Abstract
The Shiga toxin operon (stx) is composed of A and B subunit genes which are transcribed as a bicistronic mRNA from a promoter which lies 5' to the stxA gene. Northern (RNA) blot and primer extension analyses revealed the existence of a second stxB gene transcript. Recombinant plasmids which carried the stxB gene without the stx operon promoter and with the influence of a vector promoter abrogated produced STX B polypeptides, suggesting that the stxB gene mRNA was transcribed from an independent promoter and was not produced by endoribonucleotic processing of the bicistronic mRNA. Examination of the DNA sequences 5' to the stxB gene transcription initiation site which were carried by the recombinant plasmids revealed a region with high homology to the consensus for Escherichia coli promoters. Deletion and mutation of this region affected StxB and holotoxin production, establishing its role in the regulation of the stxB gene. Comparison of the promoters by using a transcription analysis vector revealed that the stxB gene promoter differed from the stx operon promoter in that was approximately sixfold less efficient and was not repressed by iron. Identification of a second promoter in the stx operon indicates that independent transcription of the stxB gene may regulate overproduction of the STX B polypeptides and may contribute to the 1A:5B subunit stoichiometry of the holotoxin.