Ibañez E, Campos E, Baldoma L, Aguilar J, Badia J
Department of Biochemistry, School of Pharmacy, University of Barcelona, 08028 Barcelona, Spain.
J Bacteriol. 2000 Aug;182(16):4617-24. doi: 10.1128/JB.182.16.4617-4624.2000.
The yiaKLMNOPQRS (yiaK-S) gene cluster of Escherichia coli is believed to be involved in the utilization of a hitherto unknown carbohydrate which generates the intermediate L-xylulose. Transcription of yiaK-S as a single message from the unique promoter found upstream of yiaK is proven in this study. The 5' end has been located at 60 bp upstream from the ATG. Expression of the yiaK-S operon is controlled in the wild-type strain by a repressor encoded by yiaJ. No inducer molecule of the yiaK-S operon has been identified among over 80 carbohydrate or derivative compounds tested, the system being expressed only in a mutant strain lacking the YiaJ repressor. The lacZ transcriptional fusions in the genetic background of the mutant strain revealed that yiaK-S is modulated by the integration host factor and by the cyclic AMP (cAMP)-cAMP receptor protein (Crp) activator complex. A twofold increase in the induction was observed during anaerobic growth, which was independent of ArcA or Fnr. Gel mobility shift assays showed that the YiaJ repressor binds to a promoter fragment extending from -50 to +121. These studies also showed that the cAMP-Crp complex can bind to two different sites. The lacZ transcriptional fusions of different fragments of the promoter demonstrated that binding of cAMP-Crp to the Crp site 1, centered at -106, is essential for yiaK-S expression. The 5' end of the yiaJ gene was determined, and its promoter region was found to overlap with the divergent yiaK-S promoter. Expression of yiaJ is autogenously regulated and reduced by the binding of Crp-cAMP to the Crp site 1 of the yiaK-S promoter.
大肠杆菌的yiaKLMNOPQRS（yiaK-S）基因簇被认为参与了一种迄今未知的碳水化合物的利用，该碳水化合物可生成中间产物L-木酮糖。本研究证实，yiaK-S作为来自yiaK上游唯一启动子的单一信息进行转录。其5'端位于ATG上游60 bp处。在野生型菌株中，yiaK-S操纵子的表达受yiaJ编码的阻遏物控制。在测试的80多种碳水化合物或衍生物化合物中，未鉴定出yiaK-S操纵子的诱导分子，该系统仅在缺乏YiaJ阻遏物的突变菌株中表达。突变菌株遗传背景中的lacZ转录融合表明，yiaK-S受整合宿主因子和环腺苷酸（cAMP）-cAMP受体蛋白（Crp）激活复合物的调节。在厌氧生长期间，诱导增加了两倍，这与ArcA或Fnr无关。凝胶迁移率变动分析表明，YiaJ阻遏物与从-50延伸至+121的启动子片段结合。这些研究还表明，cAMP-Crp复合物可以结合到两个不同的位点。启动子不同片段的lacZ转录融合表明，cAMP-Crp与位于-106的Crp位点1结合对于yiaK-S表达至关重要。确定了yiaJ基因 的5'端，发现其启动子区域与反向的yiaK-S启动子重叠。yiaJ的表达受到自身调节，并通过Crp-cAMP与yiaK-S启动子的Crp位点1结合而降低。
