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.

FIG. 1

Nucleotide sequence of the purR region from L. lactis MG1363. The nucleotide sequence of a 1,151-bp fragment is shown with the translated sequence of two open reading frames (purR and orfP) aligned with the coding sequence. Putative ribosome binding sites are underlined and marked by SD (Shine-Dalgarno). The transcriptional start site is underlined and marked with +1, and the presence of putative −10 and −35 regions is likewise indicated by underlining. The location of a PurBox sequence overlapping the −35 region is indicated by underlining. An inverted repeat which might function as a ρ-independent terminator structure for both purR and orfP transcription is shown by arrows under the nucleotide sequence. The location of the ISS1 insertion point in MK177 is shown by double overlining of the duplicated purR sequence (ATAATAAA). Primers MKP90, MKP72, and MKP104 are also shown by underlining.

FIG. 2

Physical maps of the purR regions in wild-type and mutant strains. (A) Physical map of the purR region in L. lactis MG1363. Boxes represent structural genes, and the transcriptional start site is marked with an arrow. The putative terminator of purR and orfP transcription is indicated by T. The purR-derived inserts in a number of plasmids are shown below the physical map. (B) Integration point and structure of the pGh9:ISS1 integrative plasmid after replicative insertion into the chromosome. During the insertion event, the ISS1 element is duplicated. (C) Physical map of the purR region in strain JM1010. The shaded regions indicate duplicated DNA.

FIG. 3

Comparison of PurR from L. lactis with homologous proteins. (A) Alignment of PurR from L. lactis with PurR from B. subtilis. A PRPP binding motif is underlined. Also, a region fulfilling the consensus requirements for a LysR family DNA binding motif (see text) is underlined. A region with similarity to a flexible loop found in the three-dimensional structure of the orotate phosphoribosyltransferase (OPRTase) of Salmonella typhimurium is also underlined. Asterisks and dots indicate identical amino acids and semiconservative substitutions, respectively. (B) Comparison of PurR with phosphoribosyltransferases exemplified by the adenine phosphoribosyltransferase (APRTase) from B. subtilis. The shaded regions show the extent of similarity between the two proteins. The locations of the putative LysR family DNA binding motif and the PRPP binding motif are indicated by black boxes. Also shown by a black box is a region with similarity to a flexible loop found in the three-dimensional structure of the OPRTase from S. typhimurium. The loop was inferred to make contact with PRPP. (C) Comparison of the PurR region from amino acids D130 to L169 with the homologous regions of the OPRTases from Sulfolobus solfataricus and Lactobacillus plantarum (accession no. g2065443 and e199390, respectively) and the APRTases from B. subtilis and Saccharomyces cerevisiae (accession no. U86377 and z46659, respectively). The flexible loop consensus sequence (also shown in panel B) was reported previously (28). A consensus amino acid sequence for this region is shown in the bottom row.

FIG. 4

Primer extension analysis of the purR transcriptional start site. An autoradiogram shows primer extension experiments performed with 10 μg of RNA extracted from MK219 (MG1363 purR-lacLM, lanes 1 and 2) and MK221 (MK177 purR-lacLM, lanes 3 and 4). RNA was extracted from cells growing exponentially in GSA medium (lanes 1 and 3) or in the same medium supplemented with purines (lanes 2 and 4). Lanes G, A, T, and C, sequencing reactions. Asterisks indicate the limits of the nucleotide sequence, shown on the right, with the transcriptional start site underlined. The picture was scanned at 400 dpi with a Scan Jet 4c/T (Hewlett-Packard Co.) and DeskScan II version 2.3 software. The TIF file was imported into Top Draw version 3.1 for the addition of text. Lanes 5, 6, 7, and 8 are identical to lanes 1, 2, 3, and 4, respectively, except that the image was acquired with a Packard Instant Imager. The Instant Imager measures the radioactivity over the surface of the gel and is more sensitive than autoradiography.

FIG. 5

Hypothetical bifunctional regulator as an intermediate in the evolution of PurR. The hypothetical bifunctional regulator is proposed to bind to a PurBox under all conditions, but it can change between two conformations, depending on the presence of PRPP. The conformational changes in the transit PurR upon PRPP binding might favor polymerization at low PRPP concentrations and RNA polymerase binding at high PRPP concentrations. The model incorporates data for PurR binding in both B. subtilis and L. lactis. In B. subtilis, PurR binding to PurBox DNA is only detected at low PRPP concentrations and only with an extended footprint from multiple PurR homodimers bound to the DNA. In L. lactis, PurR binds to DNA with both high and low PRPP concentrations, but activation is detected only with high PRPP concentrations. The bifunctional transit PurR protein could be the intermediary state in the evolution of an activator-regulated system into a repressor-regulated system with the same regulatory protein, promoter, and regulator binding site.