ANALYSIS OF THE MYCOBACTERIUM-TUBERCULOSIS 85A ANTIGEN PROMOTER REGION

A mycobacterial expression-secretion vector was constructed in which t he Escherichia coli alkaline phosphatase (phoA) reporter gene was plac ed under the control of the Mycobacterium tuberculosis 85A promoter an d secretion signal sequences. In recombinant Mycobacterium smegmatis a nd Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indica ting that the 85A signals can drive heterologous expression and secret ion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progres sive deletions using BAL 31 exonuclease and by primer extension analys is, Insertion and deletion mutations within the promoter region indica ted that, unlike most E. coli promoters but similar to Streptomyces pr omoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST ), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specif ic T-cell response to Sm28GST.