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Journal of Bacteriology, January 2005, p. 512-521, Vol. 187, No. 2
0021-9193/05/$08.00+0     doi:10.1128/JB.187.2.512-521.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Probing Direct Interactions between CodY and the oppD Promoter of Lactococcus lactis

Chris D. den Hengst,1,{dagger} Peter Curley,2,{dagger} Rasmus Larsen,1 Girbe Buist,1 Arjen Nauta,3 Douwe van Sinderen,2 Oscar P. Kuipers,1 and Jan Kok1*

Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Haren,1 Corporate Research, Friesland Coberco Dairy Foods, Deventer, The Netherlands,3 Alimentary Pharmabiotic Centre and Department of Microbiology, National University of Ireland, Cork, Ireland2

Received 12 July 2004/ Accepted 11 October 2004

CodY of Lactococcus lactis MG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system. These genes include pepN, pepC, opp-pepO1, and probably prtPM, pepX, and pepDA2, since the expression of the latter three genes relative to nitrogen availability is similar to that of the former. By means of in vitro DNA binding assays and DNase I footprinting techniques, we demonstrate that L. lactis CodY interacts directly with a region upstream of the promoter of its major target known so far, the opp system. Our results indicate that multiple molecules of CodY interact with this promoter and that the amount of bound CodY molecules is affected by the presence of branched-chain amino acids and not by GTP. Addition of these amino acids strongly affects the extent of the region protected by CodY in DNase I footprints. Random and site-directed mutagenesis of the upstream region of oppD yielded variants that were derepressed in a medium with an excess of nitrogen sources. Binding studies revealed the importance of specific bases in the promoter region required for recognition by CodY.

* Corresponding author. Mailing address: Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, 9751 NN Haren, The Netherlands. Phone: 31 50 363 2111. Fax: 31 50 363 2348. E-mail: j.kok{at}biol.rug.nl.

{dagger} C.D.D.H. and P.C. contributed equally to this work.

Journal of Bacteriology, January 2005, p. 512-521, Vol. 187, No. 2
0021-9193/05/$08.00+0     doi:10.1128/JB.187.2.512-521.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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