Characterization of sept of enterohemorrhagic Escherichia coli
Characterization of sept of enterohemorrhagic Escherichia coli
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DOI：
10.1128/jb.182.22.6490-6498.2000
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发表时间：
2000-11-01
影响因子：
3.2
通讯作者：
Guzmán, CA
中科院分区：
文献类型：
--
作者：
Kresse, AU;Beltrametti, F;Guzmán, CA
sept基因在肠细胞脱落位点表达，因此很可能与附着和脱落过程有关，位于该基因上下游的开放阅读框编码的产物也是如此。本研究对肠出血性大肠杆菌（EHEC）菌株EDL933的sept基因进行了分析，并对其多肽进行了表征。我们发现sept是单顺反转录的，独立于位于下游的esp操纵子，后者编码分泌蛋白EspA， d和-B。引物延伸分析使我们确定了在sept起始密码子上游83bp的单个转录起始。通过对上游区域的分析，鉴定出-5和-36之间的典型启动子序列。利用lacZ作为报告基因的翻译融合表明，sept在指数生长阶段被肠道生态位特有的刺激激活，例如37度的温度、营养丰富的环境、高渗透压和Mn2+的存在。蛋白定位研究表明，Sept存在于细胞质中，并与细菌膜组分有关。为了分析Sept蛋白在真核细胞感染过程中的功能作用，我们产生了一个框内缺失突变体。这种sept突变体在附着HeLa细胞和诱导肌动蛋白局部积聚的能力上受到严重损害。通过在trans中添加sept基因，这些缺陷得以部分修复。EDL933 Delta sepL突变体也表现出Esp蛋白的分泌受损，但没有生物合成，这是通过提供sept in trans完全补充的。这些结果表明，9在肠出血性大肠杆菌的生物循环中起着至关重要的作用。
The sept gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up- and downstream of this gene. In this study, the sept gene of the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sept is transcribed monocistronically and independently from the esp operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the sept start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions -5 and -36. Translational fusions using lacZ as a reporter gene demonstrated that sept is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37 degreesC, a nutrient-rich environment, high osmolarity, and the presence of Mn2+. Protein localization studies showed that Sept was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the Sept protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. This sept mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the sept gene in trans. The EDL933 Delta sepL mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing sept in trans. These results demonstrate the crucial role played by Sept in the biological cycle of EHEC.