Nucleotide sequence of IS492, a novel insertion sequence causing variation in extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica.

The complete nucleotide sequence of insertion element IS492, which causes reversible inactivation of extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica, is presented. Insertion of IS492 results in the EPS- phenotype, and excision results in restoration of EPS+. DNA sequencing of the site of insertion in the eps locus showed that insertion of IS492 generates a 5-base-pair repeat and that its excision is precise. IS492 is 1,202 nucleotides in length and contains one large open reading frame encoding a protein of 318 amino acids, a candidate for transposition function. No similarity between IS492 and other transposable elements has been found. Unlike the situation with other insertion sequences, no direct or inverted repeats exist at the termini of IS492.     

In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family

The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.     

Characterization of IS476 and its role in bacterial spot disease of tomato and pepper.

IS476 is an endogenous insertion sequence present in copper-tolerant strains of Xanthomonas campestris pv. vesicatoria. Sequence analysis has revealed that the element is 1,225 base pairs in length, has 26-base-pair inverted repeats, and causes a 4-base-pair target site duplication upon insertion into the avirulence gene avrBs1. Comparison of the full-length sequence with sequences in the National Biomedical Research Foundation and National Institutes of Health data bases showed that one of the predicted IS476 proteins is partially homologous to the putative transposase of IS3 from Escherichia coli, and the inverted repeats of IS476 have significant homology to the inverted repeats of the IS51 insertion sequence of Pseudomonas syringae pv. savastanoi. A transposition assay based on the insertional inactivation of the sacRB locus of Bacillus subtilis was used to demonstrate that one of the three copies of IS476 residing on the 200-kilobase copper plasmid pXVCU1 is capable of transposition in several strains of Xanthomonas campestris. The position of IS476 insertion in several avrBs1 mutants was established and was shown to influence both induction of hypersensitivity and bacterial growth in planta.     

Transposition of cyanobacterium insertion element ISY100 in Escherichia coli

The genome of the cyanobacterium Synechocystis sp. strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY100 in 22 copies is the most abundant. A typical ISY100 member is 947 bp long and has imperfect terminal inverted repeat sequences. It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS630, indicating that ISY100 belongs to the IS630 family. To determine whether ISY100 has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY100 with the chloramphenicol resistance gene, substituted for the transposase gene of ISY100, at another site and introduced the plasmid into an Escherichia coli strain already harboring a target plasmid. Mini-ISY100 transposed to the target plasmid in the presence of IPTG at a very high frequency. Mini-ISY100 was inserted into the TA sequence and duplicated it upon transposition, as do IS630 family elements. Moreover, the mini-ISY100-carrying plasmid produced linear molecules of mini-ISY100 with the exact 3' ends of ISY100 and 5' ends lacking two nucleotides of the ISY100 sequence. No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc1/mariner family elements, Tc1 and Tc3, which transpose to the TA sequence, have. These findings suggest that ISY100 transposes to a new site through the formation of linear molecules, such as Tc1 and Tc3, by excision. Some Tc1/mariner family elements leave a footprint with an extra sequence at the site of excision. No footprints, however, were detected in the case of ISY100, suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system. ISY100 transposes in E. coli, indicating that it transposes without any host factor other than the transposase encoded by itself. Therefore, it may be able to transpose in other biological systems.     

Isolation and characterization of IS elements repeated in the bacterial chromosome

Shigella sonnei contains repetitive sequences, including an insertion element IS1, which can be isolated as double-stranded DNA fragments by DNA denaturation and renaturation and by treatment with S1 nuclease. In this paper, we describe a method of cloning the IS1 fragments prepared by the S1 nuclease digestion technique into phage M13mp8 RFI DNA. Several clones contained IS1, usually with a few additional bases. We isolated and characterized five other repetitive sequences using this method. One sequence, 1264 base-pairs in length, had terminal inverted repeats and contained two open reading frames. This sequence, called IS600, showed about 44% sequence homology with IS3 and was repeated more than 20 times in the Sh. sonnei chromosome. Another sequence (named IS629, 1310 base-pairs in length), which was repeated six times, was found also to be related to IS3 and thus IS600. Two other sequences (named IS630 and IS640, 1159 and 1092 base-pairs in length, respectively), which were repeated approximately ten times, had characteristic terminal inverted repeats and contained a large open reading frame coding for a protein. The inverted repeat sequences of IS630 were similar to the sequence at one end of IS200, a Salmonella-specific IS element. The fifth sequence, repeated ten times in Sh. sonnei, had about 98% sequence homology with a portion of IS2. The method described here can be applied to the isolation of IS or iso-IS elements present in any other bacterial chromosome.     

A novel IS element,IS621, of the IS110/IS492 family transposes to a specific site in repetitive extragenic palindromic sequences in Escherichia coli

An Escherichia coli strain, ECOR28, was found to have insertions of an identical sequence (1,279 bp in length) at 10 loci in its genome. This insertion sequence (named IS621) has one large open reading frame encoding a putative protein that is 326 amino acids in length. A computer-aided homology search using the DNA sequence as the query revealed that IS621 was homologous to the piv genes, encoding pilin gene invertase (PIV). A homology search using the amino acid sequence of the putative protein encoded by IS621 as the query revealed that the protein also has partial homology to transposases encoded by the IS110/IS492 family elements, which were known to have partial homology to PIV. This indicates that IS621 belongs to the IS110/IS492 family but is most closely related to the piv genes. In fact, a phylogenetic tree constructed on the basis of amino acid sequences of PIV proteins and transposases revealed that IS621 belongs to the piv gene group, which is distinct from the IS110/IS492 family elements, which form several groups. PIV proteins and transposases encoded by the IS110/IS492 family elements, including IS621, have four acidic amino acid residues, which are conserved at positions in their N-terminal regions. These residues may constitute a tetrad D-E(or D)-D-D motif as the catalytic center. Interestingly, IS621 was inserted at specific sites within repetitive extragenic palindromic (REP) sequences at 10 loci in the ECOR28 genome. IS621 may not recognize the entire REP sequence in transposition, but it recognizes a 15-bp sequence conserved in the REP sequences around the target site. There are several elements belonging to the IS110/IS492 family that also transpose to specific sites in the repeated sequences, as does IS621. IS621 does not have terminal inverted repeats like most of the IS110/IS492 family elements. The terminal sequences of IS621 have homology with the 26-bp inverted repeat sequences of pilin gene inversion sites that are recognized and used for inversion of pilin genes by PIV. This suggests that IS621 initiates transposition through recognition of their terminal regions and cleavage at the ends by a mechanism similar to that used for PIV to promote inversion at the pilin gene inversion sites.     

Transposon Tn10: genetic organization, regulation, and insertion specificity

Transposon Tn10 is a composite element in which two individual insertion sequence (IS)-like sequences cooperate to mediate transposition of the intervening material. The two flanking IS10 elements are not identical; IS10-right is responsible for functions required to promote transposition, and IS10-left is defective in transposition functions. We suggest that the two IS10 elements were originally identical in sequence and have subsequently diverged. IS10-right is compactly organized with structural gene(s), promoters, and sites important for transposition and (presumably) its regulation all closely linked and, in some cases, overlapping. IS10 has a single major coding region that almost certainly encodes an essential transposition function. A pair of opposing promoters flank the start of this coding region. One of these promoters is responsible for expression in vivo of transposon-encoded transposition functions. We propose that the second promoter is involved in modulation of Tn10 transposition. Genetic analysis suggests that transposon-encoded function(s) may be preferentially cis-acting. Insertion of Tn10 into particular preferred target sites is due primarily to the occurrence of a particular six-base pair target DNA sequence. The properties of this sequence suggest that symmetrically disposed subunits of a single protein may be responsible for both recognition and cleavage of target DNA during insertion.     

Transposition of IS10 from the host Escherichia coli genome to a plasmid may lead to cloning artefacts

During recloning of Nicotiana tabacum L. repetitive sequence R8.3 in Escherichia coli, a modified clone that differed from the original by the insertion of an IS10 sequence was unintentionally produced. The insert was flanked by a 9-bp direct repeat derived from the R8.3 sequence, the 9-bp duplication of acceptor DNA in the site of insertion being a characteristic of IS10 transposition events. A database search using the FASTA program showed IS10 and other prokaryotic IS elements inserted into numerous eukaryotic clones. Unexpectedly, the IS10, which is not a natural component of the E. coli genome, appeared to be by far the most frequent contaminant of DNA databases among several IS sequences tested. In the GenEMBL database, the IS10 query sequence yielded positive scores with more than 500 eukaryotic clones. Insertions of shortened IS10 sequences having only one intact terminal inverted repeat were commonly found. Most full-length IS10 insertions (32 out of 40 analyzed) were flanked by 9-bp direct repeats having the consensus 5'-NPuCNN-NGPyN-3' with a strong preference for 5'-TGCTNA-GNN-3'. One insertion was flanked by an inverted repeat of more than 400 bp in length. PCR amplification and Southern analysis revealed the presence of IS10 sequences in E. coli strains commonly used for DNA cloning, including some reported to be Tn10-free. No IS10-specific PCR product was obtained with N. tabacum or human DNA. Our data suggest that transposition of IS10 elements may accompany cloning steps, particularly into large BAC vectors. This might lead to the relatively frequent contamination of DNA databases by this bacterial sequence. It is estimated that one in approximately every thousand eukaryotic clone in the databases is contaminated by IS-derived sequences. We recommend checking submitted sequences for the presence of IS10 and other IS elements. In addition, DNA databases should be corrected by removing contaminating IS sequences.     

Nucleotide sequence of insertion sequence IS3411, which flanks the citrate utilization determinant of transposon Tn3411.

The nucleotide sequences of insertion sequences IS3411L (left) and IS3411R (right), present as direct terminal repeats in the citrate utilization of citrate utilization transposon Tn3411, and of IS3411 (generated by intramolecular recombination between IS3411L and IS3411R) were determined. The three IS3411 elements (IS3411R, IS3411L, and IS3411) were 1,309 base pairs long and identical in DNA sequence. IS3411 had 27-base-pair terminal inverted repeats with three bases mismatched and one long open reading frame (240 amino acids) that was proposed to be a transposase. Three polypeptides of 29,000, 27,000, and about 10,000 molecular weight, determined by IS3411, were identified in minicells. Since Tn3411 generates a 3-base-pair repeat upon integration, the nucleotide sequences of IS3411 were compared with those of IS3.     

Structural and Functional Characterization of IS679 and IS66-Family Elements

A new insertion sequence (IS) element, IS679 (2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic Escherichia coli B171. IS679 has imperfect 25-bp terminal inverted repeats (IRs) and three open reading frames (ORFs) (here called tnpA, tnpB, and tnpC). A plasmid carrying a composite transposon (Tn679) with the kanamycin resistance gene flanked by an intact IS679 sequence and an IS679 fragment with only IRR (IR on the right) was constructed to clarify the transposition activity of IS679. A transposition assay done with a mating system showed that Tn679 could transpose at a high frequency to the F plasmid derivative used as the target. On transposition, Tn679 duplicated an 8-bp sequence at the target site. Tn679 derivatives with a deletion in each ORF of IS679 did not transpose, finding indicative that all three IS679 ORFs are essential for transposition. The tnpA and tnpC products appear to have the amino acid sequence motif characteristic of most transposases. A homology search of the databases found that a total of 25 elements homologous to IS679 are present in Agrobacterium, Escherichia, Rhizobium, Pseudomonas, and Vibrio spp., providing evidence that the elements are widespread in gram-negative bacteria. We found that these elements belong to the IS66 family, as do other elements, including nine not previously reported. Almost all of the elements have IRs similar to those in IS679 and, like IS679, most appear to have duplicated an 8-bp sequence at the target site on transposition. These elements have three ORFs corresponding to those in IS679, but many have a mutation(s) in an ORF(s). In almost all of the elements, tnpB is located in the -1 frame relative to tnpA, such that the initiation codon of tnpB overlaps the TGA termination codon of tnpA. In contrast, tnpC, separated from tnpB by a space of ca. 20 bp, is located in any one of three frames relative to tnpB. No common structural features were found around the intergenic regions, indicating that the three ORFs are expressed by translational coupling but not by translational frameshifting.     

细菌插入序列的转座酶和转座机制

插入序列（insertion sequence, IS）是细菌中最简单的移动遗传因子,由两端的反向重复序列（inverted repeats, IR）和中间的转座酶 (transposase)编码序列组成。在细菌中,因为插入序列的转座酶催化活性中心氨基酸序列不同,所以将其转座酶分为DDE转座酶、DEDD转座酶、HUH转座酶和丝氨酸转座酶。在转座过程中,根据插入序列是否有复制,将插入序列的转座分为复制型转座（replicative -ansposition）和非复制型转座（non-replicative transposition）,而将形成夏皮罗中间体（Shapiro intermediate）的非复制型转座称为保守型转座（conservative transposition）。此外,插入序列通过不同的转座机制插入到基因编码区导致基因突变、缺失和倒置;或者插入到基因上游,通过自身启动子或与基因形成杂交启动子来影响插入序列下游基因的表达,从而帮助细菌抵抗复杂的环境变化。本文主要围绕细菌插入序列的特征、转座酶、转座机制和转座影响展开综述,以期为进一步研究插入序列的机制和插入序列在细菌中所起的作用提供参考。     

The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1/ISRm7 and ISRm220-13-5 belong to a new family of insertion sequence elements

The Sinorhizobium meliloti insertion sequence (IS) elements ISRm102F34-1 and ISRm220-13-5 are 1481 and 1550 base pairs (bp) in size, respectively. ISRm102F34-1 is bordered by 15 bp imperfect terminal inverted repeat sequences (two mismatches), whereas the terminal inverted repeat of ISRm220-13-5 has a length of 16 bp (two mismatches). Both insertion sequence elements generate a 6-bp target duplication upon transposition. The putative transposase enzymes of ISRm102F34-1 and ISRm220-13-5 consist of 449 or 448 amino acid residues with predicted molecular weights of 50.7 or 51.3 kDa and theoretical isoelectric points of 10.8 or 11.1, respectively. ISRm102F34-1 is identical in 98.9% of its nucleotide sequence to an apparently inactive copy of an insertion sequence element, designated ISRm7, which flanks the left-end of the nodule formation efficiency (nfe) region of plasmid pRmeGR4b of S. meliloti strain GR4. ISRm102F34-1 and ISRm220-13-5 are closely related since they show an overall identity of 57.0% at the nucleotide sequence level and of 47.3% at the deduced amino acid level of their putative transposases. Both insertion sequence elements displayed significant similarity to the Xanthomonas campestris ISXc6 and its homolog IS1478a. Since none of these insertion sequence elements could be allocated to existing families of insertion sequence elements, a new family is proposed. Analysis of the distribution of ISRm102F34-1/ISRm7 in various local S. meliloti populations sampled from Medicago sativa, Medicago sphaerocarpa and Melilotus alba host plants at different locations in Spain revealed its presence in 35% of the isolates with a copy number ranging from 1 to 5. Furthermore, ISRm102F34-1/ISRm7 homologs were identified in other rhizobial species.     

The left end of IS2: a compromise between transpositional activity and an essential promoter function that regulates the transposition pathway

Cut-and-paste (simple insertion) and replicative transposition pathways are the two classical paradigms by which transposable elements are mobilized. A novel variation of cut and paste, a two-step transposition cycle, has recently been proposed for insertion sequences of the IS3 family. In IS2 this variation involves the formation of a circular, putative transposition intermediate (the minicircle) in the first step. Two aspects of the minicircle may involve its proposed role in the second step (integration into the target). The first is the presence of a highly reactive junction formed by the two abutted ends of the element. The second is the assembly at the minicircle junction of a strong hybrid promoter which generates higher levels of transposase. In this report we show that IS2 possesses a highly reactive minicircle junction at which a strong promoter is assembled and that the promoter is needed for the efficient completion of the pathway. We show that the sequence diversions which characterize the imperfect inverted repeats or ends of this element have evolved specifically to permit the formation and optimal function of this promoter. While these sequence diversions eliminate catalytic activity of the left end (IRL) in the linear element, sufficient sequence information essential for catalysis is retained by the IRL in the context of the minicircle junction. These data confirm that the minicircle is an essential intermediate in the two-step transposition pathway of IS2.     

Identification of insertion sequence element IS427 in pTiT37 plasmid DNA of an Agrobacterium tumefaciens T37 isolate

The isolation and characterization of an insertion sequence (IS) element, IS427, from Agrobacterium tumefaciens T37 is described. IS427 is present in three nonidentical copies on the pTiT37 plasmid. The copy that was isolated through transposition on the entrapment vector pUCD800 contains at its ends a 16-bp imperfect inverted repeat and generates a 2-bp duplication of the target DNA. IS427 does not show homology with previously characterized IS elements of A. tumefaciens, based on hybridization experiments and/or sequence comparison.     

Structural and genetic organization of IS232, a new insertion sequence of Bacillus thuringiensis.

In the Bacillus thuringiensis strains toxic for the lepidopteran larvae, the delta-endotoxin genes cryIA are frequently found within a composite transposonlike structure flanked by two inverted repeat sequences. We report that these elements are true insertion sequences and designate them IS232. IS232 is a 2,184-bp element and is delimited by two imperfect inverted repeats (28 of 37 bp are identical). Two adjacent open reading frames, overlapping for three codons, span almost the entire sequence of IS232. The potential encoded polypeptides of 50 and 30-kDa are homologous to the IstA and IstB proteins of the gram-negative insertion sequence IS21. The N-terminal part of the 50-kDa polypeptide contains a helix-turn-helix DNA-binding motif. The junctions at the insertion sites of three IS232 elements were analyzed. Each case was different, with 0, 4, or 6 bp of the target DNA being duplicated. Transposition of IS232 in Escherichia coli was demonstrated by using a genetic marker inserted upstream of the two open reading frames.     

Structure-function analysis of the inverted terminal repeats of the sleeping beauty transposon

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates.