RNA polymerases (RNAP) can be divided into two unrelated canonical families: multisubunit (ms) and single-subunit (ss) polymerases. All phage-encoded RNAPs studied so far belong to the ssRNAP family. However, giant phiKZ-like bacteriophages of Myoviridae family may encode unique non-canonical msRNAPs. Our bioinformatic search revealed that all known giant phiKZ-related phages encode two distinct sets of proteins distantly related to bacterial RNAP. These sets were referred to as “virion”(v) and “non-virion”(nv) because the proteins of the virion set were identified in virions. We studied transcription profiling during infection of a model bacteriophage phiKZ and showed that the phage development is not affected by high concentrations of rifampicin (rif). Since RNAP of the phage host is sensitive to rif, we propose that phage-encoded RNAPs have to transcribe phage genes throughout phiKZ development using phage-specific promoters. Next, a combination of bioinformatic analysis and biochemical approaches allowed us to identify putative early, middle and late phiKZ promoters. We were able to isolate the nvRNAP from the host P. aeruginosa PAO1 cells infected with phiKZ. This enzyme consists of 5 major polypeptides (4 β’-/β–like proteins from the nv-set, and one more phage protein of unknown function). It is able to transcribe in vitro from phage DNA fragments containing late phiKZ promoters. Transcription is rif-resistant and in vitro transcription start points match the ones observed in vivo.
Acknowledgement
This research is supported by the Ministry of Education and Science of Russian Federation, project 14.B25.31.0004 and by NIH R01 GM59295 for K. Severinov.