Isolation and Characterization of Phages That Bypass the Requirement for RNA-Mediated Antitermination.

Abstract

Introduction: The rpoCY75N mutation in the zinc-binding domain of the β' subunit of Escherichia coli RNA polymerase blocks the RNA-based mechanism of transcription antitermination utilized by bacteriophage HK022.

Materials and methods: Mutant phages that overcome the block imposed by the rpoCY75N mutation are described. These phages, designated "orc" (overcomes rpoC), carry mutations that create new promoters. Promoter activity was assessed by cloning the respective regions from the wild-type and orc phages into a promoterless lacZ reporter vector.

Results: Reporter assays showed that the sequence originating from orc phages had significant promoter activity when compared with the equivalent sequence cloned from the parental phage.

Conclusions: The newly created promoters facilitate the expression of phage genes that are essential for growth on the rpoCY75N strain by bypassing transcription terminators. The small plaque phenotype of orc phages, when grown on the mutant host, suggests that suppression of the rpoCY75N mutation is incomplete.