Identification of mycoplasmal promoters in Escherichia coli using a promoter probe vector with Green Fluorescent Protein as reporter system.

Abstract

A promoter probe vector, pGFPUV2, with Green Fluorescent Protein (GFP) as the reporter system was constructed to identify potential mycoplasmal promoter-containing regulatory sequences in E. coli. Libraries of M. pneumoniae and M. genitalium DNA constructed in pGFPUV2 and transformed into E. coli resulted in GFP-expressing clones. Primer extension analysis with E. coli RNA from five M. pneumoniae clones and two M. genitalium clones indicated that transcription originated from the insert DNA fragments of these promoter constructs. Primers based on the insert DNA sequences were used in primer extension reactions with total RNA isolated from M. pneumoniae or M. genitalium. Of the seven primers used, three generated products by primer extension with mycoplasmal RNA. However, only one of the DNAs had a 5' end similar to that obtained in a comparable reaction with E. coli RNA, and the start site of this transcript appeared to originate one base prior to the predicted open reading frame. These results indicate that E. coli can identify mycoplasmal promoters which have transcriptional elements resembling E. coli promoters.