IS1-mediated intramolecular rearrangements: formation of excised transposon circles and replicative deletions.

A system is described which permits visualization and analysis of a number of molecular species associated with transposition activity of the bacterial insertion sequence, IS1, in vivo. The technique involves induction of an IS1 transposase gene carried by a plasmid which also includes an IS1-based transposable element. It is, in principle, applicable to the identification of transposition intermediates as well as unstable transposition products and those which are not detectable by genetic means. Thirteen novel molecular species were detected after 4 h of induction. Five major species were characterized, based on their behaviour as a function of time, on their hybridization patterns and on the nucleotide sequences of the transposon-backbone junctions. All result from intramolecular IS1 transposition events. The two reciprocal partner products of IS1-mediated deletions, the intramolecular equivalent of co-integrates generated by intermolecular transposition, have been identified. Both carry a single copy of the transposable element and present complementary distributions of deletion endpoints. These results establish, by direct physical means, that adjacent IS1-mediated deletions are accompanied by duplication of the element. A second type of molecule identified was an excised circular copy of the transposon, raising the possibility that IS1 is capable of following an intermolecular transposition pathway, via excised transposon circles, leading to direct insertion.     

Insertional inactivation of an Escherichia coli urease gene by IS3411.

Ureolytic Escherichia coli are unusual clinical isolates that are found at various extraintestinal sites of infection, predominantly the urinary tract. The urease-positive phenotype is unstable in approximately 25% of these isolates, and urease-negative segregants are produced at a high frequency. We have studied the nature of the urease-positive-to-negative transition in one of these isolates, designated E. coli 1021. Southern hybridization experiments with genomic DNA extracted from seven independent E. coli 1021 urease-negative segregants revealed the presence of a 1.3-kb DNA insertion in the urease gene cluster. A DNA fragment containing the DNA insertion was cloned from one of the urease-negative segregants. This cloned DNA fragment was capable of mediating cointegrate formation with the conjugative plasmid pOX38, suggesting that the DNA insertion was a transposable element. The insert was identified as an IS3411 element in ureG by DNA sequence analysis. A 3-bp target duplication (CTG) flanking the insertion element was found. DNA spanning the insertion site was amplified from the other six urease-negative segregants by using the polymerase chain reaction. The DNA sequence of the amplified fragments indicated that an IS3411 element was found in an identical site in all urease-negative segregants examined. These data suggest that in E. coli 1021, IS3411 transposes at a high frequency into ureG at a CTG site, disrupting this gene and eliminating urease activity.     

Identification of shiga toxin-producing Escherichia coli possessing insertionally inactivated Shiga toxin gene

We have investigated the Shiga toxin genes of Shiga toxin-producing Escherichia coli (STEC) strains, using polymerase chain reaction (PCR) amplifying the full lengths of these genes. As a result, we found the Shiga toxin 2 gene which was insertionally inactivated by an insertion sequence (IS). This IS element was identical to IS1203v which has been also found in inactivated Shiga toxin 2 genes, and was inserted at the same site as in the previous paper. On the other hand, both Shiga toxin 2 genes were different (98.3% identity). These suggested that IS1203v independently inserted into each Shiga toxin 2 genes, and STEC strains possessing the insertionally inactivated Shiga toxin genes are most likely to have a wide distribution. Amplification of the full length of the Shiga toxin gene is one of the effective methods to detect the gene no matter where the IS element is included, i.e., the insertion can be reflected in the size of amplicon.     

The evolution of DNA sequences in Escherichia coli

It is proposed that certain families of transposable elements originally evolved in plasmids and functioned in forming replicon fusions to aid in the horizontal transmission of non-conjugational plasmids. This hypothesis is supported by the finding that the transposable elements Tn3 and gamma delta are found almost exclusively in plasmids, and also by the distribution of the unrelated insertion sequences IS4 and IS5 among a reference collection of 67 natural isolates of Escherichia coli. Each insertion sequence was found to be present in only about one-third of the strains. Among the ten strains found to contain both insertion sequences, the number of copies of the elements was negatively correlated. With respect to IS5, approximately half of the strains containing a chromosomal copy of the insertion element also contained copies within the plasmid complement of the strain.     

Transposable elements generate novel spatial patterns of gene expression in Antirrhinum majus

The pallida gene of A. majus encodes a product required for the synthesis of red flower pigment. We have shown that the unstable pallida(recurrens) mutation is due to the insertion of the Tam3 transposable element near the promoter of the gene. Imprecise excision of Tam3 alters pallida gene expression and generates new spatial patterns or different intensities of flower pigmentation. Distinct spatial patterns may also result from rearrangements induced by Tam3 that alter the relative position of the pallida gene. Changes in Tam3 structure or position result in new unstable phenotypes. These findings suggest that genes may be rendered genetically hypervariable as a consequence of transposable element insertion and excision.     

Genome-wide comparison of cyanobacterial transposable elements, potential genetic diversity indicators

Transposable elements are widely distributed in archaea, bacteria and eukarya domains. Considerable discrepancies of transposable elements in eukaryotes have been reported, however, the studies focusing on the diversity of transposable element systems in prokaryotes were scarce. Understanding the transposable element system in cyanobacteria by the genome-wide analysis will greatly improve the knowledge of cyanobacterial diversity. In this study, the transposable elements of seventeen cyanobacterial genomes were analyzed. The abundance of insertion sequence (IS) elements differs significantly among the cyanobacterial genomes examined. In particular, water bloom forming Microcystis aeruginosa NIES843 was shown to have the highest abundance of IS elements reaching 10.85% of the genome. IS family is a widely acceptable IS classification unit, and IS subfamily, based on probe sequences, was firstly proposed as the basic classification unit for IS element system, therefore both IS family and IS subfamily were suggested as the two hierarchical units for evaluating the IS element system diversity. In total, 1980 predicted IS elements, within 21 IS families and 132 subfamilies, were identified in the examined cyanobacterial genomes. Families IS4, IS5, IS630 and IS200-605 are widely distributed, and therefore supposed to be the ancestral IS families. Analysis on the intactness of IS elements showed that the percentage of the intact IS differs largely among these cyanobacterial strains. Higher percentage of the intact IS detected in the two hot spring cyanobacterial strains implied that the intactness of IS elements may be related to the genomic stabilization of cyanobacteria inhabiting in the extreme environments. The frequencies between IS elements and miniature inverted-repeat transposable elements (MITEs) were shown to have a linear positive correlation. The transposable element system in cyanobacterial genomes is of hypervariability. With characterization of easy definition and stability, IS subfamily is considered as a reliable lower classification unit in IS element system. The abundance of intact IS, the composition of IS families and subfamilies, the sequence diversity of IS element nucleotide and transposase amino acid are informative and suitable as the indicators for studies on cyanobacterial diversity. Practically, the transposable system may provide us a new perspective to realize the diversity and evolution of populations of water bloom forming cyanobacterial species.     

Natural Populations of Escherichia Coli and Salmonella Typhimurium Harbor the Same Classes of Insertion Sequences

Despite their close phylogenetic relationship, Escherichia coli and Salmonella typhimurium were long considered as having distinct classes of transposable elements maintained by either host-related factors or very restricted gene exchange. In this study, genetically diverse collections of E. coli and S. typhimurium (subgroup I) were surveyed for the presence of several classes of insertion sequences by Southern blot analysis and the polymerase chain reaction. A majority of salmonellae contained IS1 or IS3, elements originally recovered from E. coli, while IS200, a Salmonella-specific element, was present in about 20% of the tested strains of E. coli. Based on restriction mapping, the extent of sequence divergence between copies of IS200 from E. coli and S. typhimurium is on the order of that observed in comparisons of chromosomally encoded genes from these taxa. This suggests that copies of IS200 have not been recently transferred between E. coli and S. typhimurium and that the element was present in the common ancestor to both species. IS200 is polymorphic within E. coli but homogeneous among isolates of S. typhimurium, providing evidence that these species might differ in their rates of transfer and turnover of insertion sequences.     

DNA sequence at the integration sites of the insertion element IS1.

We have detected two independent occurrences of insertion mutations in the lacl gene of E. Coli, and have used small plasmids carrying the l gene to purify large amounts of DNA containing these insertions. Analyses with restriction endonucleases and DNA sequencing techniques establish that both insertions involve the previously characterized element IS1. In each case, the integration of IS1 into the l gene DNA is associated with a directly repeated sequence of 9 nucleotides appearing at each end of the insertion element. Since one of these sequences was present in the wild-type gene, the second sequence either preexisted in the IS1 before integration, or else was generated by the process of insertion itself. The 9 base repeat is different in both cases. We discuss the relevance of these findings to the mechanism of integration of transposable elements.     

Genetic organization of insertion element IS2 based on a revised nucleotide sequence

We identified a transposable element resident in the chromosome of Escherichia coli K-12 strain HB101. This is an approx. 4400-bp-long transposon flanked by two copies of insertion sequence (IS) 1 element in direct orientation. One of the IS1 elements was found to be integrated into an IS2 element between IS2 bp 139 and bp 140 with the large moiety of IS2 within the transposon. The sequence of this part of IS2 differs from the published sequence of galOP-308::IS2 at a number of positions. Restriction analysis of the published allele, however, indicated that both alleles may in fact be identical. Since six of the eight differences found alter open reading frames, the revised sequence presents a new outlook for the potential genetic organization of IS2.     

Imprecise excision of insertion element IS5 from the fliC gene contributes to flagellar diversity in Escherichia coli

Motile strains of Escherichia coli K12 carrying both a chromosomal fliC-H48 gene and a plasmid encoded fliC-H4 gene express both types of flagellins, which are coassembled into functional flagella. By using flagellar-H48-specific antiserum and a plasmid curing procedure, nonmotile mutants were found that carried an IS5 insertion in the chromosomal fliC-H48 gene. Motile revertants were isolated that showed deletions of the IS5 element together with sections of the fliC-H48 gene resulting in an altered flagellar serotype in these strains. As IS5 elements were found associated with 35 of 53 known H-types in wildtype E. coli strains, this insertion element might play a major role in serotype diversity.     

Cointegrate resolution following transposition of Tn1792 in Streptomyces avermitilis facilitates analysis of transposon-tagged genes.

The insertion sequence IS6100, belonging to the IS6 family, normally forms a cointegrate as an end product of transposition. The IS6100-based minitransposon, Tn1792, has been developed as a genetic tool to mutagenise antibiotic-producing Streptomyces. Here, we describe resolution of Tn1792 cointegrates in Streptomyces avermitilis that can facilitate both the initial isolation of Tn1792 insertion mutants and also the subsequent rescue of Tn1792-tagged sequences. This is the first reported example of cointegrate resolution for an IS6-type transposable element. As a result of mutagenesis, several putative genes involved in morphological development and antibiotic production have been isolated.     

Tyrosine protein phosphorylation in murine B lymphocytes by stimulation with lipopolysaccharide from Porphyromonas gingivalis

Abstract The sacB gene of Bacillus subtilis was successfully applied in various Arthrobacter, Brevibacterium, Corynebacterium and Rhodococcus strains for the isolation of transposable elements. Three different insertion sequence (IS) elements entrapped in sacB were isolated. The IS elements IS- Bl and IS- Cg isolated from Brevibacterium lactofermentum and Corynebacterium glutamicum , respectively, were found to be similar in size (1.45 kb) and generated target duplications of 8 bp. Their inverted repeats showed homology. In contrast, the IS element IS- Rf isolated from Rhodococcus fascians was only 1.3 kb long and generated a 3-bp target duplication. IS- Cg and IS- Rf were not restricted to their original host strains, and we also found strains harbouring more than one element.     

Insertion sequence inversions mediated by ectopic recombination between terminal inverted repeats

Transposable elements are widely distributed and diverse in both eukaryotes and prokaryotes, as exemplified by DNA transposons. As a result, they represent a considerable source of genomic variation, for example through ectopic (i.e. non-allelic homologous) recombination events between transposable element copies, resulting in genomic rearrangements. Ectopic recombination may also take place between homologous sequences located within transposable element sequences. DNA transposons are typically bounded by terminal inverted repeats (TIRs). Ectopic recombination between TIRs is expected to result in DNA transposon inversions. However, such inversions have barely been documented. In this study, we report natural inversions of the most common prokaryotic DNA transposons: insertion sequences (IS). We identified natural TIR-TIR recombination-mediated inversions in 9% of IS insertion loci investigated in Wolbachia bacteria, which suggests that recombination between IS TIRs may be a quite common, albeit largely overlooked, source of genomic diversity in bacteria. We suggest that inversions may impede IS survival and proliferation in the host genome by altering transpositional activity. They may also alter genomic instability by modulating the outcome of ectopic recombination events between IS copies in various orientations. This study represents the first report of TIR-TIR recombination within bacterial IS elements and it thereby uncovers a novel mechanism of structural variation for this class of prokaryotic transposable elements.     

Genetic and molecular analysis of spontaneous respiratory deficient (res-) mutants of Escherichia coli K-12

Respiratory deficient (res-) mutants of E. coli are slow growing microcolonial, anaerobic, catalase and benzidine negative strains whose broad phenotypic alteration may result from pleiotropic mutations in genes of the hemin biosynthetic pathway. They are easily recovered from platings of sensitive cells on concentrations of gentamicin higher than the minimal inhibitory concentration. These mutants show a dramatic change in their biochemical diagnostic profile resulting primarily from deficiencies in the active transport mechanisms of the cell. Using well-marked F- and Hfr strains, 157 mutants were analyzed from 3 different parent strains; all but 2 resulted from mutations in 3 loci of the hemin biosynthetic pathway. Of these a marked skew to hemB- mutations was seen, with more than 80% mapping there. The possibility that this hot spot resulted from transpositional activity was tested by Southern hybridization of EcoRI digests of the chromosomal DNA, using as a probe, a 2.8-kb fragment containing the hemB gene. The WT and other hemB+ control strains contained a 14.6-kb fragment. Of 18 hemB strains tested, 14 showed deletion and insertion mutations which fell into four classes based on the variation in the size of the fragment or on the absence of hybridization. The latter resulted from complete deletion of the hemB gene. An increase in fragment size from 1.5-kb to 3.4-kb was observed in some of the strains.     

Analysis of the chromosomal location of two copies of a Bordetella pertussis insertion sequence

IS481v1 and IS481v2 are two copies of a Bordetella pertussis insertion sequence element. We have shown that IS481v1 is located within 3 kbp of the start of the adenylate cyclase gene whilst IS481v2 is immediately adjacent to the end of the agglutinogen 2 gene and provides the stop codon for that gene. In addition, IS481v1 and IS481v2 were present at these two specific sites in nine strains of B. pertussis, including two Phase IV strains which expressed neither adenylate cyclase nor agglutinogen 2 and three Phase I strains which did not express agglutinogen 2. The loss of expression in these strains is not the result of DNA rearrangements at the sites of IS481v1 or IS481v2.