A TRANSCRIPTIONAL ACTIVATOR, HOMOLOGOUS TO THE BACILLUS-SUBTILIS PURRREPRESSOR, IS REQUIRED FOR EXPRESSION OF PURINE BIOSYNTHETIC GENES INLACTOCOCCUS-LACTIS

A purR::pGh9:ISS1 mutant of Lactococcus lactis was obtained following transposon mutagenesis of strain MG1363 and selection for purine auxot rophs. After determination of the nucleotide sequence and deduction of the purR reading frame, the PurR product was found to be highly simil ar to the purR-encoded repressor from Bacillus subtilis, The wild-type purR gene complemented the purine auxotrophy of a purR::ISS1 mutant, and it was shown that the purR::ISS1 mutation lowered the level of tra nscription from the purine-regulated L. lactis purD promoter. In a par allel study on the regulation of purC and purD expression in L, lactis (M. Kilstrup, S. G. Jessing, S. B. Wichmand-Jorgensen, M, Madsen, and D. Nilsson, J. Bacteriol. 180:3900-3906, 1998), we identified regions (PurBox sequences: AWWWCCGAACWWT) upstream of the promoters,vith a ce ntral G residue at exactly position -76 relative to the transcriptiona l start site. The PurBox sequences were found to be required for high- level promoter activity and purine regulation. We identified a PurBox sequence overlapping the -35 region of the L. lactis purR promoter and found, by studies of a purR-lacLM fusion plasmid, that purR is autore gulated, Because of the high degree of similarity of the PurR proteins from B, subtilis and L, lactis, we looked for PurBox sequences in the promoter regions of the PurR-regulated genes in B, subtilis and ident ified a perfectly matching PurBox sequence in the purA promoter region and slightly degenerate PurBox-like sequences in the promoter regions for the pur operon and the purR gene. Interestingly, the PurBox in th e pur operon of B, subtilis is located almost identically, with respec t to the promoter, to the PurBox sequences located in front of purC an d purD in L, lactis, We present a hypothesis to explain how an ancestr al PurR protein in B. subtilis could have evolved from an activator of the pur operon into a repressor which regulates transcription initiat ion from the same pur promoter by using the same PurR binding site and a similar response toward its effectors.