Transcriptional activation of flanking sequences by Tn1000 insertion

The carotenoid biosynthesis operon of Erwinia herbicola Eho13 consists of five genes, which are organized in the order crtE-crtX-crtY-crtI-crtB. These genes, with the exception of crtX, encode functions of beta-carotene biosynthesis and give an orange-coloured phenotype in Escherichia coli. Since crtX is not involved in the biosynthesis of beta-carotene, deletion of this gene does not alter the phenotype of pigmented cells. On the other hand, insertion of Tn1000 into crtX or into the upstream untranslated region of the operon resulted in a light-yellow, rather than an unpigmented phenotype, indicating that Tn1000 does not exert a strong polar effect when inserted in this operon. RNA analysis revealed that the sequence downstream from the insertion site was transcribed at a low level. Primer extension showed that the "-35"-like sequence in the terminal inverted repeats was not responsible for the transcription of the downstream sequence. Furthermore, primer extension and polymerase chain reaction (PCR) studies revealed that RNA transcribed from the promoters inside of Tn1000 was extended through the terminal inverted repeats into the adjacent sequences. In addition Tn1000, in either orientation, was able to generate fusion transcripts when placed upstream of a promoter-less tetracycline-resistance gene and resulted in cells resistant to the drug. These results showed that Tn1000 insertion transcriptionally activates the DNA sequences adjacent to the transposon.