Escherichia coli insertion sequence IS150: transposition via circular and linear intermediates

IS150, a member of the widespread IS3 family, contains two consecutive out-of-phase open reading frames, orfA and orfB, that partially overlap. These open reading frames encode three proteins, InsA, InsB, and the InsAB protein, which is jointly encoded by both open reading frames by means of programmed translational frameshifting. We demonstrate that the InsAB protein represents the IS150 element's transposase. In vivo, the wild-type IS150 element generates circular excision products and linear IS150 molecules. Circular and linear species have previously been detected with mutant derivatives of other members of the IS3 family. Our finding supports the assumption that these products represent true transposition intermediates of members of this family. Analysis of the molecular nature of these two species suggested that the circular forms are precursors of the linear molecules. Elimination of InsA synthesis within the otherwise intact element led to accumulation of large amounts of the linear species, indicating that the primary role of InsA may be to prevent abortive production of the linear species and to couple generation of these species to productive insertion events.     

Immunity to repeated transposition of the insertion sequence IS21

The ability of pBR325 derivatives carrying a copy of IS21-element to accept the second copy of this element from plasmid pRP19.6, a temperature-sensitive for replication mutant of RPI containing the duplicated IS21 was studied. It was shown that the frequency of IS21 transposition into plasmids pBR32S::IS21 differing by localization IS21 was lower by two orders of magnitude as compared to that of pBR325. The restriction endonuclease analysis revealed that the insertion of the second copy of IS21 resulted in the formation of pBR325 derivatives carrying the tandem repeated copies of IS21. It was also shown that the plasmids pBR325::IS21 were capable of increasing the frequency of pRP19.6 insertion into the bacterial chromosome from 3-9 to 200-300 times depending on IS21 localization. On the basis of the results obtained and literature data the possible mechanism of the transposition immunity is discussed.     

Terminal inverted repeats of insertion sequence IS30 serve as targets for transposition.

In the present study, we demonstrate that the terminal inverted repeats of the Escherichia coli insertion sequence IS30 are functional target sites for the transposition of the (IS30)2 dimer, which represents an intermediate structure in the transposition of IS30. Comparative analysis of various target regions revealed that the left and right ends differ in their "attractivity." In our experiments, the joined left and right ends, i.e., the (IS30)2 intermediate structure, was found to be the most preferred target. It was also shown that flanking sequences can influence the target activity of the terminal repeats. The functional part of the target region was localized in the inverted repeats by means of mutational analysis, and it corresponds to the binding site of IS30 transposase. Insertion of 1 bp into the right inverted repeat resulted in unusual target duplication accompanied by gene conversion. The choice of the terminal inverted repeats as targets in transposition leads to the reconstruction of the (IS30)2 structure, which may induce a cascade of further rearrangements. Therefore, this process can play a role in the evolution of the genome.     

Intramolecular transposition of insertion sequence IS91 results in second-site simple insertions

A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition.     

Two frameshift products involved in the transposition of bacterial insertion sequence IS629

IS629 is 1,310 bp in length with a pair of 25-bp imperfect inverted repeats at its termini. Two partially overlapping open reading frames, orfA and orfB, are present in IS629, and two putative translational frameshift signals, TTTTG (T4G) and AAAAT (A4T), are located near the 3'-end of orfA. With the lacZ gene as the reporter, both T4G and A4T motifs are determined to be a -1 frameshift signal. Two peptides representing the two transframe products designated OrfAB' and OrfAB, are identified by a liquid chromatography-tandem mass spectrometric approach. Results of transposition assays show that OrfAB' is the transposase and that OrfAB aids in the transposition of IS629. Pulse-chase experiments and Escherichia coli two-hybrid assays demonstrate that OrfAB binds to and stabilizes OrfAB', thus increasing the transposition activity of IS629. This is the first transposable element in the IS3 family shown to have two functional frameshifted products involved in transposition and to use a transframe product to regulate transposition.     

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.     

High-frequency transposition of IS1373, the insertion sequence delimiting the amplifiable element AUD2 of Streptomyces lividans.

IS1373 is the putative insertion sequence delimiting the amplifiable unit AUD2 of Streptomyces lividans. Two IS1373-derived thiostrepton-resistant transposons, Tn5492 and Tn5494, transposed into multiple sites of the S. lividans chromosome at frequencies as high as 0.4 and 1%, respectively. Hence, IS1373 is a functional insertion sequence and its unique open reading frame, insA, encodes the transposase.     

Identification of IS 1206, a Corynebacterium glutamicum IS3-related insertion sequence and phylogenetic analysis

Integration of plasmid pCGL320 into a Corynebacterium glutamicum ATCC21086 derivative led to tandem amplification of the inserted plasmid (Labarre et a/., 1993). One amplification event was associated with integration of an insertion sequence that we have named IS 1206. Hybridizing sequences were only found in C. glutamicum strains and at various copy numbers. IS1206 is 1290bp long, carries 32 bp imperfect inverted repeats and generates a 3bp duplication of the target DNA upon insertion. IS1206 presents the features characteristic of the IS3 family and part of the DNA sequence centering on the putative transposase region (orfB) is similar to those of IS3 and some other related elements. Phylogenetic analysis of orfB deduced protein sequences from IS 1206 and IS3-related elements contradicts the phylogeny of the species, suggesting that evolution of these elements might be complex. Horizontal transfer could be invoked but other alternatives like ancestral polymorphism or/and different rates of evolution could also be involved.     

Cloning of insertion sequence IS1485 from Enterococcus species.

We have cloned and identified an insertion sequence, IS1485, that was present in several members of the genus Enterococcus. IS1485 exists in varying copy numbers with at least 12 copies in E. durans (ATCC 11576), 3 copies in E. faecium (ATCC 19434), and one copy each in E. faecalis (ATCC 19433) and E. avium (ATCC 14024). It was also detected in clinical strains of E. gallinarum, E. casseliflavus, and E. saccharolyticus. IS1485 is 1366 bp in length, it has imperfect terminal inverted repeats with 25 of the terminal 39 residues matched, and it contains three open reading frames exceeding 50 codons, designated orfA, orfB, and orfC. The largest, orfB, was located 36 bp downstream and in the -1 reading frame relative to orfA; orfC is oriented in the opposite direction and overlaps orfA. The genetic organization of IS1485 resembles that of members of the IS3 family of transposable elements. Sequence homology exists with several members of the IS3 family especially with IS199 from Streptococcus mutans.     

Stimulation of transposition of the Mycobacterium tuberculosis insertion sequence IS6110 by exposure to a microaerobic environment

The Mycobacterium tuberculosis-specific insertion sequence IS6110/986 has been widely used as a probe because of the multiple polymorphism observed among different strains. To investigate transposition of IS6110, a series of artificially constructed composite transposons containing IS6110 and a kanamycin resistance marker were constructed. The composite transposons were inserted into a conditionally replicating, thermosensitive, Escherichia coli-mycobacterial shuttle vector and introduced into M. smegmatis mc2155. Lawns of transformants were grown at the permissive temperature on kanamycin-supplemented agar and subsequently prevented from further growth by shifting to the non-permissive temperature. Under normal atmospheric conditions, kanamycin-resistant papillae appeared after only about 5-6 weeks of incubation. However, these events were not associated with transposon mobilization. In contrast, lawns that were exposed to a 48 h microaerobic shock generated kanamycin-resistant papillae after only 6-14 days. These events were generated by conservative transposition of the IS6110 composite transposon into the M. smegmatis chromosome, with loss of the shuttle vector. In common with other IS3 family elements, transposition of IS6110 is thought to be controlled by translational frameshifting. However, we were unable to detect any significant frameshifting within the putative frameshifting site of IS6110, and the level of frameshifting was not affected by microaerobic incubation. The finding that transposition of IS6110 is stimulated by incubation at reduced oxygen tensions may be relevant to transposition of IS6110 in M. tuberculosis harboured within TB lesions.     

IS200: a Salmonella-specific insertion sequence

A new IS element (IS200) has been identified in Salmonella. The sequence was identified as an IS element by the following criteria: its insertion caused the mutation hisD984; six copies of the sequence are present in strain LT2 of S. typhimurium; and transposition of the sequence has been observed on several occasions. IS200 is found in almost all Salmonella species examined but is absent from most other enteric bacteria. The specificity of this element for Salmonella (and the absence of IS1-IS4 from Salmonella) suggest that transfer of insertion sequences between bacterial groups may be less extensive than is commonly believed. Alternatively, the distribution may suggest that these elements play a selectively important role in bacteria.     

Transposase-induced excision and circularization of the bacterial insertion sequence IS911.

We have investigated the role of three IS911-specified proteins in transposition in vivo: the products of the upstream (OrfA) and downstream (OrfB) open reading frames, and a transframe protein (OrfAB) produced by -1 translational frameshifting between orfA and orfB. The production of OrfAB alone is shown to lead both to excision and to circularization of the element and to be sufficient for intermolecular transposition into a plasmid target. Simultaneous and independent production of OrfA is shown to stimulate OrfAB-mediated intermolecular transposition while greatly reducing the appearance of transposon circles. We have not been able to detect a role for OrfB. Although under certain conditions, the vector plasmid undergoes precise resealing after IS911 excision, the data suggest that this is not normally the case and that the donor plasmid is not generally conserved. The use of IS911 derivatives carrying mutations in the terminal 2 bp suggested that circle formation represents a site-specific intramolecular transposition event. We present a model which explains both intra- and intermolecular transposition events in terms of a single reaction mechanism of the 'cut and paste' type.     

-1 frameshifting at a CGA AAG hexanucleotide site is required for transposition of insertion sequence IS1222

The discovery of programmed -1 frameshifting at the hexanucleotide shift site CGA_AAG, in addition to the classical X_XXY_YYZ heptanucleotide shift sequences, prompted a search for instances among eubacterial insertion sequence elements. IS1222 has a CGA_AAG shift site. A genetic analysis revealed that frameshifting at this site is required for transposition.     

Nucleotide sequence and structural organization of Yersinia pestis insertion sequence IS100

Abstract The L1 major protein of human papillomavirus type 16 was expressed in Sf-21 insect cells with a recombinant baculovirus vector. Virus-like particles similar in appearance to empty verions were identified by electron microscoy at densities of 1.29–1.30. Purified particles reacted with monoclonal anti-HPV-16-L1 antibody in Western blot and immuno dot blot suggesting that conformational epitopes are present in the recombinant particles. Immunodot blot assays using human sera correlated with the detection of HPV-16 DNA by the polymerase chain reaction. The results suggest that HPV-16-L1 virions produced by the baculovirus system might be useful for developing serologic tests to measure antibodies to conformational epitopes and may offer potential for vaccine development.     

Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family

A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).     

Complete nucleotide sequence of insertion element IS4351 from Bacteroides fragilis.

The nucleotide sequence and genetic analyses of one of the directly repeated sequences flanking the macrolide-lincosamide-streptogramin B drug resistance determinant, ermF, from the Bacteroides fragilis R plasmid, pBF4, suggested that this region is an insertion sequence (IS) element. This 1,155-base-pair element contained partially matched (20 of 25 base pairs) terminal-inverted repeats, overlapping, anti-parallel open reading frames, and nine promoterlike sequences, including three that were oriented outward. Analysis of this sequence revealed no significant nucleotide homology to 13 other known IS elements. Inasmuch as Southern blot hybridization analysis detected homologous sequences in chromosomal DNA and its G+C content (42 mol%) was similar to that of B. fragilis, the data suggested that this element is of Bacteroides origin. Transposition promoted by this element was demonstrated in recA E. coli. Recombinants were recovered by selecting for the activation of a promoterless chloramphenicol resistance gene on the plasmid pDH5110 and were characterized by restriction endonuclease mapping and Southern blot hybridization. We propose that this IS element be designated IS4351.