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.
Bacillus subtilis LmrA is known to be a repressor that 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 (three-out-of-four match [in lowercase type]). Gel retardation analysis indicated that out of the eight flavonoids tested, quercetin, fisetin, and catechin are most inhibitory for LmrA to DNA binding, whereas quercetin, fisetin, tamarixetin, and galangin are most inhibitory 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-mentioned in vitro results, except 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 beta-galactosidase and quercetin 2,3-dioxgenase as well as that of multidrug resistance suggested that LmrA represses the lmrAB and yxaGH operons but that YxaF represses yxaGH more preferentially.
Hirooka
Kazutake
K
Department of Biotechnology, Faculty of Life Science and Biotechnology, Fukuyama University, 985 Sanzo, Higashimura, Fukuyama 729-0292, Japan.
Kunikane
Satoshi
S
Matsuoka
Hiroshi
H
Yoshida
Ken-Ichi
K
Kumamoto
Kanako
K
Tojo
Shigeo
S
Fujita
Yasutaro
Y
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002392:Catechin / Q000737:chemistry / Q000494:pharmacology; D018983:DNA Footprinting; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism; D004187:Disaccharides / Q000737:chemistry / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D005419:Flavonoids / Q000737:chemistry / Q000494:pharmacology*; D044948:Flavonols; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D008034:Lincomycin / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D020411:Oligonucleotide Array Sequence Analysis; D009876:Operon / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D011794:Quercetin / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D018085:Regulon / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004187:Disaccharides; D005419:Flavonoids; D044948:Flavonols; D012097:Repressor Proteins; C037032:galangin; C508967:tamarixetin; D002392:Catechin; D011794:Quercetin; D008034:Lincomycin; D049308:Dioxygenases; C003281:quercetin 2,3-dioxygenase; C017875:fisetin