Yamanaka Yuki, Shimada Tomohiro, Yamamoto Kaneyoshi, Ishihama Akira
Research Center for Micro-Nano Technology, Nano Technology, Koganei, Tokyo 184-8584, Japan.
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuda, Yokohama, Japan.
Microbiology (Reading). 2016 Jul;162(7):1253-1264. doi: 10.1099/mic.0.000292. Epub 2016 Apr 25.
Genomic SELEX (systematic evolution of ligands by exponential enrichment) screening was performed for identification of the binding site of YbiH, an as yet uncharacterized TetR-family transcription factor, on the Escherichia coli genome. YbiH was found to be a unique single-target regulator that binds in vitro within the intergenic spacer located between the divergently transcribed ybiH-ybhGFSR and rhlE operons. YbhG is an inner membrane protein and YbhFSR forms a membrane-associated ATP-binding cassette (ABC) transporter while RhlE is a ribosome-associated RNA helicase. Gel shift assay and DNase footprinting analyses indicated one clear binding site of YbiH, including a complete palindromic sequence of AATTAGTT-AACTAATT. An in vivo reporter assay indicated repression of the ybiH operon and activation of the rhlE operon by YbiH. After phenotype microarray screening, YbiH was indicated to confer resistance to chloramphenicol and cefazoline (a first-generation cephalosporin). A systematic survey of the participation of each of the predicted YbiH-regulated genes in the antibiotic sensitivity indicated involvement of the YbhFSR ABC-type transporter in the sensitivity to cefoperazone (a third-generation cephalosporin) and of the membrane protein YbhG in the control of sensitivity to chloramphenicol. Taken together with the growth test in the presence of these two antibiotics and in vitro transcription assay, it was concluded that the hitherto uncharacterized YbiH regulates transcription of both the bidirectional transcription units, the ybiH-ybhGFSR operon and the rhlE gene, which altogether are involved in the control of sensitivity to cefoperazone and chloramphenicol. We thus propose to rename YbiH as CecR (regulator of cefoperazone and chloramphenicol sensitivity).
进行了基因组SELEX（指数富集配体的系统进化）筛选，以鉴定YbiH（一种尚未表征的TetR家族转录因子）在大肠杆菌基因组上的结合位点。发现YbiH是一种独特的单靶点调节因子，在体外结合于反向转录的ybiH - ybhGFSR和rhlE操纵子之间的基因间隔区。YbhG是一种内膜蛋白，YbhFSR形成膜相关的ATP结合盒（ABC）转运蛋白，而RhlE是一种核糖体相关的RNA解旋酶。凝胶迁移试验和DNase足迹分析表明YbiH有一个明确的结合位点，包括一个完整的回文序列AATTAGTT - AACTAATT。体内报告基因试验表明YbiH抑制ybiH操纵子并激活rhlE操纵子。经过表型微阵列筛选，YbiH显示出对氯霉素和头孢唑林（第一代头孢菌素）的抗性。对每个预测的YbiH调控基因参与抗生素敏感性的系统调查表明，YbhFSR ABC型转运蛋白参与对头孢哌酮（第三代头孢菌素）的敏感性，而膜蛋白YbhG参与氯霉素敏感性的控制。结合在这两种抗生素存在下的生长试验和体外转录试验，得出结论：迄今未表征的YbiH调节双向转录单元ybiH - ybhGFSR操纵子和rhlE基因的转录，它们共同参与头孢哌酮和氯霉素敏感性的控制。因此，我们建议将YbiH重新命名为CecR（头孢哌酮和氯霉素敏感性调节因子）。
