Differential roles of the transposon termini in IS91 transposition

Insertion sequence 91 (IS91) inserts specifically at GTTC or CTTG target sequences without duplication of the target. After insertion, the right inverted repeat (IRR) lies adjacent to the 3' end of the target sequences (or 5' to the complementary sequence CAAG or GAAC). We have analyzed the effects of alteration of each terminus of IS91 on transposition activity in Escherichia coli. IRR is absolutely required for transposition. Deletion analysis indicates that a 14-bp segment is not sufficient, but an 81-bp sequence within the IRR region is sufficient. Furthermore, the GTTC/CTTG target site is also required. The left inverted repeat (IRL) of IS91 is dispensable. Plasmid fusions originated by one-ended transposition of IS91 derivatives lacking IRL occur at about the same frequency as cointegrate formation observed for the wild-type element. In the one-ended-type fusions, the inserted fragment of donor DNA is flanked at one end (constant end) by IRR and at the other end by a GTTC or CTTG sequence present in the donor (variable end) in a way that usually results in multiple tandem insertions of the donor plasmid in the target site. These results are easily accommodated by a rolling-circle replicative transposition mechanism. This model also draws support from the finding that the IS91 transposase is related in sequence to the superfamily of rolling-circle replication proteins and the observation that IRR shows some conservation in sequence and secondary structure with the origins of replication of some rolling-circle replication plasmids.     

Negative and positive regulation of Tn10/IS10-promoted recombination by IHF: two distinguishable processes inhibit transposition off of multicopy plasmid replicons and activate chromosomal events that favor evolution of new transposons

Tn10 is a composite transposon; inverted repeats of insertion sequence IS10 flank a tetracycline-resistance determinant. Previous work has identified several regulatory processes that modulate the interaction between Tn10 and its host. Among these, host-specified DNA adenine methylation, an IS10-encoded antisense RNA and preferential cis action of transposase are particularly important. We now find that the accessory host protein IHF and the sequences that encode the IHF-binding site in IS10 are also important regulators of the Tn10 transposition reaction in vivo and that these determinants are involved in two distinguishable regulatory processes. First, IHF and the IHF-binding site of IS10, together with other host components (e.g., HU), negatively regulate the normal intermolecular transposition process. Such negative regulation is prominent only for elements present on multicopy plasmid replicons. This multicopy plasmid-specific regulation involves effects both on the transposition reaction per se and on transposase gene expression. Second, specific interaction of IHF with its binding site stimulates transposon-promoted chromosome rearrangements but not transposition of a short Tn10-length chromosomal element. However, additional considerations predict that IHF action should favor chromosomal transposition for very long composite elements. On the basis of these and other observations we propose that, for chromosomal events, the major role of IHF is to promote the evolution of new IS10-based composite transposons.     

A selective medium for the rapid detection by an impedance technique of Pseudomonas spp. associated with poultry meat

IS511 is an endogenous insertion sequence (IS) of the bacterium Caulobacter crescentus strain CB15 and it is the first Caulobacter IS to be characterized at the molecular level. We determined the 1266-bp nucleotide sequence of IS511 and investigated its genetic organization, relationship to other ISs, and transposition properties. IS511 belongs to a distinct branch of the IS3 family that includes ISR1, IS476, and IS1222, based on nucleotide sequence similarity. The nucleotide sequence of IS511 encodes open reading frames (orfs) designated here as orfA and orfB, and their relative organization and amino acid sequences of the predicted protein products are very similar to those of orfAs and orfBs of other IS3 family members. Nuclease S1 protection assays identified an IS511 RNA, and its 5' end maps approximately 16 nucleotides upstream of orfA and about six nucleotides downstream of a sequence that is similar to the consensus sequence of C. crescentus housekeeping promoters. Evidence is presented that IS511 is capable of precise excision from the chromosome, and transposition from the chromosome to a plasmid. Transpositional insertions of IS511 occurred within sequences with a relatively high G + C content, and they were usually, but not always, flanked by a 4-bp direct repeat that matches a sequence at the site of insertion. We also determined the nucleotide sequence flanking the four endogenous IS511 elements that reside in the chromosome of C. crescentus. Our findings demonstrate that IS511 is a transposable IS that belongs to a branch of the IS3 family.     

Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate

The Clostridium perfringens enterotoxin gene is on a transposon-like element, Tn5565, integrated in the chromosome in human food poisoning strains. The flanking IS elements, IS1470 A and B, are related to IS30. The IS element found in the transposon, IS1469, is related to IS200 and has been found upstream of cpe in all Type A strains. PCR and sequencing studies from cell extracts and plasmid isolations of C. perfringens indicate that Tn5565 can form a circular form with the tandem repeat (IS1470)2, similar to the transposition intermediates described for a number of IS elements.     

Target choice and orientation preference of the insertion sequence IS903

We have examined the targeting preference of the bacterial insertion element IS903 by determining the sites of insertion of a large number of transposition events into the 55-kb conjugative plasmid pOX38. Despite the large target size, all the insertions were clustered in four small distinct regions associated with conjugal DNA transfer. Within these regions, many different sites were used for insertion; however, there were a few sites that IS903 inserted into more than once. Alignment of the insertion sites showed that there was no consensus sequence within the 9-bp target duplication but that there were preferred sequences located symmetrically on either side of the target. This is consistent with target recognition by a dimer or multimer of transposase, with either sequence-specific or structure-specific interactions on both sides of the target. We show further that when one of these preferred regions was cloned into a second conjugative plasmid, pUB307, it was still a preferred target, implying that all the sequences necessary for target selection are contained within this DNA segment. Also, we observed a very strong preference for insertion in a single orientation in pUB307. We examined the possibility that either DNA replication from the origin of vegetative replication, oriV, or the origin of transfer, oriT, might determine this orientation effect. We find that reversing the direction of vegetative replication had no effect on the orientation of transposon insertions; however, reversing the direction of DNA transfer abolished the orientation effect. This supports the idea that conjugal DNA transfer imparts a polarity on the target that is sensed by the transposon.     

Generalized transduction for genetic linkage analysis and transfer of transposon insertions in different Staphylococcus epidermidis strains

Staphylococcus epidermidis phage 48 was used to efficiently transduce plasmid pTV1ts and a chromosomal Tn917 insertion M27 from S. epidermidis 13-1 to biofilm-producing clinical S. epidermidis isolates 1457, 9142, and 8400. The Tn917 insertion leading to the biofilm-negative phenotype of transposon mutant M10 was sequentially transduced to biofilm-producing S. epidermidis 1457 using S. epidermidis phage 48 and then, using the resulting biofilm-negative transductant 1457-M10 as a donor, into several unrelated biofilm-producing clinical S. epidermidis isolates using S. epidermidis phage 71. All resultant transductants displayed a completely biofilm-negative phenotype. In addition, S. epidermidis phage 71 was adapted to S. epidermidis 1457 and 8400, which allowed generalized transduction of transposon insertions in these wild-type strains. As Tn917 predominantly transposed into endogenous plasmids of all three strains used, an efficient system for chromosomal transposon mutagenesis was established by curing of S. epidermidis 1457 of a single endogenous plasmid p1457 by sodium dodecylsulfate treatment. After transduction of the resulting derivative, S. epidermidis 1457c with pTV1ts, insertion of transposon Tn917 to different sites of the chromosome of S. epidermidis 1457c was observed. Biofilm-producing S. epidermidis 1457c x pTV1ts was used to isolate a biofilm-negative transposon mutant (1457c-M3) with a chromosomal insertion apparently different from two previously isolated isogenic biofilm-negative transposon mutants, M10 and M11 (Mack, D., M. Nedelmann, A. Krokotsch, A. Schwarzkopf, J. Heesemann, and R. Laufs: Infect Immun 62 [1994] 3244-3253). S. epidermidis phage 71 was used to prove genetic linkage between transposon insertion and altered phenotype by generalized transduction. In combination with phage transduction, 1457c x pTV1ts will be a useful tool facilitating the study of bacterial determinants of the pathogenicity of S. epidermidis.     

The Himar1 mariner transposase cloned in a recombinant adenovirus vector is functional in mammalian cells

Mariner transposons belong to the mariner /Tc1 superfamily of class II, DNA-mediated elements. One of these transposons, Himar1 , isolated from the horn fly, is independent of host-specific factors that would limit transfer between different species, making it an ideal candidate for gene transfer technology development. To determine the activity of Himar1 transposase in mammalian cells, we introduced the Himar1 transposase gene into an adenovirus (Ad) vector under control of the phage T7 RNA polymerase promoter. Mammalian cells infected with the Ad vector carrying the Himar1 gene efficiently expressed the Himar1 transposase in the presence of T7 polymerase. In in vitro inter-plasmid transposition reactions, Himar1 transposase expressed by the Ad vector mediated precise cut-and-paste transposition and resulted in a characteristic duplication of TA at the integration site of the target plasmid. Further studies showed that this transposase was capable of catalyzing transposition between twoplasmids co-transfected into 293T7pol cells, which express T7 RNA polymerase. Combining the integration capability of mariner transposons with the transduction efficiency of Ad vectors is expected to provide a powerful tool for introducing transgenes into the host chromosome.     

Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria

A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin-resistant strain CA11 of Erwinia amylovora, was identified by its ability to move from pEa34 to different sites in plasmids pGEM3Zf(+) and pUCD800. Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 bp with 81-bp terminal inverted repeats and generates 5-bp duplications of the target DNA following insertion. Tn5393 contains open reading frames that encode a putative transposase (tnpA) and resolvase (tnpR) of 961 and 181 amino acids, respectively. The two open reading frames are separated by a putative recombination site (res) consisting of 194 bp. Two streptomycin resistance genes, strA and strB, were identified on the basis of their DNA sequence homology to streptomycin resistance genes in plasmid RSF1010. StrA is separated from tnpR by a 1.2-kb insertion element designated IS1133. The tnpA-res-tnpR region of Tn5393 was detected in Pseudomonas syringae pv. papulans Psp36 and in many other gram-negative bacteria harboring strA and strB. Except for some strains of Erwinia herbicola, these other gram-negative bacteria lacked insertion sequence IS1133. The prevalence of strA and strB could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria.     

Interaction of RecBCD enzyme with DNA at double-strand breaks produced in UV-irradiated Escherichia coli: requirement for DNA end processing

The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m2). The activity decrease was seen by an increase in survival of phage T4 2(-) of about 200-fold (phage T4 2(-) has defective duplex DNA end-protecting gene 2 protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with ssb+) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in xonA, recJ, and sbcCD, particularly in the recJ sbcCD and xonA recJ sbcCD combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.     

Organisation of the tmb catabolic operons of Pseudomonas putida TMB and evolutionary relationship with the xyl operons of the TOL plasmid pWW0

In Pseudomonas putida (Pp) TMB the genes involved in the catabolism of methyl-substituted aromatic hydrocarbons 1,2,4-trimethylbenzene, m- and p-xylene (tmb operon), are functionally and genetically homologous to the xyl genes of the plasmid pWW0, but are chromosomally encoded. We have analysed by cloning. Southern blotting and sequencing of selected regions the organisation of the tmb cluster. This analysis shows that the structural and regulatory genes of the tmb and xyl systems exhibit a high degree of homology and are similarly organised in operons. However the operons are differently arranged on the Pp TMB chromosome and on the pWW0 plasmid. Comparison of the two systems suggests that the operon arrangement found in pWW0 may have originated from that found in Pp TMB via cointegration mediated by replicative transposition or by intermolecular recombination between two copies of the insertion element IS1246.     

Delayed development of hypertension after short-term nitrendipine treatment

In its natural host, Bacillus thuringiensis, the insertion sequence IS231A is preferentially inserted into the terminal inverted repeats of the transposon Tn4430. Using a novel transposition assay, we demonstrate that the Tn4430 ends behave as insertion hot spots for IS231A in Escherichia coli. Sequence analysis reveals that IS231A insertion sites match the 5'-GGG(N)5CCC-3' consensus. However, this consensus is not the only determinant of IS231A insertion specificity. Although both Tn4430 ends have identical sequences, one is strongly preferred to the other and the orientation of insertion into this end is not random. We demonstrate that this preference is determined by the flanking regions of the site. These regions display a conserved periodic organization of their sequence which, by conferring anisotropic flexibility, would induce the DNA to bend in a roughly 'S'-shaped structure centered on the target consensus. DNA conformation analysis by polyacrylamide gel electrophoresis indeed shows that the preferred target site of IS231A is flanked by DNA segments curved in opposite directions. We present a model in which DNA bendability and curvature would contribute to the positioning of IS231A transposase on the target DNA.     

Switching from cut-and-paste to replicative Tn7 transposition

The bacterial transposon Tn7 usually moves through a cut-and-paste mechanism whereby the transposon is excised from a donor site and joined to a target site to form a simple insertion. The transposon was converted to a replicative element that generated plasmid fusions in vitro and cointegrate products in vivo. This switch was a consequence of the separation of 5'- and 3'-end processing reactions of Tn7 transposition as demonstrated by the consequences of a single amino acid alteration in an element-encoded protein essential for normal cut-and-paste transposition. The mutation specifically blocked cleavage of the 5' strand at each transposon end without disturbing the breakage and joining on the 3' strand, producing a fusion (the Shapiro Intermediate) that resulted in replicative transposition. The ability of Tn7 recombination products to serve as substrates for both the limited gap repair required to complete cut-and-paste transposition and the extensive DNA replication involved in cointegrate formation suggests a remarkable plasticity in Tn7's recruitment of host repair and replication functions.     

Structural and functional characterization of IS1358 from Vibrio cholerae

The new epidemic serovar O139 of Vibrio cholerae has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374-10378, 1995) and designated IS1358O139. We studied the distribution of the IS1358 element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS1358 was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS1358 from V. cholerae O22 and one copy from V. cholerae O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS1358 to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an Escherichia coli recA donor at a frequency of 1.2 x 10(-8). Sequence analysis revealed that IS1358 duplicates 10 bp at its insertion site.     

Assembly of a strong promoter following IS911 circularization and the role of circles in transposition

When supplied with high levels of the IS911-encoded transposase, IS911-based transposons can excise as circles in which the right and left terminal inverted repeats are abutted. Formation of the circle junction is shown here to create a promoter, p(junc), which is significantly stronger than the indigenous promoter, pIRL, and is also capable of driving expression of the IS911 transposition proteins. High transposase expression from the circular transposon may promote use of the circle as an integration substrate. The results demonstrate that IS911 circles are highly efficient substrates for insertion into a target molecule in vivo. Insertion leads to the disassembly of p(junc) and thus to a lower level of synthesis of the transposition proteins. The observation that normal levels of IS911 transposition proteins supplied by wild-type copies of IS911 are also capable of generating transposon circles, albeit at a low level, reinforces the idea that the transposon circles might form part of the natural transposition cycle of IS911. These observations form the elements of a feedback control mechanism and have been incorporated into a model describing one possible pathway of IS911 transposition.     

An analysis of a class of DNA sequence reading molecules

The bacterial insertion sequence IS21 contains two genes, istA and istB, which are organized as an operon. IS21 spontaneously forms tandem repeats designated (IS21)2. Plasmids carrying (IS21)2 react efficiently with other replicons, producing cointegrates via a cut-and-paste mechanism. Here we show that transposition of a single IS21 element (simple insertion) and cointegrate formation involving (IS21)2 result from two distinct non-replicative pathways, which are essentially due to two differentiated IstA proteins, transposase and cointegrase. In Escherichia coli, transposase was characterized as the full-length, 46 kDa product of the istA gene, whereas the 45 kDa cointegrase was expressed, in-frame, from a natural internal translation start of istA. The istB gene, which could be experimentally disconnected from istA, provided a helper protein that strongly stimulated the transposase and cointegrase-driven reactions. Site-directed mutagenesis was used to express either cointegrase or transposase from the istA gene. Cointegrase promoted replicon fusion at high frequencies by acting on IS21 ends which were linked by 2, 3, or 4 bp junction sequences in (IS21)2. By contrast, cointegrase poorly catalyzed simple insertion of IS21 elements. Transposase had intermediate, uniform activity in both pathways. The ability of transposase to synapse two widely spaced IS21 ends may reside in the eight N-terminal amino acid residues which are absent from cointegrase. Given the 2 or 3 bp spacing in naturally occurring IS21 tandems and the specialization of cointegrase, the fulminant spread of IS21 via cointegration can now be understood.     

Simple and efficient generation in vitro of nested deletions and inversions: Tn5 intramolecular transposition

We have exploited the intramolecular transposition preference of the Tn 5 in vitro transposition system to test its effectiveness as a tool for generation of nested families of deletions and inversions. A synthetic transposon was constructed containing an ori, an ampicillin resistance (Ampr) gene, a multi-cloning site (MCS) and two hyperactive end sequences. The donor DNA that adjoins the transposon contains a kanamycin resistance (Kanr) gene. Any Amprreplicating plasmid that has undergone a transposition event (Kans) will be targeted primarily to any insert in the MCS. Two different size targets were tested in the in vitro system. Synthetic transposon plasmids containing either target were incubated in the presence of purified transposase (Tnp) protein and transformed. Transposition frequencies (Ampr/Kans) for both targets were found to be 30-50%, of which >95% occur within the target sequence, in an apparently random manner. By a conservative estimate 10(5) or more deletions/inversions within a given segment of DNA can be expected from a single one-step 20 microl transposition reaction. These nested deletions can be used for structure-function analysis of proteins and for sequence analysis. The inversions provide nested sequencing templates of the opposite strand from the deletions.     

Hematuria and proteinuria as predictors of Schistosoma haematobium infection

The inducible tetracycline resistance determinant isolated from Proteus mirabilis cloned into the plasmid pACYC177 was mutagenized by insertion of a mini-Mu-lac phage in order to define the regions in the cloned sequences encoding the structural and regulatory proteins. Three different types of mutants were obtained: one lost the resistance phenotype and became Lac+; another expressed the resistance at lower levels and constitutively; the third was still dependent on induction but showed a lower minimal inhibitory concentration. The mutant phenotypes and the locations of the insertions indicate that the determinant is composed of a repressor gene and a structural gene which are not transcribed divergently as are other known tetracycline determinants isolated from Gram-negative bacteria.