocaldarius that will promote the study of unknown gene functions in this organism and makes it a suitable host for synthetic biology approaches.
Keywords: archaea, Sulfolobus, genetics, deletion mutant, expression system, in-frame deletion
Citation: Wagner M, van Wolferen M, Wagner A, Lassak K, Meyer BH, Reimann J and Albers S-V (2012) Versatile genetic tool box for the crenarchaeote Sulfolobus acidocaldarius. Front. Microbio. 3:214. doi: 10.3389/fmicb.2012.00214
Received: 06 March 2012; Paper pending published: 09 May 2012;
Accepted: 24 May 2012; Published online: 13 June 2012.
Edited by:Zvi Kelman, University of Maryland, USA
Copyright: © 2012 Wagner, van Wolferen, Wagner, Lassak, Meyer, Reimann and Albers. This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.
*Correspondence: Sonja-Verena Albers, Molecular Biology of Archaea, Max Planck Institute for terrestrial Microbiology, Karl-von-Frisch-Strasse 10, 35043 Marburg, Germany. e-mail: firstname.lastname@example.org