New tools for integrated genetic and physical analyses of the Escherichia coli chromosome

Rode, Christopher K.; Obreque, Victor; Bloch, Craig A.

doi:10.1016/0378-1119(95)00630-5

Cited by 19 publications

(34 citation statements)

References 26 publications

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“…In favor of this hypothesis is the fact that segments 66 and 69 (Fig. 1), which we did not obtain, correspond to rfb genes and the bacteriophage, respectively, which are deleted in strain MG1655 (19,25).…”

Section: X62158mentioning

confidence: 75%

Identification of Regions of the Escherichia coli Chromosome Specific for Neonatal Meningitis-Associated Strains

et al. 2000

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Specific virulence factors associated with the pathogenesis of Escherichia coli strains causing neonatal meningitis (ECNM), such as the K1 capsular polysaccharide, the S fimbriae, and the Ibe10 protein, have been previously identified. However, some other yet unidentified factors are likely to be involved in the pathogenesis of ECNM. To identify specialized unique DNA regions associated with ECNM virulence, we used the representational difference analysis technique. The genomes of two strains belonging to nonpathogenic phylogenetic group A of the ECOR reference collection were subtracted from E. coli strain C5, isolated from a case of neonatal meningitis. Strain C5 belongs to the phylogenetic group B2 as do the majority of ECNM. We have isolated and mapped 64 DNA fragments which are specific for strain C5 and not found in nonpathogenic strains. Of these clones, 44 were clustered in six distinct regions on the chromosome. The sfa and ibe10 genes were located in regions 2 and 6, respectively. A group of genes (cnf1, hra, hly, and prs) known to be present in a pathogenicity island of the uropathogenic strain E. coli J96 colocalized with region 6. The occurrence of these DNA regions was tested in a set of meningitis-associated strains and in a control group composed of non-meningitisassociated strains belonging to the same B2 group. Regions 1, 3, and 4 were present in 91, 82, and 81%, respectively, of the meningitis strains and in 40, 13, and 47% of the control strains. Together, these data suggest that regions 1, 3, and 4 code for factors associated with the ability of E. coli to invade the meninges of neonates.Escherichia coli is responsible for a third of the cases of neonatal meningitis (NM), with an incidence of 0.1 per 1,000 live births (8). Case fatality rates are still very high and range from 25 to 40%. Furthermore, the occurrence of long-term neurologic sequelae in nonfatal cases is 33 to 50% of neonates with E. coli meningitis (8, 9, 32). Understanding the pathogenesis of this disease and characterizing these pathogenic strains are prerequisites to the development of new treatments.Few specific pathogenic determinants have been described for E. coli strains causing NM (ECNM). Both expression of the K1 capsular polysaccharide (17) and production of aerobactin (21) are believed to be important for bloodstream dissemination. On the other hand, S fimbrial adhesin (sfa) (11,17,23) and Ibe10 protein (13), involved in the adhesion and invasion of brain microvascular endothelial cells, likely promote the crossing of the blood-brain barrier.Phylogenetic approaches have helped to characterize the pathogenic strains. The E. coli species has been divided into four main phylogenetic groups designated A, B1, B2, and D (12, 28). Previous studies have shown that ECNM has a clonal structure (3,27) and that strains mostly belong to the B2 group (5). Considering that only 38% of ECNM have both sfa and ibe10, it is likely that other determinants remain to be identified (5). In favor of this hypothesis is the fact that 10 pa...

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Section: X62158mentioning

confidence: 75%

Identification of Regions of the Escherichia coli Chromosome Specific for Neonatal Meningitis-Associated Strains

et al. 2000

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“…Genomic DNA was purified from 5.0-ml overnight cultures of E. coli::Tn10dRCP2 and S. flexneri::Tn10dRCP2 mutants in a manner suitable for yielding macrorestriction fragments (0.05-1.0 Mb) as described (21). After digestion of agarose-embedded DNA with I-SceI (Boehringer Mannheim) for 1 hr or with I-CeuI (Panvera, Madison, WI) overnight, according to the manufacturers' directions, reaction buffer was decanted, and dots were melted (70°C) and gently pipetted into sample wells in 1.3% agarose (Fastlane, FMC) gels for electrophoresis in a Bio-Rad DR-III pulse field gel apparatus.…”

mentioning

confidence: 99%

“Black holes” and bacterial pathogenicity: A large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli

Maurelli

Fernández²,

Bloch³

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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407

308

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Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylase (LDC) activity is present in Ϸ90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these ''black holes,'' deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.

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“…De novo insertion mutants of E. coli strain J96 containing single Tn10dKanRCP2 or Tn10dSpcRCP2 insertions were generated by electroporation with plasmid pGI290 or pGI300, respectively, as previously described for strain K-12 (36). Double insertion mutants of strain MG1655 and single and double insertion mutants of strain J96 were generated by transducing recipient strains with P1⌬damrev6 lysates of MG1655 insertion mutants (36). Each Tn10dRCP2 insertion was mapped, and the genome structure was assessed by pulsed-field gel electrophoresis (PFGE) following NotI, BlnI, or I-CeuI digestion.…”

Section: Methodsmentioning

confidence: 99%

“…In every method, however, there is the requirement for overlapping or second-dimension data that will allow the ordering of anonymous macrorestriction fragments, for example, with double enzymatic digests (12) or through hybridization analysis with cloned sequences that integrate genetic and physical data (39). Specialized transposable elements carrying a battery of rare restriction sites (29) provide the opportunity for similar overlapping data because they serve to integrate at transposon insertion sites the macrorestriction patterns generated by different rare-cutter enzymes (36). That is, the use of more than one enzyme (in this case, NotI, BlnI, and I-CeuI) for restriction of genomic DNAs of multiple insertion-bearing strains provides overlapping or second-dimension data for ordering the fragments from individual enzyme digests.…”

mentioning

confidence: 99%

Integrated Genomic Map from Uropathogenic Escherichia coli J96

Melkerson-Watson¹,

Rode²,

Zhang

et al. 2000

Infect Immun

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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.

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New tools for integrated genetic and physical analyses of the Escherichia coli chromosome

Cited by 19 publications

References 26 publications

Identification of Regions of the Escherichia coli Chromosome Specific for Neonatal Meningitis-Associated Strains

Identification of Regions of the Escherichia coli Chromosome Specific for Neonatal Meningitis-Associated Strains

“Black holes” and bacterial pathogenicity: A large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli

Integrated Genomic Map from Uropathogenic Escherichia coli J96

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