CONSTRUCTION OF PLASMID VECTORS FOR THE DETECTION OF STREPTOCOCCAL PROMOTERS

Plasmid vectors have been constructed for detecting DNA fragments that exhibit promoter activity in Streptococcus sanguis. The plasmids are able to replicate in both S. sanguis and Escherichia coli, and contain an erythromycin resistance marker which is expressed in both hosts. Selection for promoter activity is dependent upon the insertion of appropriate DNA fragments upstream from a promoterless chloramphenicol acetyl transferase gene (cat) from Staphylococcus aureus. To facilitate this insertion, a pair of vectors, pMU1327 and pMU1328, were constructed with the polylinkker from M13mp18 in either orientation. The to transcripitional terminator of phage .lambda. is present downstream from cat. Translation stop codons in all reading frames are located between the polylinker and the initiation codon of cat. These plasmids have been used to isolate DNA fragments from S. sanguis, S. lactis and S. cremoris that exhibit promoter activity in S. sanguis.