AN IMPROVED PROCEDURE AND NEW VECTORS FOR TRANSPOSON TN5 MUTAGENESIS OF THE PHOTOTROPHIC BACTERIUM RHODOSPIRILLUM-RUBRUM

A detailed examination of vectors and procedures used for Tn5 mutagenesis of the phototrophic purple non-suIfur bacterium Rhodospirillum rubrum has been performed. The mobilizable Tn5 suicide vectors currently available show a frequency of Tn5 mutagenesis for R. rubrum of approx. 10(-7)-10(-8), approx. 100-1000-fold lower than observed for the related bacteria Rhodobacter capsulatus and Rhodobacter sphaeroides. Using the blue-to-red reversion of a blue-green mutant, R. rubrum ST6, containing a single Tn5 lesion in one of the early genes for carotenoid biosynthesis, we have shown that the frequency of precise excision of a chromosomally inserted Tn5 element: to restore the wild-type phenotype in the absence of selection, is 10(-6). We have constructed three new suicide vectors for Tn5 mutagenesis, where the transposase encoded by the IS50R element was placed in the same (pSUPEG11, pSUPEG21) or in the opposing (pSUPEG22) orientation from the weak promoter of the RK2-derived terR gene. With the vector pSUPEG11, the frequency of Tn5 mutagenesis was increased to 10(-5), approx. 100-fold higher than observed previously.