Lo-Tseng T, Lee J, Isaacson R E
University of Illinois, Department of Veterinary Pathobiology, Urbana 61801, USA.
Adv Exp Med Biol. 1997;412:303-10. doi: 10.1007/978-1-4899-1828-4_50.
Expression of K99 is highly regulated being dependent on 8 K99-specific genes and several host-specific genes including cyclic AMP receptor protein (Crp) and leucine responsive protein (Lrp). The 8 K99-specific genes are organized into 3 separately regulated clusters (regions I-III) with region I and II genes being dependent on Crp. Using TnphoA tagged K99 genes, Lrp was shown to be required for expression of region I genes. Differential methylation of GATC boxes is a common method by which Lrp functions as a regulator. Two GATC boxes are present adjacent to the 5' end of fanA, the first gene. Using the restriction enzymes Dpn I and Mbo I which recognize methylated and non-methylated GATC boxes, respectively, it was shown that differential methylation was not a mechanism regulating K99 expression. Using a gel mobility shift assay and protein extracts from various strains, it was shown that a 625 bp DNA fragment adjacent to the 5' end of fanA bound protein prepared from Lrp+ strains but not from Lrp- strains. While region I genes also require CRP for expression, the same degree of gel shift was observed when the extract was prepared from a Crp- strain. The products of fanA and fanB are believed to be positive regulators. However, protein extracts from strains with or without fanA and fanB caused the same degree of gel shift. Thus, while there are a variety of regulators necessary for region I gene expression, only Lrp or Lrp regulated proteins bind to the promoter region 5' to fanA.
K99的表达受到高度调控，这依赖于8个K99特异性基因和几个宿主特异性基因，包括环腺苷酸受体蛋白（Crp）和亮氨酸反应蛋白（Lrp）。这8个K99特异性基因被组织成3个分别受调控的簇（区域I - III），其中区域I和II的基因依赖于Crp。使用TnphoA标记的K99基因，发现Lrp是区域I基因表达所必需的。GATC框的差异甲基化是Lrp作为调节因子发挥作用的常见方式。在第一个基因fanA的5'端相邻处存在两个GATC框。分别使用识别甲基化和非甲基化GATC框的限制性内切酶Dpn I和Mbo I，结果表明差异甲基化不是调节K99表达的机制。使用凝胶迁移率变动分析和来自各种菌株的蛋白质提取物，发现与fanA的5'端相邻的一个625 bp DNA片段能与从Lrp +菌株制备的蛋白质结合，但不能与从Lrp -菌株制备的蛋白质结合。虽然区域I的基因表达也需要CRP，但从Crp -菌株制备提取物时观察到相同程度的凝胶迁移。fanA和fanB的产物被认为是正调节因子。然而，来自有或没有fanA和fanB的菌株的蛋白质提取物导致相同程度的凝胶迁移。因此，虽然区域I基因表达需要多种调节因子，但只有Lrp或Lrp调节的蛋白质与fanA 5'端的启动子区域结合。
