A two-host fosmid system for functional screening of (meta)genomic libraries from extreme thermophiles

Abstract

A new cloning system is described, which allows the construction of large-insert fosmid libraries in Escherichia coli and the transfer of the recombinant libraries to the extreme thermophile Thermus thermophilus via natural transformation. Libraries are established in the thermophilic host by site-specific chromosomal insertion of the recombinant fosmids via single crossover or double crossover recombination at the T. thermophilus pyr locus. Comparative screening of a fosmid library constructed from genomic DNA from the thermophilic spirochaete, Spirochaeta thermophila, for clones expressing thermoactive xylanase activity revealed that 50% of the fosmids that conferred xylanase activity upon the corresponding T. thermophilus transformants did not give rise to xylanase-positive E. coli clones, indicating that significantly more S. thermophila genes are functionally expressed in T. thermophilus than in E. coli. The novel T. thermophilus host/vector system may be of value for the construction and functional screening of recombinant DNA libraries from individual thermophilic or extremely thermophilic organisms as well as from complex metagenomes isolated from thermophilic microbial communities.