A novel strategy for the identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites in closely related bacteria

We devised software tools to systematically investigate the contents and contexts of bacterial tRNA and tmRNA genes, which are known insertion hotspots for genomic islands (GIs). The strategy, based on MAUVE-facilitated multigenome comparisons, was used to examine 87 Escherichia coli MG1655 tRNA and tmRNA genes and their orthologues in E.coli EDL933, E.coli CFT073 and Shigella flexneri Sf301. Our approach identified 49 GIs occupying ∼1.7 Mb that mapped to 18 tRNA genes, missing 2 but identifying a further 30 GIs as compared with Islander [Y. Mantri and K. P. Williams (2004), Nucleic Acids Res., 32, D55–D58]. All these GIs had many strain-specific CDS, anomalous GC contents and/or significant dinucleotide biases, consistent with foreign origins. Our analysis demonstrated marked conservation of sequences flanking both empty tRNA sites and tRNA-associated GIs across all four genomes. Remarkably, there were only 2 upstream and 5 downstream deletions adjacent to the 328 loci investigated. In silico PCR analysis based on conserved flanking regions was also used to interrogate hotspots in another eight completely or partially sequenced E.coli and Shigella genomes. The tools developed are ideal for the analysis of other bacterial species and will lead to in silico and experimental discovery of new genomic islands

Characterising the mobile genome of Shigella

Shigella spp. are pathogenic variants of Escherichia coli that cause bacillary dysentery, resulting in over 1 million deaths per year. Across E. coli bacteria, the dynamic process of acquisition and loss of GIs, especially those associated with virulence (pathogenicity islands [PAIs]) is a driving force behind the emergence of new pathogenic strains. In Shigella only 5 GIs have been well characterised and there is currently no effective vaccine. Therefore the development of an efficient screen to detect GIs in unsequenced Shigella strains could be highly informative. Nineteen Shigella strains were probed for the presence of GIs using a high throughput PCR screen (tRIP), across 16 tRNA gene integration hotspots. Putative GIs were then investigated using a chromosome walking technique (SGSP-PCR). Representative PCR amplicons were sequenced to get a snapshot of the islands contents. Islands of particular interest were characterised further using allelic exchange and marker rescue to capture clones that harbour larger portions of the GI and subsequent sequencing and analysis. Using SGSP-PCR, 81% of the putative GI occupied tRNA loci were characterised, and sequencing analysis found they all contain island DNA, indicating that tRIP followed by SGSP-PCR is a robust strategy for GI discovery in unsequenced strains; also this method should be applicable to a broad range of microorganisms. At least 54% of the islands identified harbour phage-like integrase genes, strongly supporting the notion that many of these elements arose following acquisition of horizontally acquired integrative GIs. The frequent presence of integrase genes also highlights the potential role of bacteriophage in the original and/or ongoing dissemination of island DNA in Shigella. Only one novel GI was discovered; it has classic prophage-like features and contains completely novel DNA, indicating that while Shigella has a plastic genome, it is a highly specialised human pathogen that has undergone considerable pathoadaptive genome reduction. The major development from this study is evidence that a number of key Shigella virulence determinants are independently mobile and not only localised to a single family of islands; this significantly increases their potential to spread by HGT across Shigella and could contribute to the rapid emergence of new endemic strains

Extraction of Acanthamoeba DNA by Use of Chelex Resin▿

Extraction of Acanthamoeba DNA by Use of Chelex Resi

Genomic island discovery in Shigella by tRNA site interrogation

Introduction: Shigella spp. are pathogenic variants of Escherichia coli that cause bacillary dysentery, resulting in more than 1 million deaths per year. E. coli bacteria demonstrate extensive intra-species genodiversity. The dynamic process of acquisition and loss of genomic islands (GIs), especially those associated with virulence (pathogenicity islands [PAIs]) is a driving force behind the emergence of new pathotypes. In Shigella only 4 GIs have been well characterised and there is currently no effective vaccine against the many serotypes of this pathogen. Therefore the development of an effective screen to detect GIs in unsequenced Shigella strains could be highly informative. In this study, 16 known E. coli tRNA gene integration hotspots in 10 strains representative of the 4 ‘species’ of Shigella were probed for the presence of island DNA using a high throughput PCR screen known as tRNA site Interrogation for PAIs, prophages and other GIs (tRIP). Putative GIs were then investigated using a chromosome walking technique known as single genome-specific primer-PCR (SGSP-PCR)

A novel strategy for the identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites in closely related bacteria.

We devised software tools to systematically investigate the contents and contexts of bacterial tRNA and tmRNA genes, which are known insertion hotspots for genomic islands (GIs). The strategy, based on MAUVE-facilitated multigenome comparisons, was used to examine 87 Escherichia coli MG1655 tRNA and tmRNA genes and their orthologues in E.coli EDL933, E.coli CFT073 and Shigella flexneri Sf301. Our approach identified 49 GIs occupying ∼1.7 Mb that mapped to 18 tRNA genes, missing 2 but identifying a further 30 GIs as compared with Islander [Y. Mantri and K. P. Williams (2004), Nucleic Acids Res., 32, D55–D58]. All these GIs had many strain-specific CDS, anomalous GC contents and/or significant dinucleotide biases, consistent with foreign origins. Our analysis demonstrated marked conservation of sequences flanking both empty tRNA sites and tRNA-associated GIs across all four genomes. Remarkably, there were only 2 upstream and 5 downstream deletions adjacent to the 328 loci investigated. In silico PCR analysis based on conserved flanking regions was also used to interrogate hotspots in another eight completely or partially sequenced E.coli and Shigella genomes. The tools developed are ideal for the analysis of other bacterial species and will lead to in silico and experimental discovery of new genomic islands

Schematic representation of a range of hypothetical tRNA site configurations present in the four complete genomes (MG1655, CFT073, EDL933 and Sf301) (–)

<p><b>Copyright information:</b></p><p>Taken from "A novel strategy for the identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites in closely related bacteria"</p><p>Nucleic Acids Research 2006;34(1):e3-e3.</p><p>Published online 9 Jan 2006</p><p>PMCID:PMC1326021.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The conserved UF and DF regions flanking tRNA genes are shown as dark grey filled boxes. UF and DF boxes drawn below the line indicate inversions with respect to the reference template MG1655 ( and ). The UF and DF boxes shown in pale grey with a broken outline represent deletions with respect to MG1655 ( and ). Genomic islands, where present, are indicated as broken boxes to emphasize the relatively large size of these regions. Arrowheads shown below each sub-figure indicate the location and orientation of primers specific to the UF and DF regions. Hollow arrowheads indicate the absence of matching complementary sequence. The solid line between the arrowheads shown in () indicates a likely successful PCR amplification; while the dotted line in () indicates a successful e-PCR-based ‘amplification’ that would typically yield a product of size far in excess of that that could be generated through standard PCR. The numbers shown above each configuration after the colon symbol represent the number of examples observed in the four genomes tested based on the 87 MG1655 tRNA genes and the total complement of orthologues present in the other three genomes (). The numbers of examples observed in the five unpublished genomes ( EAEC O42, EPEC E2348/69, ETEC E24377A, HS and 53G), with respect to the subset of 20 tRNA genes only (Supplementary Table S3), are shown in parentheses. The symbols shown alongside the drawings are used in and Supplementary Table S4 to highlight tRNA loci affected by inversions and/or deletions. Examples of the various atypical configurations observed in the four genomes are shown to the right. The figure is not drawn to scale