Gene. 121 (1992) 87-94 0 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-I 119/92/$05.00 87 GENE 0674 1 Mutagenic analysis of the promoter of the Streptomyces fradiae p -lactamase-encoding gene (Streptomyces lividuns; signal peptide; transcriptional initiation; exonuclease III-mediated deletions; promoter sequence; expression; RNA colony hybridization; enzyme activity) Mats Forsman and Micael Gram&-am zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Department of Microbiology, Notional Dcfknce Research Establishment, S-901 82 Umed. Sweden Received by K.F. Chater: 3 March 1992; Accepted: 29 June 1992; Received at publishers: 16 July 1992 SUMMARY The Streptomyces fradiue P-lactamase promoter (PhlaF) 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. lividuns and S. fiudiue. 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 anal- ysis identified c&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. PhbL was shown not to be recognized by sigma-whiG or by sigma-hrdA, hrdC, or hrdD. Sequence alignment of PhlUF to sigma factor-classified Streptomwes promoters revealed little homology. Thus, P,,(,, is probably recognized by an as yet uniden- tified sigma factor. INTRODUCTION Transcription initiation plays a crucial role in the com- plex process of gene regulation. In the Gram’, mycelial soil Correspondence to: Dr. M. Forsman, FOA 4, S-901 82 Umei, Sweden. Tel. (46-90) 106669; Fax (46-90) 106800. Abbreviations: aa, amino acid(s); B., Bacillus; Bla, /I-lactamase; bluF, gene encoding a Bla from S.,fiadiae; bp, base pair(s); ccc, covalently closed circular; cpm, counts per minute; A, deletion; DOG, 2-deoxyglu- case; E., Escherichia; Exo III, exonuclease III of E. co/i; hrd, gene(s) en- coding sigma factor(s) homologous to rpoD of E. coli and B. subtilis; kb. kilobase or 1000 bp; PolIk, Klenow (large) fragment of E. coli DNA polymerase I; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; P, pro- moter; PEG, polyethylene glycol; RF, replicative form; S., Streptomyces; SDS, sodium dodecyl sulfate; Sm, streptomycin; Th; thiostrepton; tsp, transcription start point(s); tsr, gene, encoding Th resistance; whiG, gene encoding sporulation sigma (0) factor; wt, wild type; [ 1. denotes plasmid- carrier state. bacterium Streptomyces, a tremendous promoter sequence diversity is observed (Strohl, 1992). At least seven forms of RNA polymerase holoenzymes have been discovered so far in Streptomyces. The different forms of RNA polymerase are distinguished from each other by the association of the core enzyme (b/? u2) with different c~factors. Thus far, four different u factors, $a, d5, 049 and aG6have been well characterized biochemically (Westpheling et al., 1985; Buttner et al., 1988; Brown et al., 1992). Another 0 factor, cYhiG, was identified by genetic studies (Chater et al., 1989) and shown to be obligatory for sporulation in S. coelicolor A3(2). In addition, S. coelicolor contains four genes which are highly similar to the Escherichia coli rpoD gene, specifying the principal u factor (Tanaka et al., 1988), and these genes have therefore been named hrdA, hrdB, hrdC, and hrdD (homology to rpoD). The hrdB gene encodes a r~ factor, g6, which in association with the core components of the RNA polymerase complex directs transcription from the dagAp4 and Bacillus subtilis