Generation of Targeted Deletions in the Genome of Rhodothermus marinus

Abstract

ABSTRACTThe aim of this work was to develop an approach for chromosomal engineering of the thermophileRhodothermus marinus. A selection strategy forR. marinushad previously been developed; this strategy was based on complementing a restriction-negativetrpBstrain with theR. marinustrpBgene. The current work identified an additional selective marker,purA, which encodes adenylosuccinate synthase and confers adenine prototrophy. In a two-step procedure, the available Trp+selection was used during the deletion ofpurAfrom theR. marinuschromosome. The alternative Ade+selection was in turn used while deleting the endogenoustrpBgene. Since both deletions are unmarked, thepurAandtrpBmarkers may be reused. Through the double deletant SB-62 (ΔtrpBΔpurA), the difficulties that are associated with spontaneous revertants and unintended chromosomal integration of marker-containing molecules are circumvented. The selection efficiency inR. marinusstrain SB-62 (ΔtrpBΔpurA) was demonstrated by targeting putative carotenoid biosynthesis genes,crtBI, using a linear molecule containing a marked deletion with 717 and 810 bp of 5′ and 3′ homologous sequences, respectively. The resulting Trp+transformants were colorless rather than orange-red. The correct replacement of an internalcrtBIfragment with thetrpBmarker was confirmed by Southern hybridization analysis of the transformants. Thus, it appears that target genes in theR. marinuschromosome can be readily replaced with linear molecules in a single step by double-crossover recombination.