Melkerson-Watson L J, Rode C K, Zhang L, Foxman B, Bloch C A
Department of Pediatrics, School of Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA.
Infect Immun. 2000 Oct;68(10):5933-42. doi: 10.1128/IAI.68.10.5933-5942.2000.
Escherichia coli J96 is a uropathogen having both broad similarities to and striking differences from nonpathogenic, laboratory E. coli K-12. Strain J96 contains three large (>100-kb) unique genomic segments integrated on the chromosome; two are recognized as pathogenicity islands containing urovirulence genes. Additionally, the strain possesses a fourth smaller accessory segment of 28 kb and two deletions relative to strain K-12. We report an integrated physical and genetic map of the 5,120-kb J96 genome. The chromosome contains 26 NotI, 13 BlnI, and 7 I-CeuI macrorestriction sites. Macrorestriction mapping was rapidly accomplished by a novel transposon-based procedure: analysis of modified minitransposon insertions served to align the overlapping macrorestriction fragments generated by three different enzymes (each sharing a common cleavage site within the insert), thus integrating the three different digestion patterns and ordering the fragments. The resulting map, generated from a total of 54 mini-Tn10 insertions, was supplemented with auxanography and Southern analysis to indicate the positions of insertionally disrupted aminosynthetic genes and cloned virulence genes, respectively. Thus, it contains not only physical, macrorestriction landmarks but also the loci for eight housekeeping genes shared with strain K-12 and eight acknowledged urovirulence genes; the latter confirmed clustering of virulence genes at the large unique accessory chromosomal segments. The 115-kb J96 plasmid was resolved by pulsed-field gel electrophoresis in NotI digests. However, because the plasmid lacks restriction sites for the enzymes BlnI and I-CeuI, it was visualized in BlnI and I-CeuI digests only of derivatives carrying plasmid inserts artificially introducing these sites. Owing to an I-SceI site on the transposon, the plasmid could also be visualized and sized from plasmid insertion mutants after digestion with this enzyme. The insertional strains generated in construction of the integrated genomic map provide useful physical and genetic markers for further characterization of the J96 genome.
大肠杆菌J96是一种尿路致病菌，与非致病性的实验室大肠杆菌K-12既有广泛的相似性，又有显著的差异。J96菌株在染色体上整合了三个大的（>100 kb）独特基因组片段；其中两个被认为是含有尿路毒力基因的致病岛。此外，该菌株相对于K-12菌株还拥有一个28 kb的较小的第四个辅助片段和两个缺失区域。我们报告了5120 kb的J96基因组的整合物理图谱和遗传图谱。该染色体包含26个NotI、13个BlnI和7个I-CeuI宏观限制酶切位点。通过一种基于转座子的新方法快速完成了宏观限制酶切图谱绘制：对修饰的微型转座子插入进行分析，以对齐由三种不同酶产生的重叠宏观限制酶切片段（每种酶在插入片段内共享一个共同的切割位点），从而整合三种不同的消化模式并对片段进行排序。由总共54个mini-Tn10插入产生的最终图谱，通过营养缺陷型分析和Southern分析进行补充，分别指示插入中断的氨基合成基因和克隆的毒力基因的位置。因此，它不仅包含物理的、宏观限制酶切标记，还包含与K-12菌株共有的八个管家基因的位点和八个公认的尿路毒力基因的位点；后者证实了毒力基因在大的独特辅助染色体片段上的聚集。115 kb的J96质粒通过NotI酶切的脉冲场凝胶电泳进行解析。然而，由于该质粒缺乏BlnI和I-CeuI酶的限制酶切位点，只有在携带人工引入这些位点的质粒插入片段的衍生物的BlnI和I-CeuI酶切中才能观察到它。由于转座子上存在一个I-SceI位点，在用该酶消化后，也可以从质粒插入突变体中观察到并确定该质粒的大小。在构建整合基因组图谱过程中产生的插入菌株为进一步表征J96基因组提供了有用的物理和遗传标记。
