Characterization of the Flavonoid-Responsive Regulator FrrA and Its Binding Sites

Abstract

Previous microarray analyses revealed that in Bradyrhizobium japonicum, about 100 genes are induced by genistein, an isoflavonoid secreted by soybean. This includes the three genes freC, freA, and freB (systematic designations bll4319, bll4320, and bll4321), which are likely to form a genistein-, daidzein-, and coumestrol-inducible operon and to encode a multidrug efflux system. Upstream of freCAB and in the opposite orientation, FrrA (systematic designation Blr4322), which has similarity to TetRtype regulators, is encoded. A deletion of frrA leads to increased expression of freB in the absence of an inducer. We identified the correct translational start codon of frrA and showed that the gene is inducible by genistein and daidzein. The protein, which was heterologously expressed and purified from Escherichia coli, binds to two palindrome-like DNA elements (operator A and operator B), which are located in the intergenic region between freC and frrA. The replacement of several nucleotides or the insertion of additional spacer nucleotides prevented binding. Binding of FrrA was also affected by the addition of genistein. By mapping the transcription start sites, we found that operator A covers the transcriptional start site of freC and operator B is probably located between the ؊35 regions of the two divergently oriented genes. Operator A seems to be conserved in a few similar gene constellations in other proteobacteria. Our data indicate that in B. japonicum, besides NodD1 (the LysR family) and NodVW (a two-component response regulator), a third regulator type (a TetR family member) which responds to the plant signal molecules genistein and daidzein exists.

The TetR-Type MfsR Protein of the Integrative and Conjugative Element (ICE) ICE clc Controls both a Putative Efflux System and Initiation of ICE Transfer

“…4). This suggests MfsR-His6 to form homodimers in solution, which is in agreement with several observations that TetR family members occur as homodimers (31,32). Even larger protein bands were seen in cross-linked preparations (e.g., 95 kDa) ( Fig.…”

A Complex Mechanism Involving LysR and TetR/AcrR That Regulates Iron Scavenger Biosynthesis in Pseudomonas donghuensis HYS

“…These two regulators were not arranged in parallel and are both under the Gac/Rsm system; orf1 was upstream of orf12 in the biosynthesis of 7-HT in P. donghuensis HYS. Members of the TetR/AcrR family mainly act as repressors by forming homodimers and binding to the operator of target DNAs (28,29). In this study, the repression of orf9 expression via the overexpression of Orf12 may also occur through the formation of dimers; however, this speculation requires further study.…”

Transcriptional analysis of genes involved in competitive nodulation in Bradyrhizobium diazoefficiens at the presence of soybean root exudates

“…Many studies have shown that the “two-component system” regulates the specific gene expression of rhizobia by recognizing additional plant and environmental signals, which affects rhizobial ability to colonize roots. It also affects the competitive performance of rhizobia 19 , 47 , 48 . This study showed that a total of 19 genes were related to the “two-component system”, which was found to be significantly enriched in the KEGG analysis (Supplementary Table S3 ).…”