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A transcriptional activator, homologous to the Bacillus subtilis PurR repressor, is required for expression of purine biosynthetic genes in Lactococcus lactis.

ABSTRACT: 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.

SUBMITTER: Kilstrup M

PROVIDER: S-EPMC107375 | biostudies-literature | 1998 Aug

REPOSITORIES: biostudies-literature

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A transcriptional activator, homologous to the Bacillus subtilis PurR repressor, is required for expression of purine biosynthetic genes in Lactococcus lactis.

Kilstrup M M Martinussen J J

Journal of bacteriology 19980801 15

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 ...[more]

PMID: 9683488

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Project description:Bacterial MutS proteins are subdivided into two families, MutS1 and MutS2. MutS1 family members recognize DNA replication errors during their participation in the well-characterized mismatch repair (MMR) pathway. In contrast to the well-described function of MutS1, the function of MutS2 in bacteria has remained less clear. In Helicobacter pylori and Thermus thermophilus, MutS2 has been shown to suppress homologous recombination. The role of MutS2 is unknown in the Gram-positive bacterium Bacillus subtilis In this work, we investigated the contribution of MutS2 to maintaining genome integrity in B. subtilis We found that deletion of mutS2 renders B. subtilis sensitive to the natural antibiotic mitomycin C (MMC), which requires homologous recombination for repair. We demonstrate that the C-terminal small MutS-related (Smr) domain is necessary but not sufficient for tolerance to MMC. Further, we developed a CRISPR/Cas9 genome editing system to test if the inducible prophage PBSX was the underlying cause of the observed MMC sensitivity. Genetic analysis revealed that MMC sensitivity was dependent on recombination and not on nucleotide excision repair or a symptom of prophage PBSX replication and cell lysis. We found that deletion of mutS2 resulted in decreased transformation efficiency using both plasmid and chromosomal DNA. Further, deletion of mutS2 in a strain lacking the Holliday junction endonuclease gene recU resulted in increased MMC sensitivity and decreased transformation efficiency, suggesting that MutS2 could function redundantly with RecU. Together, our results support a model where B. subtilis MutS2 helps to promote homologous recombination, demonstrating a new function for bacterial MutS2.ImportanceCells contain pathways that promote or inhibit recombination. MutS2 homologs are Smr-endonuclease domain-containing proteins that have been shown to function in antirecombination in some bacteria. We present evidence that B. subtilis MutS2 promotes recombination, providing a new function for MutS2. We found that cells lacking mutS2 are sensitive to DNA damage that requires homologous recombination for repair and have reduced transformation efficiency. Further analysis indicates that the C-terminal Smr domain requires the N-terminal portion of MutS2 for function in vivo Moreover, we show that a mutS2 deletion is additive with a recU deletion, suggesting that these proteins have a redundant function in homologous recombination. Together, our study shows that MutS2 proteins have adapted different functions that impact recombination.

Dataset Information

A transcriptional activator, homologous to the Bacillus subtilis PurR repressor, is required for expression of purine biosynthetic genes in Lactococcus lactis.

Publications

A transcriptional activator, homologous to the Bacillus subtilis PurR repressor, is required for expression of purine biosynthetic genes in Lactococcus lactis.

Similar Datasets

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