Cloning of bacillus subtilis genomic DNA fragments that show promoter activity in escherichia coli and bacillus subtilis

Abstract

To compare the promoter recognition ability between Escherichia coli and Bacillus subtilis as well as to screen a strong promoter of B. subtilis, we have constructed a promoter library of B. subtilis in E. coli using a promoter probe vector p602/8. The library was screened for the presence of chloramphenicol resistance. About eighty chloramphenicol resistant clones were confirmed. The recombinant plasmids isolated from chloramphenicol resistant lines contained chromosomal inserts with size of 130-500 base pairs. Relative strengths of chloramphenicol resistance of each clones in E. coli were compared with control promoters of tact and SPOII on the solid media. The level of chloramphenicol resistance ranged from 80 to 700 ug/ml. Transferring p602/8 derivatives containing cloned DNA fragments into B. subtilis, promoter donor cell, promoter activity of cloned DNA fragments were analyzed with chloramphenicol resistance, SDS polyacrylamide gel electrophoresis and assay of chloramphenicol acetyltransferase activity. All cloned DNA fragments showed promoter activity in B. subtilis also. B. subtilis cells harvouring the cloned DNA fragments displayed chlorampheni col resistance from 10 to 150ug/ml on solid media and chloramphenicol acetyltransferase activity from 31 miliunits to 5 units. Some of the cloned Bacillus chromosomal DNA fragments such as BJ 2, BJ 27, BJ 29, showed promoter activity similar to SPOII promoter which is known to a strong promoter in B. subtilis, and promoted synthesis of chloramphenicol acetyl transferase from 20 to 25\% of total cell proteins. These promoters have a potential to make foreign genes express efficiently in B. subtilis.