Gamma delta-mediated deletions of chromosomal segments on F-prime plasmids

Deleted derivatives of F lac+ proC+ tsx+/- purE+ plasmids ORF203 and F13 were isolated and physically characterized. Among 31 deletions, 24 were adjacent to the gamma delta element on F, four were associated with IS2 or IS3 elements normally present on F, and three displayed additional DNA rearrangements. With the genetic selection employed, the deletion endpoints in the chromosomal segment could fall anywhere within a 210 kb (5 min) region between proC and lac. The distribution of endpoints in this region was not random: the endpoints primarily occurred in an extended region near purE, and a 50 kb segment between tsx and purE was devoid of deletion endpoints. Deletion termini for mutants obtained from F13, which contains an additional 48 kb-segment interposed between gamma delta and the target region on ORF203, displayed a distribution similar to that seen for ORF203. Among simple deletions, there was no marked tendency for the chromosomal deletion endpoints to fall at IS1, IS3, or IS5 elements normally present in this chromosomal region. Point mutations and mutations caused by gamma delta or IS transposition into lac appeared in a small proportion of all plasmids studied.     

Transposition and target specificity of the typical IS30 family element IS1655 from Neisseria meningitidis

We have analysed the transposition and target selection strategy of IS1655, a typical IS30 family member resident in Neisseria meningitidis. We have redefined IS1655 as a 1080 bp long element with 25 bp imperfect inverted repeats (IRs), which generates a 3 bp target duplication and have shown that it transposes using an intermediate with abutted IRs separated by 2 bp. IS1655 exhibits bipartite target specificity inserting preferentially either next to sequences similar to its IRs or into an unrelated but well defined sequence. IR-targeting leads to the formation of a new junction in which the targeted IR and one of the donor IRs are separated by 2 bp. The non-IR targets were characterized as an imperfect 19 bp palindrome in which the central five positions show slight GC excess and the distal region is AT-rich. Artificial targets designed according to the consensus were recognized by the element as hot spots for insertion. The organization of IS1655 is similar to that of other IS30 family members. Moreover, it shows striking similarity to IS30 in transposition strategy even though their transposases differ in their N-terminal regions, which, for IS30, appears to determine target specificity. Comparative analysis of the transposases and the evolutionary aspects of sequence variants are also briefly discussed.     

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.     

Plasmid vectors for selecting IS1-promoted deletions in cloned DNA: sequence analysis of the omega interposon.

We have constructed two plasmid vectors which allow selection for in vivo deletions within cloned DNA fragments. The plasmids are derivatives of pBR322 which carry the Escherichia coli rpsL (strA) gene, known to confer a dominant streptomycin (Sm)-sensitivity phenotype to the host cell, and a copy of the IS1 transposable element. Sm-resistant strains that harbor these plasmids display sensitivity to Sm. Spontaneous IS1-promoted deletions across the rpsL gene can be isolated simply by selection for Sm resistance. Hence, nested sets of deletions of a cloned DNA can be obtained and sequenced with an IS1-specific primer. Using this approach, we have determined the complete nucleotide sequence of the omega interposon [Prentki and Krisch, Gene 29 (1984) 303-313].     

Atypical deletions generated by mutated IS102 elements

Summary The element IS102 potentially codes for two polypeptide chains. We have introduced several mutations in the larger one near the COOH terminus and determined the residual ability of the mutated elements to generate deletions in order to assign a role to this polypeptide chain. We show that in these elements, deletions still occur, although at a reduced level, but that in all cases examined so far the ends of the element are no longer recognized as the fixed endpoint of IS-mediated deletions, even though some other structural features of normal deletions formation are still present.     

The role of IS-elements of Yersinia pestis (Lehmann, Neumann) in the emergence of calcium-independent mutations

Calcium independent mutants of two Yersinia pestis strains were studied. Insertions of IS100 element at three different sites of plasmid pCad within calcium dependence region were detected in Y. pestis EV, as well as two extensive deletions covering the whole region. It was shown that IS100 carries no HindIII sites. Novel IS element of Y. pestis designated IS101 was discovered in strain 358, in addition to IS100. It is distinguished by a slightly smaller size, HindIII site presence and high specificity of integration.     

Transposition of IS2 into the hemB gene of Escherichia coli K-12.

Genetic studies of the hemB gene in Escherichia coli have resulted in the recovery of both stable and unstable mutant strains. The stable strains have been shown to result from large deletions. This study demonstrates that unstable strains result from the insertion of transposable element IS2 primarily into the 5' region of the structural gene; the instability results from precise excision of the element, producing strains with both high and low frequencies of reversion. This first report of IS2 insertion into hemB suggests that this gene may be a preferred target for insertion of this transposable element.     

Insertion sequences in the IS1111 family that target the attC recombination sites of integron-associated gene cassettes

Members of the recently identified IS 1111 family differ from the majority of insertion sequences (IS) in that they target specific sites in an orientation-specific manner. However, the way in which target selection is achieved is not known. ISKpn4 is representative of a new subgroup of the IS 1111 family whose members are found in the attC sites (59-be) of the gene cassettes associated with integrons. The transposases of this subgroup are closely related (over 75% identity), confirming that closely related IS usually share a common target. However, among more distant relatives encoding a transposase <45% identical to those of the ISKpn4 group, one IS, ISPa25, was found that also targets attC sites. It appears that the targeting determinant of the ISKpn4 group has become associated with a transposase gene from a different group, and this allowed us to localize the region that is likely to be required for target selection to a long noncoding region found downstream of the transposase gene in all IS 1111 family members. This region may determine an RNA used to guide the IS to its specific target.     

Insertion sequence-caused large-scale rearrangements in the genome of Escherichia coli

A majority of large-scale bacterial genome rearrangements involve mobile genetic elements such as insertion sequence (IS) elements. Here we report novel insertions and excisions of IS elements and recombination between homologous IS elements identified in a large collection of Escherichia coli mutation accumulation lines by analysis of whole genome shotgun sequencing data. Based on 857 identified events (758 IS insertions, 98 recombinations and 1 excision), we estimate that the rate of IS insertion is 3.5 × 10⁻⁴ insertions per genome per generation and the rate of IS homologous recombination is 4.5 × 10⁻⁵ recombinations per genome per generation. These events are mostly contributed by the IS elements IS1, IS2, IS5 and IS186. Spatial analysis of new insertions suggest that transposition is biased to proximal insertions, and the length spectrum of IS-caused deletions is largely explained by local hopping. For any of the ISs studied there is no region of the circular genome that is favored or disfavored for new insertions but there are notable hotspots for deletions. Some elements have preferences for non-coding sequence or for the beginning and end of coding regions, largely explained by target site motifs. Interestingly, transposition and deletion rates remain constant across the wild-type and 12 mutant E. coli lines, each deficient in a distinct DNA repair pathway. Finally, we characterized the target sites of four IS families, confirming previous results and characterizing a highly specific pattern at IS186 target-sites, 5′-GGGG(N6/N7)CCCC-3′. We also detected 48 long deletions not involving IS elements.     

A novel type of transposon generated by insertion element Is102 present in a psc101 derivative

We describe a novel type of transposon in the tetracycline resistance plasmid pYM103, a derivative of pSC101 carrying a single copy of an insertion element IS102. The new transposons we found were identified as DNA segments, approximately 6 kb (Tn1021) and 10 kb (Tn1022) in length, able to mediate the cointegration of pYM1O3 with plasmid Col E1. The resulting cointegrate contains either of these pYM1O3 segments duplicated in a direct orientation at the junctions of the parent plasmids. A direct duplication of a 9 bp sequence at the target site in Col E1 is found at the junctions for cointegration. Both transposons have IS1O2 at one end and also contain different lengths of the pYM103 DNA adjacent to IS102, including the tetracycline resistance gene. Each transposon contains terminal inverted repeats of a short nucleotide sequence. These results and the fact that IS102 can itself mediate plasmid cointegration, giving rise to a duplication of a 9 bp target sequence, indicate that IS102 is responsible for generation of Tn1021 and Tn1022. They are quite different from the common IS-associated transposons, which are always flanked by two copies of an IS element, and may be similar to transposons such as those of the Tn3 family and phage Mu.     

Instability in tyrR Strains of Plasmids Carrying the Tyrosine Operon: Isolation and Characterization of Plasmid Derivatives with Insertions or Deletions

The transformation of tyrR strains of Escherichia coli with multicopy plasmids which carry the tyrosine operon gave rise to modified plasmids with either insertions or deletions. The effect of each of these insertions or deletions was to decrease the level of expression of this operon. It is proposed that plasmid instability arose as a direct consequence of the metabolic effects of an overproduction of the enzymes coded for by the tyrosine operon. The results have significant implications for the cloning of genes that are repressed by the product of a regulatory gene. Since the predominant plasmid modification observed was the insertion of an IS1 element near the regulatory region of the tyrosine operon, the results also suggest a role for IS1 elements in the regulation of gene expression.     

Evidence for a role of translational frameshifting in the expression of transposition activity of the bacterial insertion element IS1

The bacterial insertion element IS1 contains two essential open reading frames, insA and insB, arranged in tandem. We have introduced a number of site-specific mutations into the region including the 3'-terminal region of insA, the region between insA and insB, and the ATG codon at the start of insB. Relative transposition activities of mutant and wild-type elements were determined using a modified in vivo cointegration assay. The results support the hypothesis that a translational (-1) frameshift occurring in the 3'-terminal region of insA and linking insB to insA is responsible for the synthesis of the active IS1 transposition enzyme. Further results with IS1 elements containing internal deletions are in agreement with a role of the normally terminated insA product as an inhibitor of transposition.     

Genomic changes arising in long-term stab cultures of Escherichia coli

Genomic scans of clones isolated from long-term stab cultures of Escherichia coli K-12 showed the loss of two large segments of the genome, with each lost segment being approximately 20 kb long. A detailed analysis of one of the deletions, located between 5.4 and 5.9 min, revealed that similar deletions had arisen in several other stab cultures. All deletions of this type exhibited a right terminus ending precisely at an IS5A element and a left terminus that varied over an approximately 5-kb range but was bordered in all but two cases by sequences belonging to the preferred consensus target sequence for IS5, YTAR. The ubiquity of such deletions in independent stab cultures and the increase in their frequency over time argue that they have a selective advantage. It is speculated that the loss of the crl locus is responsible for the selective advantage of the deletions.     

Large-Scale Genomic Analysis Suggests a Neutral Punctuated Dynamics of Transposable Elements in Bacterial Genomes

Insertion sequences (IS) are the simplest and most abundant form of transposable DNA found in bacterial genomes. When present in multiple copies, it is thought that they can promote genomic plasticity and genetic exchange, thus being a major force of evolutionary change. The main processes that determine IS content in genomes are, though, a matter of debate. In this work, we take advantage of the large amount of genomic data currently available and study the abundance distributions of 33 IS families in 1811 bacterial chromosomes. This allows us to test simple models of IS dynamics and estimate their key parameters by means of a maximum likelihood approach. We evaluate the roles played by duplication, lateral gene transfer, deletion and purifying selection. We find that the observed IS abundances are compatible with a neutral scenario where IS proliferation is controlled by deletions instead of purifying selection. Even if there may be some cases driven by selection, neutral behavior dominates over large evolutionary scales. According to this view, IS and hosts tend to coexist in a dynamic equilibrium state for most of the time. Our approach also allows for a detection of recent IS expansions, and supports the hypothesis that rapid expansions constitute transient events—punctuations—during which the state of coexistence of IS and host becomes perturbated.     

Physical Analysis of Tn10- and Is10-Promoted Transpositions and Rearrangements

We have investigated by Southern blot hybridization the rate of IS10 transposition and other Tn10/IS10-promoted rearrangements in Escherichia coli and Salmonella strains bearing single chromosomal insertions of Tn10 or a related Tn10 derivative. We present evidence for three primary conclusions. First, the rate of IS10 transposition is approximately 10(-4) per cell per bacterial generation when overnight cultures are grown and plated on minimal media and is at least ten times more frequent than any other Tn10/IS10-promoted DNA alteration. Second, all of the chromosomal rearrangements observed can be accounted for by two previously characterized Tn10-promoted rearrangements: deletion/inversions and deletions. Together these rearrangements occur at about 10% the rate of IS10 transposition. Third, the data suggest that intramolecular Tn10-promoted rearrangements preferentially use nearby target sites, while the target sites for IS10 transposition events are scattered randomly around the chromosome.     

A comparison of intramolecular rearrangements promoted by transposons Tn5 and Tn10.

The bacterial transposon Tn10 has previously been shown to move to other genomic sites by a conservative mechanism, whereby the transposon is excised by double-strand breaks and inserted between a pair of staggered nicks at the target. Other transposons, like Tn3, have been shown to transpose by a replicative mechanism that involves symmetrical nicking of the element and formation of the 'Shapiro intermediate', which can mature into either a cointegrate or a simple insert. The situation with respect to Tn5 is unclear; it was originally reported to use a conservative mechanism, but other evidence suggests that the mechanism might be replicative. In this paper, rearrangements of adjacent DNA promoted by Tn10 and Tn5 have been compared using positive selection for galactose-resistance to detect such rearrangements. Tn10 promoted the formation of adjacent deletions (that started from an inside end of Tn10), deletion/inversions and simple IS10 insertions, but no cointegrates. This behaviour is fully consistent with a conservative mechanism. In contrast, Tn5 was found to promote formation of adjacent deletions (that started mainly from an outside end of Tn5), IS50 insertions (that were frequently accompanied by inversions of adjacent DNA) and cointegrates. These characteristics seem compatible with a replicative, rather than a conservative, mode of transposition. Clearly, Tn5 and Tn10 exhibit some significant differences in their transposition. These results, and results of some previous experiments, have been interpreted to mean that Tn5 could use a replicative mechanism for its transposition.     

Bidirectional deletions associated with IS4

Summary A new type of chromosomal rearrangements associated with a transposable element has been described for IS4. These rearrangements are deletions, in which the transposable element IS4 and DNA adjacent to it on either side is lost. These deletions are at least ten times more frequent in the presence of IS4, than when the element is absent from that region. The formation of bidirectional deletions is more than 1,000 times more frequent than precise excision of IS4.     

Chromosomal rearrangements by an IS2 insertion in phage Mu-1

We have isolated and characterized a mutant of temperate phage Mu-1 carrying an IS2 insertion in the middle of its β region. This mutant gives rise spontaneously to secondary mutants which have deletions of different sizes adjacent to IS2. One particular derivative however, was found to have acquired an additional insertion sequence adjacent to IS2. This derivative gave rise to tertiary mutants carryinh a deletion next to the tandem insertion. The tandem insertion was located at the same place in the Mu β region as another 2.6 kb insertion independently isolated by Chow et al. (1977) and was found to be homologous to that insertion. The properties of this particular secondary mutant show that Mu phage particles lacking their S end are defective for growth and lysogenisation.     

A simple and rapid procedure for sequencing long (40-kb) DNA fragments

A simple procedure has been developed for sequencing long (40-kb) DNA fragments by the dideoxy method. The fragment is cloned in the sequencing cosmid pAA113X by in vitro packaging, and subdivided by a series of overlapping IS1-promoted deletions. The deletions are isolated by positive selection for galactose resistance. Plasmids from several thousand galactose-resistant colonies are fractionated on an agarose gel, and DNA from each fraction is restricted with enzymes (such as SphI and SalI) to shorten each deletion from the opposite end. As a result, a series of short overlapping segments, spread across the entire length of the fragment, are fused to IS1. The plasmids are extracted by a rapid method, arranged according to size, and used for supercoil sequencing with an IS1 primer. Sequences of IS1-promoted deletions contain extensive overlaps that are connected further by restriction enzyme-generated deletions to give the complete 40-kb sequence.