Distribution Of Insertion Sequences Research Articles

Putative classification of clades of enterohemorrhagic Escherichia coli O157 using an IS-printing system.

Enterohemorrhagic Escherichia coli O157 (O157) strains can be classified in clades by single nucleotide polymorphisms (SNPs), but this analysis requires significant laboratory effort. As the distribution of insertion sequence (IS) 629 insertions has been reported to be biased among different clades, O157 isolates can be putatively classified in clades by comparison with an IS629 distribution database. A database of the IS629 distribution in O157 strains isolated in Chiba Prefecture and their classification in clades was determined by SNP analysis and IS-printing, an easy and quick analytical tool for IS629 in the O157 genome. The IS629 distribution in O157 strains isolated in Fukuoka and Yamagata Prefectures was determined by IS-printing. These strains were putatively classified in clades by Relative Likelihood calculations that compared the IS-printing data and the IS629 distribution database. Concordance Ratios were calculated, which compared the number of strains putatively classified in a clade by Relative Likelihood to the number of strains classified in that clade by SNP analysis. For the Fukuoka and Yamagata strains, the Concordance Ratios for clades 3, 6 and 8 were 97-100%, for clade 7 about 88%, and for clades 2 and 12 over 90%. In conclusion, O157 clade 2, 3, 6, 7, 8 and 12 strains could be putatively classified by IS-printing. This study demonstrated that enterohemorrhagic E.coli O157 (O157) strains could be putatively classified in clades using an IS-printing system. IS-printing was previously developed as a relatively quick and easy tool for analysis of insertion sequence 629 in the O157 genome. Since most local government public health institutes in Japan carry out IS-printing for early detection of O157 outbreaks, these data should be useful for putative classification of O157 strains in each area.

Insertion sequence elements in Lactococcus garvieae

Insertion sequences are the simplest intracellular Mobile Genetic Elements which can occur in very high numbers in prokaryotic genomes, where they play an important evolutionary role by promoting genome plasticity. As such, the studies on the diversity and distribution of insertion sequences in genomes not yet investigated can contribute to improve the knowledge on a bacterial species and to identify new transposable elements. The present work describes the occurrence of insertion sequences in Lactococcus garvieae, an opportunistic emerging zoonotic and human pathogen, also associated with different food matrices. To date, no insertion elements have been described for L. garvieae in the IS element database. The analysis of the twelve published L. garvieae genomes identified 15 distinct insertion sequences that are members of the IS3, IS982, IS6, IS21 and IS256 families, including five new elements. Most of the insertion sequences in L. garvieae show substantial homology to the Lactococcus lactis elements, suggesting the movement of IS between these two species phylogenetically closely related. ISLL6 elements belonging to IS3 family were most abundant, with several copies distributed in 9 of the 12 genomes analyzed. An alignment analysis of two complete genomes carrying multi-copies of this insertion sequence indicates a possible involvement of ISLL6 in chromosomal rearrangement.

Linkage disequilibrium of the IS629 insertion among different clades of enterohemorrhagic Escherichia coli O157:H7/H-strains

The distribution of insertion sequence (IS) 629 was investigated among enterohemorrhagic Escherichia coli O157:H7/H-(O157) strains in different clades. Minimum spanning tree analysis showed that most strains in each clade clustered in a separate branch, indicating biased distribution of the IS629 insertion in different clades. The standardized index of association of the IS629 distribution data showed linkage disequilibrium in the strains in every clade, indicating that IS629 distribution data could be used for population genetic analysis. For this population genetic analysis, the ΦPT value, an analogue of FST, was calculated and indicated that clade 7 strains could be split into two clades based on their lineages. The degree of pairwise linkage disequilibrium was quite different among these two groups. The clade 7 split was in agreement with the model of O157 paraphyletic evolution and a new designation of the lineage II clades was proposed. The prevalence of strains with an IS629 insertion in certain loci was significantly different in different clades. Some of these significant differences were in loci in strains in branches of clades that were close in the O157 phylogenetic model, suggesting that IS629 insertion/deletion was not directly related to the divergence of O157 clades.

Biased distribution of IS 629 among strains in different lineages of enterohemorrhagic Escherichia coli serovar O157

The distribution of insertion sequence (IS) 629 among strains of enterohemorrhagic Escherichia coli serovar O157 (O157) was investigated and compared with the strain lineages defined by lineage specific polymorphism assay-6 (LSPA-6) to demonstrate the effectiveness of IS 629 analysis for population genetics analysis. Using pulsed-field gel electrophoresis and variable-number tandem repeat typing, 140 strains producing both VT1 and VT2 and 98 strains producing only VT2 were selected from a total of 592 strains isolated from patients and asymptomatic carriers in Chiba Prefecture, Japan, during 2003–2008. By LSPA-6 analysis, six strains had atypical amplicon sizes in their Z5935 loci and five strains had atypical amplicon sizes in their arp- iclR intergenic regions. Sequence analyses of PCR amplified DNAs showed that five of the six loci used for LSPA-6 analysis had tandem repeats and the allele changes were due to changes in the number of tandem repeats. Subculturing and long-term incubation was found to have no detectable effect on the lineages defined by LSPA-6 analysis, demonstrating the robustness of LSPA-6 analysis. Minimum spanning tree analysis reconstruction revealed that strains in lineage I, I/II, and II clustered on separate branches, indicating that the distribution of IS 629 was biased among O157 strains in different lineages. Strains with LSPA-6 codes 231111, 211113, and 211114 had atypical amplicon sizes and were clustered in lineage I/II branch, and strains with LSPA-6 codes 212114, 221123, 221223, 222123, 222224, 242123, 252123, and 242222 had atypical amplicon sizes and clustered in lineage II branches. Linkage disequilibrium was observed in strains in every lineage when the standardized index of association was calculated using IS 629 distribution data. Therefore, the distribution analysis of IS 629 may be effective for population genetics analysis of O157 due to the biased IS 629 distribution among strains in the three O157 lineages.

The distribution of insertion sequences in the genome ofShigella flexneristrain 2457T

Shigella flexneri, which causes shigellosis in humans, evolved from Escherichia coli. The sequencing of Shigella genomes has revealed that a large number of insertion sequence (IS) elements (over 200 elements) reside in the genome. Although the presence of these elements has been noted previously and summarized, more detailed analyses are required to understand their evolutionary significance. Here, the genome of S. flexneri strain 2457T is used to investigate the spatial distribution of IS copies around the chromosome and the location of elements with respect to genes. It is found that most IS isoforms occur essentially randomly around the genome. Two exceptions are IS91 and IS911, which appear to cluster due to local hopping. The location of IS elements with respect to genes is biased, however, revealing the action of natural selection. The non-coding regions of the genome (no more than 21%) carry disproportionally more IS elements (at least 28%) than the coding regions, implying that selection acts against insertion into genes. Of the genes disrupted by ISs, those involved in signal transduction, intracellular trafficking, and cell motility are most commonly targeted, suggesting selection against genes in these categories.

Occurrence of insertion sequences within the genomes and Tn 1546-like elements of glycopeptide-resistant enterococci isolated in Brazil, and identification of a novel element, IS Efa5

Insertion sequences (IS) occur widely within the Tn 1546-like elements responsible for VanA glycopeptide resistance in enterococci from several countries. As such insertions can be used as epidemiological markers and for studying horizontal transfer of gene clusters, we investigated the distribution of IS 6770, IS 1542, IS 1216V, IS 1476, and IS 1251 elements in 26 VanA Enterococcus faecium and 21 VanA Enterococcus faecalis from Brazil. PCR, using genomic DNA as a template, indicated that most of the isolates contained IS 6770 (97%), IS 1216V (87%) and IS 1476 (72%) elements. IS 1251 was also detected, but at a higher frequency in E. faecium (80%) than in E. faecalis (14%). None of the isolates harboured IS 1542. Only two of 47 isolates had IS elements within their Tn 1546-like elements; one possessed IS 1251 between vanS and vanH, as reported in the United States; another possessed a novel IS element, designated IS Efa5, located between vanX and vanY. This novel element was found in the genomic DNA of 25 (96%) E. faecium and 11 (52%) E. faecalis. In stability studies, no IS-mediated changes were detected in the Tn 1546-like elements of 25 vancomycin-resistant enterococci (VRE) monitored over 11 months. These results suggest that the occurrence of IS in Brazilian isolates is similar to that reported in American isolates, but that these elements occur rarely within the vanA gene clusters. As patterns of IS carriage did not correlate with the PFGE type of the VRE, the prevalence of IS elements in genomic DNA of VRE is not a useful epidemiological marker. However, the presence of IS-modified Tn 1546-like elements, which appear to be rare in Brazil, could be a useful molecular marker in local epidemiological studies to monitor the evolution and horizontal transmission of VanA elements.

Wide-range distribution of insertion sequences identified in B. halodurans among bacilli and a new transposon disseminated in alkaliphilic and thermophilic bacilli.

All of the insertion sequences (ISs) except for IS663 and a group II intron identified in the alkaliphilic Bacillus halodurans C-125 genome were also detected in nine other strains of the same species by PCR and Southern blot analysis. The transposase of IS 653 identified in the genomes of the 10 strains of B. halodurans was found to have become the most diversified of all ISs identified in the genomes of 10 strains. A new IS element designated IS661 belonging to the IS1380 family with inverted repeats (IRs) 17 bp in length was present within IS658 identified in the genome of B. halodurans A59. In addition, a new transposon designated Tn3271bh was identified within the IS642 element in the A59 genome, which is similar to a transposon identified in thermophilic Geobacillus stearothermophilus T-6. The new transposon, Tn3271bh, generated an 8-bp duplication of the target site sequence and carries a 21-bp IR. On the other hand, all kinds of ISs except for IS643 and IS658 were distributed in the genome of obligately alkaliphilic Bacillus alcalophilus. Three ISs (IS652, IS653, and IS660) and a group II intron (Bh.Int) were widely dispersed in other Bacillus species without a correlation with the phylogenetic placement based on 16S rDNA sequences.

Evolution of the bacterial species Lactobacillus delbrueckii: a partial genomic study with reflections on prokaryotic species concept.

The species Lactobacillus delbrueckii consists at present of three subspecies, delbrueckii, lactis and bulgaricus, showing a high level of DNA-DNA hybridization similarity but presenting markedly different traits related to distinct ecological adaptation. The internal genetic heterogeneity of the bacterial species L. delbrueckii was analyzed. Phenotypic and several genetic traits were investigated for 61 strains belonging to this species. These included 16S rDNA sequence mutations, expression of beta-galactosidase and of the cell wall-anchored protease, the characterization of the lactose operon locus and of the sequence of lacR gene, galactose metabolism, and the distribution of insertion sequences. The high genetic heterogeneity of taxa was confirmed by every trait investigated: the lac operon was completely deleted in the subsp. delbrueckii, different mutation events in the repressor gene of the operon led to a constitutive expression of lacZ in the subsp. bulgaricus. Structural differences in the same genetic locus were probably due to the presence of different IS elements in the flanking regions. The different expression of the cell wall-anchored protease, constitutive in the subsp. bulgaricus, inducible in the subsp. lactis, and absent in the subsp. delbrueckii was also a consequence of mutations at the gene level. The galT gene for galactose metabolism was found only in the subsp. lactis, while no specific amplification product was detected in the other two subspecies. All these data, together with the absence of a specific IS element, ISL6, from the major number of strains belonging to the subsp. bulgaricus, confirmed a deep internal heterogeneity among the three subspecies. Moreover, this evidence and the directional mutations found in the 16S rDNA sequences suggested that, of the three subspecies, L. delbrueckii subsp. lactis is the taxon closer to the ancestor. Limitations of the current prokaryotic species definition were also discussed, based on presented evidences. Our results indicate the need for an accurate investigation of internal heterogeneity of bacterial species. This study has consequences on the prokaryotic species concept, since genomic flexibility of prokaryotes collides with a stable classification, necessary from a scientific and applied point of view.

Detection and distribution of insertion sequence 1 (IS 1)-containing bacteria in the freshwater environment

The distribution of insertion sequence 1 (IS 1)-containing bacteria was investigated in Windermere (Cumbria, UK), a freshwater body impacted by treated sewage discharge and run-off from the surrounding catchment. Culturable IS 1-containing bacteria were recovered from the water column at three depths in Windermere North Basin (WNB) and South Basin (WSB), and from sediment at both sites (at the sediment surface in WSB and to a depth of 12–13 cm in WNB). Polymerase chain reaction amplification of IS 1 and the Escherichia coli/ Shigella sp. specific gene uidA, from community DNA from shallow sediments, extended the detection limit beyond that of culture at both sites. This detection was extended further into deep sediment extracted from WNB as IS 1 and uidA were detected in sub-samples to a depth of 4.7 and 2.3 m, respectively. Analysis of a representative subset of 90 IS 1-carrying isolates recovered from water and sediment at both sites demonstrated 21 heterogeneous IS 1 profiles with estimated copy numbers ranging from 1 to 16. Identification of the host bacteria showed that the element was confined mainly to Enterobacter spp. However, this study showed IS 1 to be present in Citrobacter freundii for the first time. Plasmids were carried by 75.3% of enterobacterial isolates and four plasmids (2.6%) carried IS 1. DNA sequence analysis of five IS 1 clones demonstrated that IS 1 isoforms from this study were similar (>89% nucleotide identity) to known IS 1 isoforms. Two isoforms of IS 1 from a single Enterobacter cloacae isolate differed by 6.7% at the nucleotide level suggesting that they had been acquired independently.

Detection and distribution of insertion sequence 1 (IS1)-containing bacteria in the freshwater environment(1).

The distribution of insertion sequence 1 (IS1)-containing bacteria was investigated in Windermere (Cumbria, UK), a freshwater body impacted by treated sewage discharge and run-off from the surrounding catchment. Culturable IS1-containing bacteria were recovered from the water column at three depths in Windermere North Basin (WNB) and South Basin (WSB), and from sediment at both sites (at the sediment surface in WSB and to a depth of 12-13 cm in WNB). Polymerase chain reaction amplification of IS1 and the Escherichia coli/Shigella sp. specific gene uidA, from community DNA from shallow sediments, extended the detection limit beyond that of culture at both sites. This detection was extended further into deep sediment extracted from WNB as IS1 and uidA were detected in sub-samples to a depth of 4.7 and 2.3 m, respectively. Analysis of a representative subset of 90 IS1-carrying isolates recovered from water and sediment at both sites demonstrated 21 heterogeneous IS1 profiles with estimated copy numbers ranging from 1 to 16. Identification of the host bacteria showed that the element was confined mainly to Enterobacter spp. However, this study showed IS1 to be present in Citrobacter freundii for the first time. Plasmids were carried by 75.3% of enterobacterial isolates and four plasmids (2.6%) carried IS1. DNA sequence analysis of five IS1 clones demonstrated that IS1 isoforms from this study were similar (>89% nucleotide identity) to known IS1 isoforms. Two isoforms of IS1 from a single Enterobacter cloacae isolate differed by 6.7% at the nucleotide level suggesting that they had been acquired independently.

Molecular typing of Shigella strains using pulsed field gel electrophoresis and genome hybridization with insertion sequences

The genomes of 18 independent Shigella isolates (9 Shigella sonnei, 5 Shigella dysenteriae and 4 Shigella flexneri) as well as of 4 epidemic S. flexneri strains were analysed by pulsed field gel electrophoresis (PFGE) and by the distribution of insertion sequences (IS1, IS2 and IS911). Despite the close relatedness observed among the 9 independent S. sonnei, all of them could be differentiated from each other. The 4 independent S. flexneri isolates showed clearly distinguishable DNA profiles. Nearly complete genetic identity was detected within the 4 epidemic S. flexneri when analysed by PFGE or for IS1 and IS2 patterns. However, IS911 was found to be too mobile in these epidemic S. fluxneri to be used as a typing probe. The 5 S. dysenteriae isolates could also be distinguished by the techniques used. The diversity found within this species is striking: of the 5 investigated isolates, 3 completely different DNA profiles were revealed. In conclusion, both PFGE and IS probing demonstrated their potential usefulness in molecular epidemiology and in typing of Shigella strains. The degree of differentiation given by these two methods was generally comparable, although IS probes showed better discrimination of the isolates.

Distribution of transposable elements in prokaryotes

We consider models for the distribution of the number of elements per host genome for families of transposable elements (TEs). The hosts are assumed to be prokaryotes. These models assume (1) a constant rate of infection of uninfected hosts by TEs, (2) replicative transposition within each host, and (3) a reduction of the fitness of a host dependent on the number of TEs it contains. No provision was made for the deletion of individual TEs within a host or for recombination, since both are relatively rare events in prokaryotes. These models mostly assume that the TE performs no fuction for the host, and that the reduction in fitness with increased copy number is due to effects such as the impairment of beneficial genes by transposition or homologous recombination. We also consider a model in which the TEs can convey a selective advantage to the host. The equilibrium distributions of copy number are determined for these models, and are of a variety of classical types. Relevant parameters of the models are estimated using data on the distribution of insertion sequences in natural isolates of Escherichia coli.