Institute of Genetics, Dresden University of Technology, Dresden, Germany.
J Bacteriol. 2012 May;194(9):2363-70. doi: 10.1128/JB.06567-11. Epub 2012 Mar 2.
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 TetR-type 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.
先前的微阵列分析表明，大豆分泌的异黄酮染料木黄酮能诱导约 100 个基因在根瘤菌中表达。这包括三个基因 freC、freA 和 freB（系统命名为 bll4319、bll4320 和 bll4321），它们可能形成一个染料木黄酮、大豆苷元和黄豆黄素诱导型操纵子，并编码一个多药外排系统。在 freCAB 的上游，以相反的方向编码了 FrrA（系统命名为 Blr4322），它与 TetR 型调控因子具有相似性。frrA 的缺失导致在没有诱导剂的情况下 freB 的表达增加。我们确定了 frrA 的正确翻译起始密码子，并表明该基因可被染料木黄酮和大豆苷元诱导。该基因从大肠杆菌中异源表达和纯化的蛋白质与两个回文样 DNA 元件（操纵子 A 和操纵子 B）结合，这些元件位于 freC 和 frrA 之间的基因间区。替换几个核苷酸或插入额外的间隔核苷酸会阻止结合。FrrA 的结合也受到染料木黄酮的影响。通过绘制转录起始位点，我们发现操纵子 A 覆盖了 freC 的转录起始位点，而操纵子 B 可能位于两个不同方向基因的-35 区之间。操纵子 A 似乎在其他几种类似的原核生物的类似基因排列中保守。我们的数据表明，在根瘤菌中，除了 NodD1（LysR 家族）和 NodVW（双组分响应调节剂）之外，还存在第三种调控类型（TetR 家族成员），它对植物信号分子染料木黄酮和大豆苷元作出反应。
