The rhizosphere is the surface region of soil that is directly influenced by root secretions and associated soil microorganisms. Specific flavonoids possess antibacterial activity quercetin inhibits bacterial DNA gyrase, which induces DNA cleavage. To stay away from this kind of damaging effects, some bacteria have a program for degradation of flavonoids that detoxifies them.

A gram optimistic soil bacterium, Bacillus subtilis, possesses a quercetin 2,3 dioxygenase that converts quercetin to 2 protocatechuoyl phloroglucinol carboxylic acid and carbon monoxide. So far, quercetin 2,3 hts screening dioxygenase has been isolated from several bacteria and fungi therefore, this enzyme seems to be extensively distributed and to perform a major part in flavonoid degradation in soil microorganisms. In B. subtilis, the yxaG gene encoding quercetin 2,3 dioxygenase is a member of an operon containing the yxaH gene encoding a membrane protein with an unknown function. Because it is not uncommon for flavonoids to function as signaling molecules for communication between soil bacteria and plants, it was anticipated that, in addition to the LmrA/YxaF regulon, B.

subtilis possesses genes involved in flavonoid degradation or an additional physiological function for intercellular communication through flavonoids, which are underneath the handle of unknown transcriptional regulators in response to flavonoids. In this study, in order to elucidate the complete regulatory system for the expression of the genes responsive NSCLC to flavonoids in B. subtilis, we attempted to recognize further genes that are significantly induced by flavonoid addition by means of DNA microarray analysis. Among the new candidate flavonoid inducible genes found, we targeted on the yetM gene encoding a putative flavin adenine dinucleotide dependent monooxygenase and on its transcriptional regulatory mechanism. DNA microarray examination involving the wild variety strain and a yetL disruptant, performed in the framework of the Japan Functional Assessment Network for B.

Paclitaxel subtilis , suggested that the product of the yetL gene, which encodes a putative transcriptional regulator of the MarR household and is situated quickly upstream of the yetM gene in the opposite course, negatively regulates yetM transcription, which is induced by certain flavonoids. DNA binding experiments involving recombinant YetL showed that Paclitaxel binds to the corresponding single internet sites in the yetL and yetM promoter regions, with particularly increased affinity for the latter region. The DNA binding of YetL was inhibited successfully by flavonoids such as kaempferol, apigenin, and luteolin, and its weaker interaction with flavonoids such as quercetin and fisetin seems to be distinct from the interaction of LmrA/YxaF.

To date, the flavonoid responsive transcriptional regulators of several microorganisms have LY364947 been reported. Even so, to our knowledge, this is the 1st demonstration that a MarR family member exclusively responds to flavonoids, which gives a clue for elucidation of the whole regulatory mechanism for flavonoid induced gene expression. The B. subtilis strains used in this study are listed in Table 1. B. subtilis strain 168 was utilised as the normal strain. Strain YETLd was constructed by integration of plasmid pMUTIN2 into the yetL gene of strain 168. Strain FU1033 carrying a yetL deletion was constructed as follows. Longflanking homology PCR was performed to develop a antigen peptide fragment in which the chloramphenicol acetyltransferase gene was in the identical orientation as the unique yetL gene and sandwiched by the upstream and downstream areas of the yetL gene.

The areas upstream and downstream of yetL have been both amplified by PCR with the genomic DNA of strain 168 as the template and two primer pairs.