Purification ofEscherichia coliChromosomal Segments without Cloning

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

doi:10.1006/bbrc.1996.0853

Cited by 17 publications

(12 citation statements)

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“…The Janus m13 vector (10) was employed to isolate shotgun library clones from a mechanically sheared DNA fragment containing the relevant portion of the E. coli genome. The fragment, isolated from a pulsed-field gel, was defined by I-SceI sites introduced into the genome on mini-Tn10 transposons (5,36). After initial assembly at fivefold coverage of E. coli DNA sequence, selected Janus clones were flipped to establish closure and minimal depth of coverage three times at each point, including at least one determination on each strand.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12

et al. 1997

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We recently identified a pathogenicity island (SPI2) located at 30.7 centisomes on the Salmonella typhimurium chromosome. SPI2 contains genes encoding a type III secretion system whose function is distinct from that of the type III secretion system encoded by a pathogenicity island (SPI1) at 63 centisomes which is involved in epithelial cell entry. An analysis of the boundaries of SPI2 and comparison with the corresponding region of the Escherichia coli chromosome revealed that SPI2 inserted adjacent to the tRNA Val gene. The E. coli chromosome contains 9 kb of DNA at the region corresponding to the SPI2 insertion point which appears to be absent in S. typhimurium. The distribution of SPI1 and SPI2 was examined in various Salmonella isolates. In contrast to type III secretion system genes of SPI1, those of SPI2 are not present in Salmonella bongori, which diverged at the first branch point in the Salmonella lineage. These and other data indicate that SPI2 was acquired by a Salmonella strain already harboring SPI1 by horizontal transfer from an unknown source.Diseases caused by Salmonella spp. range from self-limiting gastroenteritis to typhoid fever, which can be fatal (28). A useful tool for investigation of the systemic form of salmonellosis is the murine model of typhoid-like illness caused by Salmonella typhimurium (full taxonomical name, Salmonella enterica subsp. enterica serotype Typhimurium). Many virulence factors required at different stages of S. typhimurium infection have been characterized at the molecular level. The genes encoding these virulence factors are distributed on the Salmonella chromosome and the 92-kb virulence plasmid (20).An important stage in S. typhimurium pathogenesis is invasion of the gut epithelium. A large number of genes is required for epithelial cell invasion, and it has been shown that these encode the structural components of a type III (contact-dependent) secretion system, the secreted effector proteins, and associated regulatory proteins (17). These genes are clustered at 63 centisomes (cs) on the S. typhimurium chromosome, and recent analysis of this locus revealed that it constitutes a pathogenicity island (PAI) (29). PAIs comprise large, sometimes unstable chromosomal regions harboring clusters of virulence genes and are often either flanked by insertion sequence elements (16) or appear to have inserted in or adjacent to tRNA genes (6, 12). The locus for enterocyte effacement of enteropathogenic Escherichia coli is a PAI; like the invasion locus of S. typhimurium, it contains genes for a type III secretion system. Homologs of these genes are also found on the virulence plasmids of Yersinia spp. (for a review, see reference 13) and Shigella spp. (for a review, see reference 32). The DNA base composition of PAIs often differs from those of the bacterial chromosomes in which they are located, indicating that they have probably been acquired by horizontal gene transfer (for a review, see reference 11).During a search for new virulence genes of S. typhimurium by signature-t...

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Section: Methodsmentioning

confidence: 99%

Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12

et al. 1997

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“…Whole genomes have been analyzed in the manner traditionally available only for plasmid-sized DNAs, allowing strains to be characterized (i) by physical mapping for the analysis of genomic organization and gene locus relationships (4,6,8,31,37), (ii) through genomic macrorestriction fingerprinting for population analysis and epidemiologic studies (36), (iii) through intraand interspecies comparative genomics (9,37), and (iv) by nucleotide sequence after fragmentation of the genome into contiguous and/or nonoverlapping segments (5,7,30). Ultimately, a macrorestriction map from a given strain integrated with the species' genetic map may provide a critical step in studying that strain's uniqueness.…”

Section: Discussionmentioning

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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“…Targeted insertion was verified by PCR in three of four cases. One colony harboring the two flanking insertions was grown further, and the ϳ35-kb LEE was excised by I-SceI and isolated from a pulsed-field gel for sequencing, as previously described (2). To demonstrate the use of the plasmid system in deletion studies, the entire LEE was deleted in vivo.…”

Section: ϫ6mentioning

confidence: 99%

“…These insertions can serve as "landmarks" in physical mapping and strain comparisons (12), allow direct isolation of preselected chromosomal segments for sequencing (2,11,14), provide a means for obtaining precise genomic deletions, and permit plasmid rescue of a genomic segment. Suicide plasmids are appropriate vehicles for delivery of such insertions into the chromosome (7).…”

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Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome

et al. 1997

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A system of versatile insertion plasmids was constructed that permits efficient delivery of the target sites of an ultra-rare-cutting endonuclease and the recombinase FLP into preselected sites of the bacterial genome. With the help of this system, the pathogenicity island LEE of the Escherichia coli O157:H7 genome was excised and isolated in vitro, deleted in vivo, rescued as a plasmid, and transferred into another strain.Characterization and manipulation of bacterial genomes can be facilitated by creating genomic insertions with ultrarare restriction sites and recognition sites for specific recombinases. These insertions can serve as "landmarks" in physical mapping and strain comparisons (12), allow direct isolation of preselected chromosomal segments for sequencing (2,11,14), provide a means for obtaining precise genomic deletions, and permit plasmid rescue of a genomic segment. Suicide plasmids are appropriate vehicles for delivery of such insertions into the chromosome (7). However, efficient introduction of plasmids into the target cell is frequently hindered by restriction, methylation sensitivity, plasmid incompatibility, and other factors.We constructed two sets of small, suicidal insertion plasmids that combine several useful features: rare and ultrarare restriction sites (NotI and I-SceI), the target site (FRT) for the recombinase FLP, the T7 and SP6 phage promoters, a choice of three antibiotic resistance genes (ampicillin [Ap], kanamycin [Kn], and chloramphenicol [Cm], and either of two conditional replication systems (R6K or pSC101 ts ). The plasmids can be recombined by a single crossover into preselected sites of the genome with the aid of DNA fragments that are cloned into them and are homologous to the targeted sites. Such homologous fragments can be obtained by PCR with available sequence information from the target cell or from related organisms. The two sets of suicide plasmids permit two insertions to be obtained sequentially in the same genome. The genomic segment flanked by these insertions can then be excised by I-SceI in vitro and can be isolated directly from a pulsed-field gel. In vivo deletion or inversion of the segment, depending on the relative orientation of the FRT sites, can also be achieved by expressing FLP from a helper plasmid. Since FLP-mediated deletion is a very effective process, no direct selection of the clone carrying the deletion is necessary. In addition, the chromosomal segment, excised and circularized by FLP, can be isolated as a plasmid and transformed into another host, where it can be either maintained as a plasmid or inserted into the genome.The insertion plasmids are introduced into the target cell by electroporation. If the transformation efficiency is sufficiently high and a large number of transformants can be obtained, the plasmids are used in "direct suicide" (nonreplicating) mode, and recombinants are selected by their antibiotic resistance. In strains that are difficult to transform, the plasmids are used in "conditional suicide" mode (6). In this ap...

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Purification ofEscherichia coliChromosomal Segments without Cloning

Cited by 17 publications

References 0 publications

Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12

Analysis of the boundaries of Salmonella pathogenicity island 2 and the corresponding chromosomal region of Escherichia coli K-12

Integrated Genomic Map from Uropathogenic Escherichia coli J96

Versatile insertion plasmids for targeted genome manipulations in bacteria: isolation, deletion, and rescue of the pathogenicity island LEE of the Escherichia coli O157:H7 genome

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