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首页> 外文期刊>Molecular Microbiology >Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1.
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Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1.

机译:铜绿假单胞菌PAO1中2-酮葡萄糖酸利用操纵子的表征。

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摘要

The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif. Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P. aeruginosa 2-ketogluconate-negative mutant. In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.
机译:铜绿假单胞菌蛋白PtxS通过与其基因上游区域的结合来负调控自身的合成。我们最近在ptxS上游区域内鉴定了一个14 bp回文序列作为PtxS操纵位点(OP1)。在这项研究中,我们搜索了铜绿假单胞菌基因组序列,以确定该14 bp序列是否存在于铜绿假单胞菌染色体的其他区域。另一个PtxS操作员位点(OP2)位于ptxS下游47 bp。 DNA凝胶移位实验证实,PtxS特异性结合了携带OP2的520 bp片段。 OP2的DNA片段3'包含四个开放阅读框(ORF1-ORF4),分别编码29、32、48和35 kDa蛋白质。通过T7表达实验确认了ORF 2和3的产物的分子量。计算机分析表明,ORF2编码一个ATP依赖激酶; ORF3,转运蛋白;和ORF4,一种脱氢酶。 ORF1的预测产物显示与先前鉴定的蛋白质没有同源性,并且包含醛糖1-表异构酶蛋白质基序内的所有保守氨基酸。 ptxs-ORF1基因间区域的检查(使用启动子融合实验)显示不存在潜在的启动子。在铜绿假单胞菌菌株PAO1中构建了一个ORF1缺陷的同基因突变体。与它的亲本菌株相反,该突变体无法在其中2-酮葡萄糖酸酯为唯一碳源的基本培养基上生长。同样,以前构建的PAO1的ptxS等基因突变体在含有2-酮葡萄糖酸作为唯一碳源的基本培养基中也不生长。此外,带有包含ptxS和ORF 1-4的片段的质粒补充了先前描述的铜绿假单胞菌2-酮葡糖酸酯阴性突变体的缺陷。在10 mM 2-酮葡萄糖酸存在下,PtxS与携带OP1或OP2的DNA片段的体外结合受到抑制。这些结果表明:(i)ptxS与其他四个ORF一起构成了铜绿假单胞菌的2-酮葡萄糖酸利用操纵子(kgu)。因此,ORF 1-4分别命名为kguE,kguK,kguT和kguD。 (ii)PtxS通过绑定操纵子中的两个操纵子(OP1和OP2)来调节kgu操纵子的表达; (iii)2-酮葡糖酸酯是kgu操纵子的分子诱导剂或PtxS的分子效应子。

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