Multiple novel promoters from the early region in the Streptomyces temperate phage φC31 are activated during lytic development

Abstract

Evidence is presented that transcription of most of the early genes in the Streptomyces coelicolor A3(2) phage phi C31 is from a series of unusual promoters that depend on a function expressed early in the phi C31 lytic cycle. Primer extension analysis on the 5' ends of three early mRNAs, from samples prepared 10 min after induction of a thermosensitive phi C31 lysogen, showed that the 5' ends all mapped close to highly similar sequences, which are proposed to be an important part of phage-specific promoters. In a shotgun cloning experiment, a fragment containing one of these sequences strongly activated transcription of the xyIE reporter gene in plaques of a phi C31-derived promoter-probe vector. Another of the sequences was inserted into a xyIE-containing promoter-probe plasmid vector, and promoted xyIE expression only when the host was supporting the lytic cycle of phi C31. This suggested that a transcription factor needed for activity of the promoters was present only in phi C31-infected cells. Examination of published and unpublished phi C31 sequence data revealed several more sequences that closely resemble the conserved region of the characterized promoters. Most of these are found in positions close to apparent transcription start sites mapped previously by low-resolution S1 mapping. An overall consensus sequence for the conserved region suggests a general organization (though not a primary sequence) resembling that of promoters recognized in other bacteria by the sigma 54 form of RNA polymerase.

Genomics ofActinobacteria: Tracing the Evolutionary History of an Ancient Phylum

“…All three sequenced phages belong to the Siphoviridae family and resemble Sfi21-like prophages in their modular organization (57). phiC31 has a remarkable system of prophage repression, with repressor targets in each of the multiple promoters spread through the early region, and these promoters have an unusual, and phagespecific, character (196,480). phiC31 has provided valuable systems for gene cloning and molecular analysis in streptomycetes (225), and its site-specific integration system is widely used in higher-organism genetics (102).…”

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

“…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.…”

“…(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).…”

“…The efficiency of plating of C31c⌬25 was poor on TK24(pCHT304) compared with TK24(pCHO405) because of a phage development limitation phenotype observed when certain phage promoters are present in the host in multiple copies (26a). pCHT304 and pCHO405 were transformed into S. coelicolor J1501C31cts1 lysogens and J1501 nonlysogens (8,10). Heat induction of cts lysogens containing pCHT304 resulted in induction of catechol 2,3-dioxygenase with a peak of activity at 40 min postinduction (Fig.…”

“…(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).…”