MUTAGENIC ANALYSIS OF THE PROMOTER OF THE STREPTOMYCES-FRADIAE BETA-LACTAMASE-ENCODING GENE

The Streptomyces fradiae beta-lactamase promoter (P(blaF)) was sequenced and characterized by promoter probing, primer extension, and exonuclease III-mediated deletions. The transcription start point (tsp) was the same in both S. lividans and S. fradiae. Oligodeoxyribonucleotide-directed random mutations and site-specific mutations were introduced in the promoter region. The effects of these mutations on transcription were assayed by an RNA colony hybridization method. This analysis identified cis-acting sequence determinants located similarly to the -10 and -35 regions of a typical Escherichia coli promoter. Also, a change in the distance between these regions from 19 to 17 bp drastically reduced promoter activity. P(blaF) was shown not to be recognized by sigma-whiG or by sigma-hrdA, hrdC, or hrdD. Sequence alignment of P(blaF) to sigma factor-classified Streptomyces promoters revealed little homology. Thus, P(blaF) is probably recognized by an as yet unidentified sigma factor.