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Journal of Bacteriology, August 1998, p. 3900-3906, Vol. 180, No. 15
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

Mogens Kilstrup,1,* Stine G. Jessing,1 Stephanie B. Wichmand-Jørgensen,1 Mette Madsen,2 and Dan Nilsson2

Department of Microbiology, Technical University of Denmark, DK2800 Lyngby,1 and Department of Physiology and Metabolism, Chr. Hansen A/S, DK2870 Hørsham,2 Denmark

Received 23 March 1998/Accepted 28 May 1998

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 sigma 70 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.

* Corresponding author. Mailing address: Department of Microbiology, Technical University of Denmark, Building 301, DK2800 Lyngby, Denmark. Phone: 45 45 25 25 28. Fax: 45 45 88 26 60. E-mail: mk{at}im.dtu.dk.

Journal of Bacteriology, August 1998, p. 3900-3906, Vol. 180, No. 15
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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