Dual Regulation of the Bacillus subtilis Regulon Comprising the lmrAB and yxaGH Operons and yxaF Gene by Two Transcriptional Repressors, LmrA and YxaF, in Response to Flavonoids

Department of Biotechnology, Faculty of Life Science and Biotechnology, Fukuyama University, 985 Sanzo, Higashimura, Fukuyama 729-0292, Japan; Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan

^* To whom correspondence should be addressed. Email: yfujita{at}bt.fubt.fukuyama-u.ac.jp.

	Abstract

Bacillus subtilis LmrA is known to be a repressor which regulates the lmrAB and yxaGH operons; lmrB and yxaG encode a multidrug resistance pump and quercetin 2,3-dioxygenase, respectively. DNase I footprinting analysis revealed that LmrA and YxaF, which are paralogous to each other, bind specifically to almost the same cis-sequences, LmrA/YxaF boxes, located in the promoter regions of the lmrAB operon, the yxaF gene, and the yxaGH operon for their repression and containing a consensus sequence of AWTATAtagaNYGgTCTA, where W, Y, and N stand for A or T, C or T, and any base, respectively (3 out of 4 match, in lower-case). Gel retardation analysis indicated that out of the 8 flavonoids tested, quercetin, fisetin and catechin are most inhibitory for LmrA as to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are for YxaF. Also, YxaF bound most tightly to the tandem LmrA/YxaF boxes in the yxaGH promoter region. The lacZ-fusion experiments essentially supported the above in vitro results, except for that galangin did not activate the lmrAB and yxaGH promoters, probably due to its poor incorporation into cells. Thus, the LmrA/YxaF regulon presumably comprising the lmrAB operon, the yxaF gene, and the yxaGH operon is induced in response to certain flavonoids. The in vivo experiments to examine the regulation of the synthesis of the reporter -galactosidase and quercetin 2,3-dioxgenase as well as that of the multidrug resistance suggested that LmrA well represses the lmrAB and yxaGH operons, but YxaF represses yxaGH more preferentially.