Mapping of insertion element IS5 in the Escherichia coli K-12 chromosome. Chromosomal rearrangements mediated by IS5

We identified phage clones containing insertion element IS5 in a set of 476 lambda phage clones carrying chromosomal segments that cover almost the entire chromosome of Escherichia coli K-12 W3110. Precise locations and orientations of IS5 were then determined by cleavage analysis of phage DNAs containing them. We mapped 23 copies of IS5 (named is5A to is5W) on the W3110 chromosome. Among them, ten were identified as the common elements present at the same locations in both chromosomes of W3110 and another E. coli K-12 strain, JE5519. While most of the mapped IS5 elements were scattered over the W3110 chromosome, four copies of IS5 (designated is5L, is5M, is5N and is5O) were in a region representing tandem duplication of a DNA segment flanked by two copies of IS5. Interestingly, one unit of this DNA segment as well as a portion of it was seen also in a tandem array in a different region where two copies of IS5 (designated is5P and is5Q) were present. In particular two pairs of the mapped IS5 elements may have been involved in inversion of the chromosomal segments in two of the E. coli K-12 derivatives.     

Mapping of insertion element IS30 in the Escherichia coli K12 chromosome

Summary We identified seven phage clones containing the insertion element IS30 in a phage library mini-set, which includes 476 clones carrying chromosomal segments that cover almost the entire chromosome ofEscherichia coli K12 W3110 (Kohara et al. 1987). We could assign locations and orientations to four copies of IS30 (namedis30A tois30D) on the W3110 chromosome by restriction analysis of phage DNAs containing them. These IS30s were present at the same locations in chromosomes of both W3110 and anotherE. coli K12 strain JE5519, and thus are assumed to be present in otherE. coli K12 derivatives, including early isolates. Among the IS30 copies found, one (is30B) contained a large deletion and possessed only a 181 by stretch of the right terminal region of IS30.EMBL Accession Number: The EMBL accession number of the sequence reported in this paper is X17345     

IS150: distribution, nucleotide sequence and phylogenetic relationships of a new E. coli insertion element.

Recently we identified the new insertion (IS) sequence IS150 in various strains of Escherichia coli K-12. We have screened other strains of E. coli and Salmonella typhimurium for the presence of homologous sequences. The strains of E. coli K-12 and W tested contain one or more copies of homology to IS150. We have also determined the complete nucleotide sequence of a copy of IS150 inserted into IS1. Comparison of nucleotide and deduced amino acid sequences of IS150, IS2, IS3, IS51, IS600 and IS629 reveals significant homologies suggesting that these elements are members of a family of phylogenetically related insertion sequences.     

IS2 insertion is a major cause of spontaneous mutagenesis of the bacteriophage P1: non-random distribution of target sites

Insertion mutations arising spontaneously in the P1 prophage and affecting vegetative phage reproduction were screened for the presence of insertion sequence 2 (IS2). Filter hybridization identified 28 out of 44 independent insertions as IS2. Their target specificity is not random. A region that amounts to < 2% of the phage genome had trapped 15 of the 28 IS2 elements. However, precise mapping of nine mutants in this hot spot segment revealed no preferred insertion site. Rather, the nine IS2 are distributed over the whole target segment and IS2 are found in both orientations. Sequence data indicate that at least two sequence variants of IS2 participated in mutagenesis of the phage genome. The detectable transposition of IS2 from the host chromosome to the prophage occurs with a frequency of 3 x 10(-5) per cell per generation under the particular experimental conditions. It is concluded that IS2, a natural resident of Escherichia coli K12 strains, is an important agent for spontaneous mutagenesis and exerts this action non-randomly along the genome.     

Four types of IS1 with differences in nucleotide sequence reside in the Escherichia coli K-12 chromosome.

The Escherichia coli K-12 chromosome contains six copies of insertion element IS1 at loci is1A-is1F. We determined their nucleotide (nt) sequences and found that they were classified into four types. Two copies of IS1 which flank a chromosomal segment containing the argF gene (IS1B and IS1C) have identical nt sequences. Another identical pair are IS1A and IS1E. Comparison of their nt sequences with the IS1 in plasmid R100 revealed seven nt mismatches for IS1A (or IS1E), two for IS1B (or IS1C), four for IS1D, and 75 for IS1F. The fact that the IS1s flanking the argF segment are identical supports the idea that the segment, together with the particular pair of IS1s, has constituted a composite transposon and transposed after genetic transfer from another bacterial species into E. coli K-12. Duplicated sequences were not observed in the regions flanking each of four copies of IS1, indicating that rearrangements have occurred in these chromosomal regions after IS1 elements had been inserted into several target sites. The four types of IS1 present in the E. coli K-12 chromosome were essentially similar to IS1s in plasmid R100 and in the chromosomes of Shigella strains. This and the above results suggest that they have been transferred horizontally from other Enterobacteriaceae, including Shigella, into E. coli K-12.     

alphabeta sequence of F is IS31.

Previous studies have shown that there is a deoxyribonucleic acid (DNA) segment, of length 1.3 kb and denoted as the alphabeta sequence, which occurs twice on the F plasmid at corrdinates 93.2 to 94.5/OF kb and 13.7 to 15.0F kb. In the present investigation, heteroduplexes were prepared between a phage DNA carrying the insertion sequence IS3 and suitable F-prime DNAs. The hybrids formed show that IS3 is the same as alphabeta. This result plus previous studies support the view that: (i) the insertion sequence IS2 and IS3 occur on F and, in multiple copies, on the main bacterial chromosome of Escherichia coli K-12; and (ii)these IS sequences on the main bacterial chromosomes are hot spots for Hfr formation by reciprocal recombination with the corresponding sequences of F.     

IS1397 is active for transposition into the chromosome of Escherichia coli K-12 and inserts specifically into palindromic units of bacterial interspersed mosaic elements

We demonstrate that IS1397, a putative mobile genetic element discovered in natural isolates of Escherichia coli, is active for transposition into the chromosome of E. coli K-12 and inserts specifically into palindromic units, also called repetitive extragenic palindromes, the basic element of bacterial interspersed mosaic elements (BIMEs), which are found in intergenic regions of enterobacteria closely related to E. coli and Salmonella. We could not detect transposition onto a plasmid carrying BIMEs. This unprecedented specificity of insertion into a well-characterized chromosomal intergenic repeated element and its evolutionary implications are discussed.     

A new insertion sequence, IS121, is found on the Mu dI1 (Ap lac) bacteriophage and the Escherichia coli K-12 chromosome

We have discovered a new insertion sequence, now designated IS121, as a component of the Mu dI1 (Ap lac) phage. This sequence is 1.2 kilobases long and contains single recognition sites for the HincII, Bg1II, and HindIII restriction endonucleases. IS121 is present in at least three copies in the chromosome of several Escherichia coli K-12 strains. When present in the nonconjugative plasmid pBR322, IS121 can mediate cointegrate formation with an F' lac plasmid and transfer of pBR322 sequences to suitable recipients. IS121 is also capable of precise or nearly precise excision. As part of the study of IS121, we have determined the physical structure of the Mu dI1 (Ap lac) phage and established an extensive restriction endonuclease map of this phage. A revised schema for the formation of the Mu dI1 (Ap lac) phage is presented.     

Transposition in Shigella dysenteriae: isolation and analysis of IS911, a new member of the IS3 group of insertion sequences.

Twenty-nine clear-plaque mutants of bacteriophage lambda were isolated from a Shigella dysenteriae lysogen. Three were associated with insertions in the cI gene: two were due to insertion of IS600, and the third resulted from insertion of a new element, IS911. IS911 is 1,250 base pairs (bp) long, carries 27-bp imperfect terminal inverted repeats, and generates 3-bp duplications of the target DNA on insertion. It was found in various copy numbers in all four species of Shigella tested and in Escherichia coli K-12 but not in E. coli W. Analysis of IS911-mediated cointegrate molecules indicated that the majority were generated without duplication of IS911. They appeared to result from direct insertion via one end of the element and the neighboring region of DNA, which resembles a terminal inverted repeat of IS911. Nucleotide sequence analysis revealed that IS911 carries two consecutive open reading frames which code for potential proteins showing similarities to those of the IS3 group of 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.     

Thermosensitive vector derived from RP1 plasmid for detection of transposable elements

The involvement of the transposable DNA element of E. coli K12 chromosome in integrative recombination of RP1 plasmid was studied. Using temperature sensitive for replication plasmid RP1ts12--the derivative of RP1 which contains mutated transposon Tnl, it was shown that integration of RP1 into host chromosome and Hfr formation may occur according to a mechanism mediated by chromosome IS-elements. Plasmids that are desintegrated from the chromosome of these Hfrs contain discrete DNA segments (IS-elements) and possess elevated frequency of integration into chromosome of rec+ cells. The latter was used for selection of RP1ts12 recombinants carrying chromosome IS. For identification of IS involved in RP1 integration the number of independent RP1ts 12 recombinants was subjected to restriction and heteroduplex analysis. By analysing recombinants integrated into bacterial chromosome with frequency 5 X 10(-3), a new IS-element of E. coli K12 designated IS111 was discovered. IS111-element is about 1500bp of length, contains Smal, Pst1 and BamH1 restriction endonuclease sites and was found in the same position on the plasmid RP1 in two different orientations. IS-elements that have been revealed in a number of other RP1ts12 recombinants were preliminary identified as IS1-like elements. One recombinants plasmid was found to have an IS5-like elements. The activity of IS-elements inserted into RP1ts12 in recA-dependent integrative recombination was estimated. From the data of absolute and relative RP1ts12 integration frequencies mediated by IS111, IS1- and IS5-like elements a conclusion was made about the absence of E. coli K12 chromosome IS-elements in RP1 plasmid. The Hfr-formation and chromosomal gene transfer by recombinant plasmids RP1ts12: IS111 were studied. The possibility to use insertion RP1ts12 derivatives for the estimation of copies number, mapping and definition of orientation of IS-elements in bacterial chromosome and the possibilities for detection of transposable DNA elements using RP1ts12 in a wide range of gram-negative bacteria are discussed.     

IS861, a group B streptococcal insertion sequence related to IS150 and IS3 of Escherichia coli.

A 1,442-base-pair (bp) insertion sequence (IS861) was identified in the type III group B streptococcal (GBS) strain COH-1. It is flanked by 26-bp imperfect inverted repeats and contains two open reading frames, 1 and 2, encoding 141- and 277-amino-acid proteins, respectively. A 3-bp target sequence, ACA, is duplicated and flanks each inverted repeat. IS861 shares greater than 30% homology with IS3 and IS150 of Escherichia coli, primarily in the region of their putative transposases. Northern (RNA) analysis revealed that RNA is actively transcribed in vivo by IS861 and 17- and 36-kilodalton proteins were synthesized in E. coli maxicell assays. Multiple copies of IS861 were observed throughout the chromosome of COH-1, and one of the copies is located near genes involved in GBS capsule synthesis. IS861 is the first insertion sequence identified in GBS. Its role in GBS and the significance of its relationship to the phylogenetically similar insertion sequences typified by IS150 and IS3 of E. coli are unknown.     

The nucleotide sequence of IS5 from Escherichia coli

A 3-kb fragment of Haemophilus haemolyticus DNA which carries the HhaII restriction (r) and modification (m) genes has been cloned into the PstI site of pBR322 (Mann et al., 1978). When propagated in Escherichia coli, it was observed that spontaneous insertions of IS5 inactivated the restriction gene, producing r- mutants at a frequency of 10(-6). Electron microscopy, restriction-site mapping and sequence analysis of two r- plasmids have demonstrated the presence of IS5 at a single target site in both possible orientations. The complete nucleotide sequence of IS5 has been determined. It is 1195 bp long and has inverted terminal repeats of 16 bp. The target site for IS5 in this plasmid is 5'-CTAG. Approx. ten copies of IS5 were found to be present at about the same locations on the E. coli chromosome in various K-12 strains, using Southern hybridization analysis.     

IS421, a new insertion sequence in Escherichia coli

The nucleotide sequence of a new insertion sequence (IS) in Escherichia coli, IS421, was determined. It is 1340 bp long and contains inverted repeats of 22 bp at its termini. It is flanked by 13 bp direct repeats apparently generated upon insertion. There are two ORFs longer than 200 bp in IS421. One can encode a polypeptide of 371 amino acids (aa) and the other, which is on the other strand, can encode a polypeptide of 102 aa. The C-terminal part of the 371 aa polypeptide shows some homology to that of transposases encoded in some other known IS elements. The copy number of IS421 in chromosomal DNA was 4 for E. coli K-12 and B, and 5 for E. coli C, as determined by the Southern hybridization of restriction fragments.     

Completion of the IS map in E. coli: IS186 positions on the E. coli K12 chromosome

Summary Insertion sites of the transposable element IS186 were physically mapped in the genome of E. coli K12 strain BHB2600. This strain maintains four IS186 copies of which three, assigned to 0.3, 14.1 and 51.8 map min., share common map positions with the three IS186 copies in strains W3110 and HB101. The fourth, unique IS copy in BHB2600 maps at 49.3 min. The IS186 data complete the BHB2600 map for all chromosomal sites of known K12-associated IS types.     

Cloning and sequencing of IS1086, an Alcaligenes eutrophus insertion element related to IS30 and IS4351.

A new insertion sequence (IS), designated IS1086, was isolated from Alcaligenes eutrophus CH34 by being trapped in plasmid pJV240, which contains the Bacillus subtilis sacB and sacR genes. The 1,106-bp IS1086 element contains partially matched (22 of 28 bp) terminal-inverted repeats and a long open reading frame. Hybridization data suggest the presence of one copy of IS1086 in the strain CH34 heavy-metal resistance plasmid pMOL28 and at least two copies in its chromosome. Analysis of the IS1086 nucleotide sequence revealed striking homology with two other IS elements, IS30 and IS4351, suggesting that they are three close members in a family of phylogenetically related insertion sequences. One open reading frame of the Spiroplasma citri phage SpV1-R8A2 B was also found to be related to this IS family but to a lesser extent. Comparison of the G+C contents of IS30 and IS1086 revealed that they conform to their respective hosts (46 versus 50% for IS30 and Escherichia coli and 64.5% for IS1086 and A. eutrophus). The pressure on the AT/GC ratio led to a very different codon usage in these two closely related IS elements. Results suggesting that IS1086 transposition might be activated by some forms of stress are discussed.     

Evolution of transposable elements: an IS10 insertion increases fitness in Escherichia coli

Strains of Escherichia coli carrying Tn10, a transposon consisting of two IS10 insertion sequences flanking a segment encoding for a tetracycline-resistance determinant, gain a competitive advantage in chemostat cultures. All Tn10-bearing strains that increase in frequency during competition have a new IS10 insertion that is found in the same location in the genome of those strains. We mapped, by a gradient of transmission, the position of the new IS10 insertion. We examined 11 isolates whose IS10 insertion was deleted by recombinational crossing- over, and in all cases the competitive fitness of the isolates was decreased. These results show that the IS10-generated insertion increases fitness in chemostat cultures. We named the insertion fit::IS10 and suggest that transposable elements may speed the rate of evolution by promoting nonhomologous recombination between preexisting variations within a genome and thereby generating adaptive variation.      

Target specificity of insertion element IS30

The Escherichia coli resident mobile element IS 30 has pronounced target specificity. Upon transposition, the element frequently inserts exactly into the same position of a preferred target sequence. Insertion sites in phages, plasmids and in the genome of E. coli are characterized by an exceptionally long palindromic consensus sequence that provides strong specificity for IS 30 insertions, despite a relatively high level of degeneracy. This 24-bp-long region alone determines the attractiveness of the target DNA and the exact position of IS 30 insertion. The divergence of a target site from the consensus and the occurrence of 'non-permitted' bases in certain positions influence the target activity. Differences in attractiveness are emphasized if two targets are present in the same replicon, as was demonstrated by quantitative analysis. In a system of competitive targets, the oligonucleotide sequence representing the consensus of genomic IS 30 insertion sites proved to be the most efficient target. Having compared the known insertion sites, we suppose that IS 30 -like target specificity, which may represent an alternative strategy in target selection among mobile elements, is characteristic of the insertion sequences IS 3 , IS 6 and IS 21 , too.