Iron-regulated transcription of the pvdA gene in Pseudomonas aeruginosa: Effect of fur and PvdS on promoter activity
Iron-regulated transcription of the pvdA gene in Pseudomonas aeruginosa: Effect of fur and PvdS on promoter activity
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DOI：
10.1128/jb.178.8.2299-2313.1996
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发表时间：
1996-04-01
影响因子：
3.2
通讯作者：
Visca, P
中科院分区：
文献类型：
--
作者：
Leoni, L;Ciervo, A;Visca, P
pvdA基因编码l -鸟氨酸n -5加氧酶，在铜绿假单胞菌中催化pyoverdin生物合成途径的关键步骤。通过启动子探针载体的表达研究，可以在pvdA上游5.9 kb的DNA片段中鉴定出三个铁紧密调控的启动子区域。调控pvdA表达的启动子位于pvdA翻译起始位点上游的154-bp序列中，RNA分析显示pvdA的表达在转录水平上受到铁调控。引物延伸和S1定位实验显示pvdA转录物有2个5‘端，分别为68 bp （T1）和43 bp (T2) 5’， pvdA转录物呈单胞性，T1占pvdA mRNA的90%。在pvdA转录起始位点的5′区明显缺失毛盒状序列。在TI的5'处发现了一个类似于algu依赖启动子-10六聚体的序列基序和一个由sigma(E)样因子PvdS控制的pyoverdin基因的铁饥饿盒。网站开始。铁调控启动子活性所需的最小DNA区域从bp -41到-154，相对于pvdA的ATG翻译起始位点，我们使用pvdA'::lacZ转录融合和Northern （RNA）分析来研究Pur和PvdS在铁调控pvdA表达中的作用。P. aeruginosa的两个皮毛突变体对pvdA表达的铁依赖性调节的反应远低于野生型PAO1。pvdA启动子的转录在异源宿主中不发生，除非存在反式pvdS基因，并且在铜绿假单胞菌pvdS突变体中被取消。在体内通过Pur滴定法证实了Fur抑制因子与包含pvdS启动子的150 bp片段的相互作用，并在体外用部分纯化的Fur制剂进行了凝胶阻滞实验。相反，pvdA的启动子区域不与Fur相互作用。我们的研究结果支持了P. aeruginosa Fur抑制因子通过中间sigma因子PvdS间接控制pvdA转录的假设；在铁含量充足的情况下，皮毛阻断pvdS启动子，从而阻止pvdS的表达，从而阻止pvdA和其他pyoverdin生物合成基因的转录。
The pvdA gene, encoding the enzyme L-ornithine N-5-oxygenase, catalyzes a key step of the pyoverdin biosynthetic pathway in Pseudomonas aeruginosa. Expression studies with a promoter probe vector made it possible to identify three tightly iron-regulated promoter regions in the 5.9-kb DNA fragment upstream of pvdA. The promoter governing pvdA expression was located within the 154-bp sequence upstream of the pvdA translation start site, RNA analysis showed that expression of PvdA is iron regulated at the transcriptional level. Primer extension and S1 mapping experiments revealed two 5' termini of the pvdA transcript, 68 bp (T1) and 43 bp (T2) 5' of the PvdA initiation, The pvdA transcripts were monocystronic, with T1 accounting for 90% of the pvdA mRNA. Fur box-like sequences were apparently absent in the regions 5' of pvdA transcription start sites. A sequence motif resembling the -10 hexamer of AlgU-dependent promoters and the iron starvation box of pyoverdin genes controlled by the sigma(E)-like factor PvdS were identified 5' of the TI. start site. The minimum DNA region required for iron-regulated promoter activity was mapped from bp -41 to -154 relative to the ATG translation start site of pvdA, We used pvdA'::lacZ transcriptional fusions and Northern (RNA) analyses to study the involvement of Pur and PvdS in the iron-regulated expression of pvdA. Two fur mutants of P. aeruginosa were much less responsive than wild-type PAO1 to the iron-dependent regulation of pvdA expression. Transcription from the pvdA promoter did not occur in a heterologous host unless in the presence of the pvdS gene in trans and was abrogated in a pvdS mutant of P. aeruginosa. Interaction of the Fur repressor with a 150-bp fragment encompassing the pvdS promoter was demonstrated in vivo by the Pur titration assay and confirmed in vitro by gel retardation experiments with a partially purified Fur preparation. Conversely, the promoter region of pvdA did not interact with Fur. Our results support the hypothesis that the P. aeruginosa Fur repressor indirectly controls pvdA transcription through the intermediary sigma factor PvdS; in the presence of sufficient iron, Fur blocks the pvdS promoter, thus preventing PvdS expression and consequently transcription of pvdA and other pyoverdin biosynthesis genes.