Conditions for gene disruption by homologous recombination of exogenous DNA into the Sulfolobus solfataricus genome

Sonja-Verena Albers,Arnold J M Driessen

doi:10.1155/2008/948014

Abstract

The construction of directed gene deletion mutants is an essential tool in molecular biology that allows functional studies on the role of genes in their natural environment. For hyperthermophilic archaea, it has been difficult to obtain a reliable system to construct such mutants. However, during the past years, systems have been developed for Thermococcus kodakarensis and two Sulfolobus species, S. acidocaldarius and derivatives of S. solfataricus 98/2. Here we describe an optimization of the method for integration of exogenous DNA into S. solfataricus PBL 2025, an S. solfataricus 98/2 derivative, based on lactose auxotrophy that now allows for routine gene inactivation.

Highlights

For many years, the functional analysis of genes and gene products of hyperthermophilic archaea was hampered by the lack of suitable generic genetic tools such as expression vectors, methods for the directed deletion of target genes and efficient gene transformation protocols that allow a high recovery of transformed cells
A shuttle vector system has been successfully used for pyrococcal species (Lucas et al 2002), whereas a targeted gene deletion system has been described for Thermococus kodakarensis and Sulfolobus solfataricus (Worthington et al 2003, Sato et al 2005)
Selection of gene disruption strains after electroporation of plasmid DNA

Summary

Introduction

The functional analysis of genes and gene products of hyperthermophilic archaea was hampered by the lack of suitable generic genetic tools such as expression vectors, methods for the directed deletion of target genes and efficient gene transformation protocols that allow a high recovery of transformed cells. Virus-based vector systems have been developed for Sulfolobus species (Stedman et al 1999) and used in promoter studies (Jonuscheit et al 2003), homologous and heterologous expression of proteins in Sulfolobus (Albers et al 2006, Aucelli et al 2006) and the complementation of deletion mutants (Bartolucci et al 2003, Jonuscheit et al 2003). A shuttle vector system has been successfully used for pyrococcal species (Lucas et al 2002), whereas a targeted gene deletion system has been described for Thermococus kodakarensis and Sulfolobus solfataricus (Worthington et al 2003, Sato et al 2005). In S. acidocaldarius, a form of conjugation has been described that generates prototrophic recombinants from pairs of auxotrophic mutations (Hansen et al 2005). Extensive testing of different conditions revealed that even the electroporation of synthetic oligonucleotides resulted in their recombination into the chromosome (Kurosawa and Grogan 2005)

Methods

Results

Discussion

Conclusion