Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR

Abstract

Background: Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements. Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient. Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E. coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced.

Results: Genomic DNA from strain CGSC6300 was digested with a four-base cutter Rsa I and the resulting restriction fragments ligated onto vectorette units. Using IS-specific primers directed outward from the extreme ends of each IS and a vectorette primer, flanking DNA fragments were amplified from all but one of the 37 IS elements identified in the genomic sequence of MG1655. Purification and sequencing of the PCR products confirmed that they are IS-associated flanking DNA fragments corresponding to the known IS locations in the MG1655 genome. Seven additional insertions were found in strain CGSC6300 indicating that very closely related isolates of the same laboratory strain (the K12 isolate) may differ in their IS complement. Two other E. coli K12 derivatives, TD2 and TD10, were also analyzed by vectorette PCR. They share 36 of the MG1655 IS sites as well as having 16 and 18 additional insertions, respectively.

Conclusion: This study shows that vectorette PCR is a swift, efficient, reliable method for typing microbial strains and identifying and mapping IS insertion sites present in microbial genomes. Unlike Southern hybridization and inverse PCR, our approach involves only one genomic digest and one ligation step. Vectorette PCR is then used to simultaneously amplify all IS elements of a given type, making it a rapid and sensitive means to survey IS elements in genomes. The ability to rapidly identify the IS complements of microbial genomes should facilitate subtyping closely related pathogens during disease outbreaks.

Figure 1

Vectorette PCR for amplification of IS franking sequences. The shadowed area represents the IS sequence. The solid lines indicate the flanking DNA sequences. ∇ indicates the restriction site. A and B are the outward IS-specific primers located at the ends of the IS. V is a vectorette primer.

Figure 2

PCR amplification of IS flanking DNA from E. coli strains CGSC6300, TD2 and TD10. Results for IS1, 2, 3, and 5 and 186 are shown. Genomic DNA was digested with Rsa I, ligated with vectorette units and amplified by vPCR. Each panel shows the PCR products generated by two outward IS-specific primers (arrows) of an IS in combination with the vectorette primer. Flanking DNA fragments from both sides of each IS location were amplified. The PCR products were excised, purified, sequenced and identified from the genome sequence of E. coli strain MG1655 [17]. A PCR fragment flanking a known IS site in MG1655 is indicated by the element's name followed by an identifying numeral; for example, IS1-1 is one of 7 IS1 elements in the MG1655 genome. Additional flanking DNAs not found in MG1655 are labeled with the b# of the gene in which the IS is located. PCR products were separated in 1.4% agarose gels and stained with ethidium bromide. Intense bands in the 100 kb ladder correspond to 500 and 1000 bp.

Figure 3

Estimation of IS flanking DNA likely to be resolved and missed. A. The maximum number of fragments likely to be resolved, m, can be estimated by plotting the number of bands observed against the genomic copy number. Only a finite number of bands can be visualized on a gel. Consequently, the likelihood that two amplified fragments comigrate increases with the number of IS copies in the genome. B. The number of amplified flanking sequences likely to be missed rapidly increases when 10 or more bands are visualized. Genomic digests with a single restriction enzyme should be restricted to IS elements with fewer than 10 copies per genome. Genomes with more than 10 copies of an IS element should be screened using high resolution agarose gels and/or using a second restriction enzyme to allow all IS copies to be identified.

Figure 4

PCR amplification of IS2 flanking DNA from genomic DNA digested with Bst UI. Flanking DNA fragments IS2-3A and IS2-6A (left hand side) and IS2-2B (right hand side), masked by other amplified fragments when genomic DNA was digested with Rsa I (see Fig. 2), were recovered with Bst UI.