The Pseudomonas aeruginosa exotoxin A regulatory gene, ptxS: evidence for negative autoregulation.
The Pseudomonas aeruginosa exotoxin A regulatory gene, ptxS: evidence for negative autoregulation.
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铜绿假单胞菌外毒素 A 调节基因 ptxS：负性自动调节的证据。
DOI：
10.1128/jb.181.16.4890-4895.1999
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发表时间：
1999
影响因子：
3.2
通讯作者：
Hamood,AN
中科院分区：
文献类型：
--
作者：
Swanson,BL;Colmer,JA;Hamood,AN
我们先前已经描述了aPseudomonasaerodynosagene，ptxR，其在转录水平上增强外毒素A的产生。我们还描述了另一个基因ptxS，它从mptxR分化转录，并通过mptxR干扰外毒素A合成的增强。然而，ptxR和/或ptxS的调节机制尚不清楚。在这项研究中，我们试图（通过使用DNA凝胶迁移试验），以确定ifP。aerobiosaccharides含有一种潜在的调节蛋白，其特异性地结合到ptxRorptxSupstream区域。在初步分析中，当含有ptxR-ptx Sintergenic区域的探针与P. aerodynosaPAO1.用代表theptxSupstream区域的较小片段检测到最强的结合活性。额外的缺失分析定位的结合到一个52 bp的片段直接上游的ptxS。当52-bp片段与ptxSisogenic突变体PAO 1：：ptxS的裂解物温育时，未检测到凝胶移位带。然而，当携带ptxSintact的质粒被引入PAO 1：：ptxS时，结合带再生。此外，当52 bp片段与从T7启动子过量表达ptxS的大肠杆菌裂解物温育时，检测到凝胶移位条带。用aptxS-lacZ融合质粒检测PtxS对PtxS表达的影响。PAO 1：：ptxS表达的β-半乳糖苷酶活性比PAO 1表达的高4 ~ 5倍。使用DNA酶I足迹分析，结合区域被指定为20-bp片段。在该片段内，存在一个14 bp的回文序列，其可以作为PtxS结合位点发挥作用。这些结果表明PtxS通过与PtxSupstream区域内的特定序列结合来自动调节其合成。
We have previously described aPseudomonas aeruginosagene,ptxR, which enhances exotoxin A production at the transcriptional level. We have also described another gene,ptxS, which is transcribed divergently fromptxRand interferes with the enhancement of exotoxin A synthesis byptxR. However, the mechanisms through whichptxRand/orptxSare regulated is not known. In this study, we attempted (by using the DNA gel shift assay) to determine ifP. aeruginosacontains a potential regulatory protein that binds specifically to theptxRorptxSupstream region. In the initial analysis, different-sized gel shift bands were detected when a probe containing theptxR-ptxSintergenic region was incubated with the lysate ofP. aeruginosaPAO1. The strongest binding activity was detected with a smaller fragment that represents theptxSupstream region. Additional deletion analysis localized the binding to a 52-bp fragment immediately upstream ofptxS. The gel shift band was not detected when the 52-bp fragment was incubated with the lysate of theptxSisogenic mutant PAO1::ptxS. However, the binding band was regenerated when a plasmid carryingptxSintact was introduced into PAO1::ptxS. In addition, the gel shift band was detected when the 52-bp fragment was incubated with a lysate ofEscherichia coliin whichptxSwas overexpressed from the T7 promoter. The effect of PtxS onptxSexpression was examined by using aptxS-lacZfusion plasmid. The level of β-galactosidase activity produced by PAO1::ptxScarrying the fusion plasmid was four- to fivefold higher than that produced by PAO1 carrying the same plasmid. Using DNase I footprinting analysis, the binding region was specified to a 20-bp fragment. Within the fragment, a 14-bp palindromic sequence exists that may function as a PtxS binding site. These results suggest that PtxS autoregulates its synthesis by binding to a specific sequence within theptxSupstream region.