Genetic and biochemical analysis of CodY-binding sites in Bacillus subtilis

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA

^* To whom correspondence should be addressed. Email: Boris.Belitsky{at}tufts.edu.

	Abstract

CodY is a global transcriptional regulator that is known to control directly expression of at least two dozen operons in Bacillus subtilis, but the rules that govern binding of CodY to its target DNA have been unclear. Using DNase I footprinting experiments, we identified CodY-binding sites upstream of the B. subtilis ylmA and yurP genes. The protected regions overlapped versions of a previously proposed CodY-binding consensus motif, AATTTTCWGAAAATT. Multiple single mutations were introduced into the CodY-binding sites of the ylmA, yurP, dppA and ilvB genes. The mutations affected both affinity of CodY for its binding sites in vitro and expression in vivo of lacZ fusions that carry these mutations in their promoter regions. Our results show that versions of the AATTTTCWGAAAATT motif, first identified for Lactococcus lactis CodY, with up to 5 mismatches play an important role in interaction of B. subtilis CodY with DNA.