Probing direct interactions between CodY and the oppD promoter of Lactococcus lactis.
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.
den Hengst
Chris D
CD
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, 9751 NN Haren, The Netherlands.
Curley
Peter
P
Larsen
Rasmus
R
Buist
Girbe
G
Nauta
Arjen
A
van Sinderen
Douwe
D
Kuipers
Oscar P
OP
Kok
Jan
J
D000597:Amino Acids, Branched-Chain / Q000032:analysis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000032:analysis; D013294:Lactococcus lactis / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding
D000597:Amino Acids, Branched-Chain; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006160:Guanosine Triphosphate; D003850:Deoxyribonuclease I