Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon
Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon
复制标题
DOI：
10.1128/jb.180.5.1277-1286.1998
复制
发表时间：
1998-03-01
影响因子：
3.2
通讯作者：
Wanner, BL
中科院分区：
文献类型：
--
作者：
Haldimann, A;Daniels, LL;Wanner, BL
大肠杆菌基因受环境无机磷酸盐（P-i）水平的调节形成磷酸盐（Pho）调节子，这一调节需要7种蛋白质，它们的合成受自身控制，包括反应调节子PhoB、它的配偶体、组氨酸传感激酶PhoR、P-i特异性转运（Pst）系统的所有4个组分（PstA、PstB、PstC和PstS），以及功能未知的蛋白质PhoU，在这里，我们通过将每一个置于阿拉伯糖调节的P-araB启动子或鼠李糖调节的P-araB启动子的严格控制下，研究了与正常对照解偶联的PhoB合成和PhoR合成的影响。为了做到这一点，我们制备了等位基因置换质粒，其通常可用于构建P-araB或P-rhaB融合体并用于将它们重组到E.使用携带这种单拷贝融合体的菌株，我们表明P-rhaB融合体比P-araB融合体受到更严格的调节，因为P-rhaB-phoR（+）融合体而不是P-araB-phoR（+）融合体在不存在其特异性诱导物的情况下显示无效表型。然而在不存在诱导的情况下，P-araB-phoB（+）和P-rhaB-phoB（+）融合体都显示无效表型。这些数据表明，Pho调节子的转录激活所需的PhoR比PhoB少，这与它们各自的作用模式一致。我们还使用这些融合体来研究PhoU。斯蒂德和B。L. Wanner，J. Bacteriol，175：6797-6809，1993）。它们也容易产生在phoB、phoR或pst基因中具有损伤的补偿突变体，这使得它们的研究特别困难。在这里，我们发现，通过使用P-araB-phoB（+）、P-rhaB-phoB（+）或P-rhaB-phoR（+）融合体，我们能够克服Pst（+）Δ phoU突变体的极其有害的生长缺陷，生长缺陷显然是一个高层次的Pst合成的结果，在没有PhoU的情况下，从自控制的PhoB和PhoR的合成。
Escherichia coli genes regulated by environmental inorganic phosphate (P-i) levels form the phosphate (Pho) regulon, This regulation requires seven proteins, whose synthesis is under autogenous control, including response regulator PhoB, its partner, histidine sensor kinase PhoR, all four components of the P-i-specific transport (Pst) system (PstA, PstB, PstC, and PstS), and a protein of unknown function called PhoU, Here we examined the effects of uncoupling PhoB synthesis and PhoR synthesis from their normal controls by placing each under the tight control of the arabinose-regulated P-araB promoter or the rhamnose-regulated P-rhaB, promoter, To do this, we made allele replacement plasmids that may be generally useful for construction of P-araB or P-rhaB fusions and for recombination of them onto the E. coli chromosome at the araCBAD or rhaRSBAD locus, respectively, Using strains carrying such single-copy fusions, we showed that a P-rhaB fusion is more tightly regulated than a P-araB fusion in that a P-rhaB-phoR(+) fusion but not a P-araB-phoR(+) fusion shows a null phenotype in the absence of its specific inducer, Yet in the absence of induction, both P-araB-phoB(+) and P-rhaB-phoB(+) fusions exhibit a null phenotype. These data indicate that less PhoR than PhoB is required for transcriptional activation of the Pho regulon, which is consistent with their respective modes of action, We also used these fusions to study PhoU, Previously, we had constructed strains with precise Delta phoU mutations, However, we unexpectedly found that such Delta phoU mutants have a severe growth defect (P. M. Steed and B. L. Wanner, J. Bacteriol, 175:6797-6809, 1993). They also readily give rise to compensatory mutants with lesions in phoB, phoR, or a pst gene, making their study particularly difficult, Here we found that, by using P-araB-phoB(+), P-rhaB-phoB(+), or P-rhaB-phoR(+) fusions, we were able to overcome the extremely deleterious growth defect of a Pst(+) Delta phoU mutant, The growth defect is apparently a consequence of high-level Pst synthesis resulting from autogenous control of PhoB and PhoR synthesis in the absence of PhoU.