TEMPERATURE SENSING IN YERSINIA-PESTIS - REGULATION OF YOPE TRANSCRIPTION BY LCRF
TEMPERATURE SENSING IN YERSINIA-PESTIS - REGULATION OF YOPE TRANSCRIPTION BY LCRF
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DOI：
10.1128/jb.174.13.4275-4286.1992
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发表时间：
1992-07-01
影响因子：
3.2
通讯作者：
GOGUEN, JD
中科院分区：
文献类型：
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作者：
HOE, NP;MINION, FC;GOGUEN, JD
在大肠杆菌中，发现在含有lcrF位点的鼠疫耶尔森菌质粒pCD1克隆的BamHI G片段存在时，yopE=lacZ融合受温度调节。增加lcrF拷贝数相对于yopE报告基因拷贝数对诱导比的影响可以忽略不计（26℃vs 37℃），但会导致yopE转录的绝对水平大幅降低。我们通过监测BamHI G缺失衍生物的诱导表型来定位lcrF基因。测序显示一个开放阅读框能够编码一个30.8 kDa的蛋白质。在T7表达系统中检测到这种大小的蛋白产物，并观察到lcrf依赖的yope特异性DNA结合活性。正如预期的那样，LcrF与小肠结肠炎耶尔森菌的VirF具有98%的同源性，并且与AraC转录调节蛋白家族其他成员的羧基端具有显著的同源性。这些蛋白质根据功能可分为两类：一类是参与碳和能源分解代谢的调控操纵子，另一类是参与调控毒力基因的。构建lcrF::lacZ转录融合体并分析其在鼠疫菌和大肠杆菌中的表达。融合体的活性不受原生pCD1毒力质粒、完整的lcrF基因或温度的影响。因此，lcrF转录的诱导对于温度依赖性的yopE转录激活并不是必需的。部分LcrF被发现与大肠杆菌的膜组分有关；然而，脉冲追踪实验表明，这一结果是一个工件的分馏。
In Escherichia coli, a yopE=lacZ fusion was found to be regulated by temperature in the presence of the cloned BamHI G fragment of Yersinia pestis plasmid pCD1, which contains the lcrF locus. Increasing the copy number of lcrF relative to that of the yopE reporter had a negligible effect on the induction ratio (26 versus 37-degrees-C) but caused large reductions in the absolute levels of yopE transcription. We localized the lcrF gene by monitoring the induction phenotype of BamHI G deletion derivatives. Sequencing revealed an open reading frame capable of encoding a protein of 30.8 kDa. A protein product of this size was detected in a T7 expression system, and LcrF-dependent yopE-specific DNA binding activity was observed. As expected, LcrF exhibited 98% homology to VirF of Yersinia enterocolitica and significant homology to the carboxy termini of other members of the AraC family of transcriptional regulatory proteins. These proteins could be divided into two classes according to function: those regulating operons involved in catabolism of carbon and energy sources and those involved in regulating virulence genes. lcrF::lacZ transcriptional fusions were constructed and analyzed in Y. pestis and E. coli. The activity of the fusions was not affected by the native pCD1 virulence plasmid, an intact lcrF gene, or temperature. Thus, induction of lcrF transcription is not essential for temperature-dependent activation of yopE transcription. A portion of LcrF was found associated with the membrane fraction in E. coli; however, pulse-chase experiments indicated that this result is an artifact of fractionation.