Morales Sergio E, Lewis Thomas A
Department of Microbiology and Molecular Genetics, University of Vermont, 95 Carrigan Dr., Burlington, VT 05405, USA.
Appl Environ Microbiol. 2006 Nov;72(11):6994-7002. doi: 10.1128/AEM.01518-06. Epub 2006 Aug 25.
In order to gain an understanding of the molecular mechanisms dictating production of the siderophore and dechlorination agent pyridine-2,6-bis(thiocarboxylic acid) (PDTC), we have begun characterization of a gene found in the pdt gene cluster of Pseudomonas stutzeri KC predicted to have a regulatory role. That gene product is an AraC family transcriptional activator, PdtC. Quantitative reverse transcription-PCR and expression of transcriptional reporter fusions were used to assess a role for pdtC in the transcription of pdt genes. PdtC and an upstream, promoter-proximal DNA segment were required for wild-type levels of expression from the promoter of a predicted biosynthesis operon (P(pdtF)). At least two other transcriptional units within the pdt cluster were also dependent upon pdtC for expression at wild-type levels. The use of a heterologous, Pseudomonas putida host demonstrated that pdtC and an exogenously added siderophore were necessary and sufficient for expression from the pdtF promoter, i.e., none of the PDTC utilization genes within the pdt cluster were required for transcriptional signaling. Tests using the promoter of the pdtC gene in transcriptional reporter fusions indicated siderophore-dependent negative autoregulation similar to that seen with other AraC-type regulators of siderophore biosynthesis and utilization genes. The data increase the repertoire of siderophore systems known to be regulated by this type of transcriptional activator and have implications for PDTC signaling.
为了了解决定铁载体和脱氯剂吡啶 - 2,6 - 双(硫代羧酸)(PDTC)产生的分子机制,我们已开始对在施氏假单胞菌KC的pdt基因簇中发现的一个预测具有调控作用的基因进行表征。该基因产物是一种AraC家族转录激活因子,PdtC。采用定量逆转录 - PCR和转录报告融合体的表达来评估pdtC在pdt基因转录中的作用。野生型水平的预测生物合成操纵子(P(pdtF))启动子表达需要PdtC和一个上游的、启动子近端DNA片段。pdt簇内至少还有另外两个转录单元在野生型水平表达时也依赖于pdtC。使用异源恶臭假单胞菌宿主表明,pdtC和外源添加的铁载体对于pdtF启动子的表达是必要且充分的,即pdt簇内的PDTC利用基因对于转录信号传导不是必需的。在转录报告融合体中使用pdtC基因的启动子进行测试表明,铁载体依赖性负自调节类似于其他铁载体生物合成和利用基因的AraC型调节因子所观察到的情况。这些数据增加了已知受此类转录激活因子调控的铁载体系统的种类,并对PDTC信号传导有影响。