GENETIC AND PHYSICAL ANALYSIS OF THE IMMUNITY REGION OF BACILLUS SUBTILIS PHAGE PHI-105.

摘要

I have performed physical and genetic analyses on the immunity region of the Bacillus subtilis temperate phage (phi)105. It has been possible to identify a (phi)105 gene and gene product which appear to be responsible for the maintenance of repression during lysogeny. A segment of the (phi)105 immunity region was isolated by cloning restriction fragments from (phi)105 genomic DNA into B.subtilis cloning vectors. Analysis of cells which contained these hybrid plasmids showed that they were immune to superinfection by (phi)105. Examination of the DNA sequence of the phage DNA fragments responsible for this immune phenotype revealed several open reading frames. It was found that one of the proteins predicted from this DNA sequence roughly corresponded in molecular weight to a protein expressed by the hybrid plasmids in B.subtilis minicells and E.coli maxicells. The hypothesis that the 16,521 dalton (16.5K) protein so identified actually is repressor protein and is responsible for the maintenance of lysogeny is supported by two lines of evidence. First, the DNA segment which contains the 16.5K protein was found by DNA sequence and restriction analyses to be deleted in all clear-plaque (phi)105 deletion mutants examined, and second, a (phi)105 lysogen was shown to synthesize an RNA that hybridizes to the DNA strand which codes for this protein. The promoter responsible for the maintenance of (phi)105 repression (PM) was identified by locating the 5'-terminus of lysogen-specific RNA on the DNA sequence. The PM region contains RNA polymerase recognition sequences which are found in most bacterial promoters. Located immediately upstream of the 16.5K repressor coding region is an eight base sequence complementary to the 3'-end of the 16S rRNA of B.subtilis. In addition to identifying the (phi)105 repressor I also identified the (phi)105 structural polypeptides and the essential J protein.