Sellars Laura E, Bryant Jack A, Sánchez-Romero María-Antonia, Sánchez-Morán Eugenio, Busby Stephen J W, Lee David J
Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41080, Seville, Spain.
BMC Microbiol. 2017 Aug 3;17(1):170. doi: 10.1186/s12866-017-1079-2.
In bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. To investigate the subcellular location of DNA bound proteins in the folded bacterial nucleoid, fluorescent reporters have been developed which can be targeted to specific DNA operator sites. Such Fluorescent Reporter-Operator System (FROS) probes consist of a fluorescent protein fused to a DNA binding protein, which binds to an array of DNA operator sites located within the genome. Here we have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome.
We developed a new DNA binding fluorescent reporter, consisting of the Escherichia coli MalI protein fused to the mCherry fluorescent protein. This was used in combination with a Lac repressor:green fluorescent protein fusion to examine the spatial positioning and possible co-localisation of target genes, regulated by the Escherichia coli AraC protein. We report that induction of gene expression with arabinose does not result in co-localisation of AraC-regulated transcription units. However, measurable repositioning was observed when gene expression was induced at the AraC-regulated promoter controlling expression of the araFGH genes, located close to the DNA replication terminus on the chromosome. Moreover, in dividing cells, arabinose-induced expression at the araFGH locus enhanced chromosome segregation after replication.
Regions of the chromosome regulated by AraC do not colocalise, but transcription events can induce movement of chromosome loci in bacteria and our observations suggest a role for gene expression in chromosome segregation.
在细菌中，许多转录激活蛋白和阻遏蛋白调控多个转录单元，这些转录单元通常在细菌基因组上呈远距离分布。为了研究折叠的细菌类核中与DNA结合的蛋白质的亚细胞定位，已开发出可靶向特定DNA操纵位点的荧光报告基因。这种荧光报告基因-操纵子系统（FROS）探针由与DNA结合蛋白融合的荧光蛋白组成，该DNA结合蛋白与基因组内的一系列DNA操纵位点结合。在这里，我们利用大肠杆菌MaI转录因子与mCherry荧光蛋白融合，开发了一种新的FROS探针。我们将其与基于LacI阻遏蛋白::GFP蛋白的FROS探针结合使用，以评估大肠杆菌基因组上远距离的共同调控转录单元的细胞定位。
我们开发了一种新的与DNA结合的荧光报告基因，它由与mCherry荧光蛋白融合的大肠杆菌MaI蛋白组成。将其与Lac阻遏蛋白-绿色荧光蛋白融合体结合使用，以研究受大肠杆菌AraC蛋白调控的靶基因的空间定位和可能的共定位。我们报告说，用阿拉伯糖诱导基因表达不会导致AraC调控的转录单元共定位。然而，当在控制位于染色体DNA复制终点附近的araFGH基因表达的AraC调控启动子处诱导基因表达时，观察到了可测量的重新定位。此外，在分裂细胞中，araFGH位点处阿拉伯糖诱导的表达增强了复制后的染色体分离。
由AraC调控的染色体区域不会共定位，但转录事件可诱导细菌中染色体位点的移动，我们的观察结果表明基因表达在染色体分离中发挥作用。
