Integrated genomic map from uropathogenic Escherichia coli J96

Escherichia call J96 is a uropathogen having both broad similarities to and striking differences from nonpathogenic, laboratory E. coli K-12. Strain 5 96 contains three large (>100-kb) unique genomic segments integrated on the chromosome; two are recognized as pathogenicity islands containing uroviru lence genes. Additionally, the strain possesses a fourth smaller accessory segment of 28 kb and two deletions relative to strain K-12. We report an in tegrated physical and genetic map of the 5,120-kb 596 genome. The chromosom e contains 26 NotI, 13 BlnI, and 7 I-CeuI macrorestriction sites. Macrorest riction mapping was rapidly accomplished by a novel transposon-based proced ure: analysis of modified minitransposon insertions served to align the ove rlapping macrorestriction fragments generated by three different enzymes (e ach sharing a common cleavage site within the insert), thus integrating the three different digestion patterns and ordering the fragments. The resulti ng map, generated from a total of 54 mini-Tn10 insertions, was supplemented with auxanography and Southern analysis to indicate the positions of inser tionally disrupted aminosynthetic genes and cloned virulence genes, respect ively. Thus, it contains not only physical, macrorestriction landmarks but also the loci for eight housekeeping genes shared with strain K-12 and eigh t acknowledged urovirulence genes; the latter confirmed clustering of virul ence genes at the large unique accessory chromosomal segments. The 115-kb 5 96 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 derivativ es 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 o f the 596 genome.