Transcription of Bacillis subtilis plasmid pBD64 and expression of bacteriophage SPO1 genes cloned therein

Abstract

Plasmid pBD64, a vector which is useful for cloning in Bacillis subtilis ( T. J. Gryczan, A. G. Shivakumar, and D. Dubnau (1980), J. Bacteriol. 141, 246–253), has at least three substantial transcription units. Two of these include the single EcoRl, XbaI, and BamHI sites, while the other includes the single BglII site. Each of these transcripts was synthesized in the counter-clockwise direction, relative to the pBD64 restriction map. No transcripts were detected in the opposite direction. Infection by bacteriophage SPO1 caused a substantial decrease in each of these transcripts. No new pBD64 transcripts were detected during SPOT infection. Various SPO1 genes, cloned at several of these pBD64 sites, were tested for expression by observing their capacity to complement SPO1 mutants. Several middle and late genes were expressed substantially, regardless of the orientation in which the fragments were inserted. Since transcription from the vector could cause expression only in one orientation, this argues that the necessary transcription originated at SPO1 promoters, and, thus, that SPO1 middle and late promoters can be active in thymine-containing DNA.