Use of an inducible regulatory protein to identify members of a regulon: Application to the regulon controlled by the leucine-responsive regulatory protein (Lrp) in Escherichia coli
Use of an inducible regulatory protein to identify members of a regulon: Application to the regulon controlled by the leucine-responsive regulatory protein (Lrp) in Escherichia coli
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DOI：
10.1128/jb.179.20.6254-6263.1997
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发表时间：
1997-10-01
影响因子：
3.2
通讯作者：
Blumenthal, RM
中科院分区：
文献类型：
--
作者：
Bhagwat, SP;Rice, MR;Blumenthal, RM
程序开发，以促进属于一个给定的调节子和表征其响应的调节器的基因的识别。大肠杆菌亮氨酸响应调节蛋白（Lrp）控制的调节子进行了研究，通过分离随机转录融合lacZ，使用λ placMu 53和一个菌株，其中Lrp是下异丙基硫代-β-D-吡喃半乳糖苷（IPTG）诱导控制。通过在lacZ处同源重组整合自杀载体pIVET 1，然后通过自连接消化的染色体DNA，克隆表现出IPTG响应性β-半乳糖苷酶活性的融合体。在使用质粒克隆产生具有相同序列的中断和不中断拷贝的部分二倍体菌株后，我们验证了lacZ表达的模式。如果部分二倍体表达模式与原始融合菌株所示的表达模式相比没有变化，则克隆的融合体负责感兴趣的调控模式;表达模式的差异可能表明原始菌株携带多个融合体，或者存在中断融合基因的自体效应。使用这些程序，我们产生了类似于5 × 10（6）菌株的融合文库;筛选了类似于3，000个这些菌株，产生了84个Lrp响应性融合体，并且84个中的10个是表型稳定的并进行了表征。在一个给定的操纵子在体内Lrp滴定不同的融合的反应揭示了插入的位置在表达上的变化。在新鉴定的调节子成员中，有一个位于rpiA和serA之间的开放阅读框架（orf 3）。此外，表达的融合下游的dinF被认为是Lrp依赖性仅在稳定期。
Procedures were developed to facilitate the identification of genes that belong to a given regulon and characterization of their responses to the regulator. The regulon controlled by the Escherichia coli leucine-responsive regulatory protein (Lrp) was studied by isolating random transcriptional fusions to lacZ, using lambda placMu53 and a strain in which lrp is under isopropylthio-beta-D-galactopyranoside (IPTG)-inducible control. Fusions exhibiting IPTG-responsive beta-galactosidase activity were cloned by integrating the suicide vector pIVET1 via homologous recombination at lacZ, followed by self-ligating digested chromosomal DNA. We verified the patterns of lacZ expression after using the plasmid clones to generate merodiploid strains with interrupted and uninterrupted copies of the same sequence. If the merodiploid expression pattern was unchanged from that shown by the original fusion strain, then the cloned fusion was responsible for the regulatory pattern of interest; a difference in the expression pattern could indicate that the original strain carried multiple fusions or that there were autogenous effects of having interrupted the fused gene. Using these procedures, we generated a fusion library of similar to 5 x 10(6) strains; similar to 3,000 of these strains were screened, yielding 84 Lrp-responsive fusions, and 10 of the 84 were phenotypically stable and were characterized. The responses of different fusions in a given operon to in vivo Lrp titrations revealed variations in expression with the position of insertion. Among the newly identified members of the regulon is an open reading frame (orf3) between rpiA and serA. Also, expression of a fusion just downstream of dinF was found to be Lrp dependent only in stationary phase.