Activation control of pur gene expression in Lactococcus lactis: proposal for a consensus activator binding sequence based on deletion analysis and site-directed mutagenesis of purC and purD promoter regions

Abstract

A comparison of the purC and purD upstream regions from Lactococcus lactis revealed the presence of a conserved ACCGAACAAT decanucleotide sequence located precisely between -79 and -70 nucleotides upstream from the transcriptional start sites. Both promoters have well-defined -10 regions but lack sequences resembling -35 regions for sigma70 promoters. Fusion studies indicated the importance of the conserved sequence in purine-mediated regulation. Adjacent to the conserved sequence in purC is a second and similar region required for high-level expression of the gene. A consensus PurBox sequence (AWWWCCGAACWWT) could be proposed for the three regions. By site-directed mutagenesis we found that mutation of the central G in the PurBox sequence to C resulted in low levels of transcription and the loss of purine-mediated regulation at the purC and purD promoters. Deletion analysis also showed that the nucleotides before the central CCGAAC core in the PurBox sequence are important. All results support the idea that purC and purD transcription is regulated by a transcriptional activator binding to the PurBox sequence.

FIG. 1

purC (A) and purD (B) promoter regions from L. lactis CHCC285. Transcriptional start sites (+1), putative −10 regions, putative operator sites (PurBox), start codons, and ribosome binding sites (Shine-Dalgarno [SD]) are shown in boldface, underlined, and marked above the nucleotide sequence, while the amino acids specified by the structural genes are shown below the sequence . End points of inserts in promoter fusion plasmid pAK80 (marked with the plasmid designations pLN95 to pLN97) are shown below the sequence as arrows, with the ending nucleotide aligned with the wavy line. Oligonucleotide primers (marked by an MKP designation) used for the generation of PCR products are also shown below the sequence, with the wavy lines aligned with the nucleotides present. The nucleotide sequence of the purD promoter region was modified from that given in reference .

FIG. 2

Primer extension analysis of purC and purD transcriptional start sites. Primer extension experiments were performed by using 10 μg of RNA extracted from CHCC285 (purC) and primer MKP58 (A), SW1 (purC-lacLM) and primer PAK80 (B), and SH1 (purD-lacLM) and primer PAK80 (C). RNA was extracted from cells growing exponentially in GSA medium (lanes 2) or in the same medium supplemented with purines (lanes 1). Lanes G, A, T, and C, sequencing reactions with the same primers as in the primer extension experiments and with PCR-generated DNA as a template. Asterisks indicate the positions of the flanking nucleotides shown in the sequences adjacent to the lanes with the extension products. The picture was scanned at 300 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.

FIG. 3

Expression of β-galactosidase from purC-lacLM (A) and purD-lacLM (B) fusions. The locations of the DNA fragments inserted in each fusion plasmid are shown below the structures of the promoter regions. Black boxes correspond to structural genes, while open and gray boxes correspond to intact and mutated PurBox sequences, respectively. Specific β-galactosidase activities are given as micromoles of ONP produced per minute per milligram of protein. The strains (MG1363 transformed with the indicated plasmids) were grown in GSA medium containing erythromycin (−Pur) or in the same medium containing guanosine, adenine, and hypoxanthine (+Pur). MG1363 transformed with vector pAK80 showed a low and constant level of background activity (<2 U/mg of protein) under both growth conditions. Fold regulation is given as the values for −Pur/+Pur.