Integrated Genomic Map from Uropathogenic Escherichia coli J96

Abstract

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.

Analysis of Genome Plasticity in Pathogenic and Commensal Escherichia coli Isolates by Use of DNA Arrays

“…In order to systematically assess the genetic variability of bacteria, several genome comparison techniques, e.g., macrorestriction analysis, PFGE, and genomic subtraction, have been used (9,39,49,58). DNA array technology is a powerful new tool for comparative genomics which has recently been used for the analysis of genome variability among bacterial species or closely related bacteria (2,4,18,19,43,50,53), as well as for detection of specific groups of trait-conferring genes (12,21).…”

Molecular characterization of extraintestinal pathogenic (ExPEC) pathogenicity islands in F165-positive strain from a diseased animal

“…Moreover a S-related type, foc operon encoding F1C pili, is present on a 47-kb island close to serX (23.64 min) in E. coli CFT073 [32]. In UPEC strain J96 and newborn sepsis-associated E. coli RS218, the foc operon was mapped near the ptsG gene (25 min) [33]. Thus, S and S-related fimbrial operons are inserted in different regions of the E. coli chromosome.…”

Molecular characterization of extraintestinal pathogenicEscherichia coli(ExPEC) pathogenicity islands in F165-positiveE. colistrain from a diseased animal

“…Moreover a S‐related type, foc operon encoding F1C pili, is present on a 47‐kb island close to serX (23.64 min) in E. coli CFT073 [32]. In UPEC strain J96 and newborn sepsis‐associated E. coli RS218, the foc operon was mapped near the ptsG gene (25 min) [33]. Thus, S and S‐related fimbrial operons are inserted in different regions of the E .…”