Organization of the pE194 genome

Summary pE194 is a 3.5 kilobase erythromycin resistance plasmid which was originally isolated from Staphylococcus aureus and has been introduced into Bacillus subtilis. This plasmid specifies at least five polypeptides, detectable in minicell extracts, one of which (E3) is inducible by erythromycin and is required for the expression of erythromycin resistance. We have constructed a detailed restriction endonuclease cleavage site map of pE194, and have localized the DNA sequences which code for the five polypeptides on the map. Four of the five polypeptides (E2, 3, 4 and 5) are specified by a region of the genome which has half the coding capacity required if these proteins were specified by contiguous genes. The determinant of E3 inducibility is located in the same segment. Based on the deficiency in coding capacity and on additional evidence including peptide mapping, we suggest that either the genes for these four polypeptides overlap, and are read in the same frame, or that some of these proteins represent degradation products. The latter alternative appears less likely since E3 is regulated independently of the other three proteins. The fifth protein (E1) is probably transcribed in the opposite direction. Strand separation and hybridization experiments confirm that both strands are transcribed. Hybridization of labeled RNA from a plasmid-carrying strain to restriction fragments of pE194 reveals that the expected plasmid sequences are transcribed in vivo, as is a region of the genome which is near the replication origin and which does not specify any known polypeptide. The map locations of 3–4 RNA polymerase binding sites are presented. Two of them are found in the segm ent which is predicted to contain the E1 and the E2-5 promoters, and a third is in the region of the replication origin.