Characterization of a late promoter from the Streptomyces temperate phage phi C31

Abstract

An operon expressed late in the lytic cycle of the Streptomyces temperate phage C31 was shown to be transcribed from an inducible promoter, lp (phage late promoter), which resembled the previously reported early promoters. mRNAs initiated at lp were processed at the 3 end (and possibly also the 5 end) of a tRNA Thr -like sequence, resulting in leaderless polycistronic mRNAs.Vectors developed from the temperate Streptomyces phage C31 are widely used to study the molecular genetics of these commercially important, antibiotic-producing bacteria (2,3,14,15). There is considerable scope for the further exploitation of the genetic components of C31, and it is one of our aims to design expression systems based on the regulation of the phage promoters. To date, two types of promoters from C31 have been characterized. (i) The immediate-early promoters are active in nonlysogens and in uninduced lysogens and must therefore be recognized by the host RNA polymerase (11,22,24,25,27). (ii) The phage-specific early promoters are active only after induction into the lytic cycle and presumably require the expression of at least one immediate-early phage gene, probably encoding a transcription factor such as sigma factor or an activator (10,12,27). Early promoters are characterized by a 21-bp consensus sequence located Ϫ9/Ϫ10 to Ϫ30/Ϫ31 bp from the transcription initiation point (10). Transcription of early genes was maximal at 10 min, and expression of a reporter gene fused to an early promoter peaked at 20 min after induction of a temperature-sensitive lysogen of Streptomyces coelicolor (10, 12). The purpose of this work was to characterize a late promoter.Late gene expression occurs after the initiation of phage DNA synthesis, i.e., at least 10 min into the lytic cycle (18). Late transcripts, detectable at 20 min and later during lytic growth, map to the 18-kbp region to the left of the repressor gene and overlapping the cos ends (26). A late operon is transcribed from the extreme right-hand end of the C31 genome, through cos and at least 3.6 kbp into the left-hand end (9, 26). Low-resolution S1 mapping using a 1.3-kbp probe that included the region shown in Fig. 1A demonstrated the presence of two major RNA 5Ј endpoints, 720 and 540 bp to the left of the KpnI restriction site adjacent to the right-hand cos end (9) (Fig. 1A). Analysis of the DNA sequence showed an 18 out of 21 bp match (at coordinates 173 to 194 according to reference 9) to the consensus early promoters, close to the position of the upstream 5Ј endpoint in the RNA (Fig. 1A). We show here that a DNA fragment containing this sequence, hereafter called lp (phage late promoter), confers late promoter activity.In order to demonstrate promoter activity, a DNA fragment containing the locations of the 5Ј endpoints of both mRNAs mapped by S1 nuclease and one containing only the position of the downstream endpoint (Fig. 1A) were amplified by PCR and inserted upstream of the promoterless xylE gene in pIJ4083 (5) to make pCHT304 and pCHO405, respectively. Primers 4.4 (5Ј T...

Gene expression in the cos region of the Streptomyces temperate actinophage øC31

“…A sequence starting at nt 174 contained an 18/21 bp match to the phage-specific early promoter sequences (; Ingham et al ., 1993). DNA fragments containing this sequence have been shown to encode a functional late promoter (Howe & Smith, 1996). Two matches to the highly conserved inverted repeat (CIR) sequences were found at coordinates 640 (CIR7) and 2323 (CIR8; ).…”