Byrne A M, Olsen R H
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109-0620, USA.
J Bacteriol. 1996 Nov;178(21):6327-37. doi: 10.1128/jb.178.21.6327-6337.1996.
Burkholderia pickettii PKO1 metabolizes toluene and benzene via a chromosomally encoded toluene-3-monooxygenase pathway. Expression of the toluene-3-monooxygenase operon (tbuA1UBVA2C) is activated by the regulator, TbuT, in the presence of toluene. We have identified the TbuT coding region downstream of the toluene-3-monooxygenase structural genes by nucleotide sequence analysis and have shown that although TbuT is similar to XylR and DmpR, two members of the NtrC family of transcriptional activators which control toluene-xylene and (methyl)phenol catabolism, respectively, it is significantly different in the domain associated with effector specificity. Using a tbuA1-lacZ fusion reporter system, we determined that TbuT is activated not only by aromatic effectors but also the chlorinated aliphatic hydrocarbon trichloroethylene. Expression of tbuT and that of the tbuA1UBVA2C operon were found to be linked by readthrough transcription of tbuT from the toluene-3-monooxygenase promoter. As a result, transcription of tbuT is low when the toluene-3-monooxygenase operon is uninduced and high when expression of tbuA1UBVA2C is induced by toluene. Thus, the toluene-3-monooxygenase promoter drives the cascade expression of both the toluene-3-monooxygenase operon and tbuT, resulting in a positive feedback circuit. Examination of the nucleotide sequence upstream of the toluene-3-monooxygenase operon for promoter-like sequences revealed a -24 TGGC, -12 TTGC sequence, characteristic of sigma54 (rpoN)-dependent promoters. Primer extension and tbuA1-lacZ fusion analyses demonstrated that this -24, -12 promoter sequence, referred to as PtbuA1, was the toluene-3-monooxygenase promoter. Upstream of PtbuA1, a DNA region with dyad symmetry exhibited homology with the XylR-binding site present upstream of the Pu promoter. Deletions within this DNA sequence resulted in complete loss of expression from PtbuA1, suggesting that this region may serve as the TbuT-binding site.
皮氏伯克霍尔德菌PKO1通过染色体编码的甲苯-3-单加氧酶途径代谢甲苯和苯。在甲苯存在的情况下，甲苯-3-单加氧酶操纵子（tbuA1UBVA2C）的表达由调节因子TbuT激活。我们通过核苷酸序列分析确定了甲苯-3-单加氧酶结构基因下游的TbuT编码区，并表明尽管TbuT与XylR和DmpR相似，它们分别是控制甲苯-二甲苯和（甲基）苯酚分解代谢的NtrC家族转录激活因子的两个成员，但在与效应物特异性相关的结构域中存在显著差异。使用tbuA1-lacZ融合报告系统，我们确定TbuT不仅被芳香族效应物激活，还被氯化脂肪烃三氯乙烯激活。发现tbuT的表达与tbuA1UBVA2C操纵子的表达通过从甲苯-3-单加氧酶启动子对tbuT的通读转录而联系在一起。因此，当甲苯-3-单加氧酶操纵子未被诱导时，tbuT的转录较低，而当tbuA1UBVA2C的表达被甲苯诱导时，tbuT的转录较高。因此，甲苯-3-单加氧酶启动子驱动甲苯-3-单加氧酶操纵子和tbuT的级联表达，形成一个正反馈回路。对甲苯-3-单加氧酶操纵子上游核苷酸序列进行启动子样序列检查，发现了一个-24 TGGC、-12 TTGC序列，这是σ54（rpoN）依赖性启动子的特征。引物延伸和tbuA1-lacZ融合分析表明，这个-24、-12启动子序列，称为PtbuA1，是甲苯-3-单加氧酶启动子。在PtbuA1上游，一个具有二重对称的DNA区域与Pu启动子上游存在的XylR结合位点具有同源性。该DNA序列内的缺失导致PtbuA1完全失去表达，表明该区域可能作为TbuT结合位点。
