A transcriptional activator, homologous to the Bacillus subtilis PurR repressor, is required for expression of purine biosynthetic genes in Lactococcus lactis.
A purR::pGh9:ISS1 mutant of Lactococcus lactis was obtained following transposon mutagenesis of strain MG1363 and selection for purine auxotrophs. After determination of the nucleotide sequence and deduction of the purR reading frame, the PurR product was found to be highly similar 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 transcription from the purine-regulated L. lactis purD promoter. In a parallel 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 with a central G residue at exactly position -76 relative to the transcriptional 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 autoregulated. 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 identified 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 the pur operon of B. subtilis is located almost identically, with respect to the promoter, to the PurBox sequences located in front of purC and purD in L. lactis. We present a hypothesis to explain how an ancestral PurR protein in B. subtilis could have evolved from an activator of the pur operon into a repressor which regulates transcription initiation from the same pur promoter by using the same PurR binding site and a similar response toward its effectors.
Department of Microbiology, Technical University of Denmark, DK2800 Lyngby, Denmark. mk@im.dtu.dk
M
M
Kilstrup
J
J
Martinussen
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011687:Purines / Q000096:biosynthesis*; D016364:Reading Frames; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000096:biosynthesis
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C065825:PurR protein, Bacteria; D011687:Purines; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D001616:beta-Galactosidase