Marolda C L, Valvano M A
Department of Microbiology and Immunology, University of Western Ontario, London, Canada.
J Bacteriol. 1998 Jun;180(12):3070-9. doi: 10.1128/JB.180.12.3070-3079.1998.
We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wbEcO7). Typical -10 and -35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wbEcO7 cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes. We observed another predicted stem-loop, located immediately downstream from the wbEcO7 transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes. wbEcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg2+ concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the wbEcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.
我们报告了大肠杆菌O7特异性脂多糖（LPS）基因簇（wbEcO7）启动子区域的鉴定结果。典型的-10和-35序列位于galF和wbEcO7簇的第一个基因rlmB之间的间隔区域。核糖核酸酶保护实验的数据揭示了一个173 bp的未翻译前导mRNA片段的存在，其中包括JUMPStart和两个ops序列。我们使用Mfold程序以及与无启动子lac操纵子转录融合的嵌套和内部缺失组合来表征该前导mRNA的结构。我们的结果表明，前导mRNA可能折叠成一系列复杂的茎环结构，其中之一包含JUMPStart元件。我们还发现其中一个ops序列位于预测的茎上，另一个位于环区域，并且我们证实这些序列对于RfaH介导的O多糖簇调控至关重要。在编码waaQGPSBIJYZK基因的LPS核心操纵子的启动子区域可以预测到非常相似的茎环结构。我们观察到另一个预测的茎环，位于wbEcO7转录起始位点的紧邻下游，它似乎参与转录的过早终止。这种假定的茎环在许多其他O多糖基因簇中很常见，但在核心寡糖基因中不存在。单拷贝数的wbEcO7-lac转录融合也用于确定各种环境信号对O多糖合成转录调控的影响。未检测到温度、渗透压、Mg2+浓度和诱导DNA超螺旋变化的药物的影响。因此，我们得出结论，wbEcO7启动子活性可能是组成型的，并且调控以RfaH介导的方式在mRNA延伸水平发生。
