Complete sequence of an IS element present in pSC101

Recently a new insertion element (IS102)b ha been described in plasmid pSC101. We have determined its complete sequence: it consists of 1057 bp; 338 bp at one end are identical to those already determined for the kanamycin resistance transposon Tn903. It is not flanked by any direct repeat. Its coding capabilities are discussed, and compared to those of IS903.     

F factor mobilization of non-conjugative chimeric plasmids in Escherichia coli: General mechanisms and a role for site-specific recA-independent recombination at oriV1

Plasmid pSC101 is neither self-transmissible nor efficiently mobilized (made to transfer) by the Escherichia coli F factor. When fragments of F factor DNA were inserted into pSC101 the resulting chimeric plasmids were mobilized by the F factor at enhanced frequencies. These chimeric plasmids, which were not self-transmissible, fell into three classes according to their relative ability to be mobilized by an autonomous or integrated F factor: (1) class I pSC101-F chimeric plasmids contain the origin of transfer of the F factor (oriT) and were mobilized in trans at an efficiency nearly equal to that of F factor transfer; (2) class II pSC101-F chimeric plasmids lacked both oriT and the origin of vegetative F replication (oriV1), and were mobilized in cis via fusion with the F factor in a recA-dependent recombination to yield a transferable co-integrated plasmid; (3) class III pSC101-F chimeric plasmids lacked oriT but contained oriV1 and were mobilized in cis via co-integration with the F factor probably at oriV1 in a recA-independent recombination. A fourth class of mobilization event, not exhibited by pSC101-F chimeric plasmids, was also observed. Mobilization of pBR322 and pSC101 occurred in cis via transposon-mediated recA-independent fusion with F. On the basis of these results we present a general classification scheme of non-conjugative plasmids and also suggest mechanisms for their mobilization.     

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.     

Stem Loop Sequences Specific to Transposable Element IS605 Are Found Linked to Lipoprotein Genes in Borrelia Plasmids

Background

Plasmids of Borrelia species are dynamic structures that contain a large number of repetitive genes, gene fragments, and gene fusions. In addition, the transposable element IS605/200 family, as well as degenerate forms of this IS element, are prevalent. In Helicobacter pylori, flanking regions of the IS605 transposase gene contain sequences that fold into identical small stem loops. These function in transposition at the single-stranded DNA level.

Methodology/Principal Findings

In work reported here, bioinformatics techniques were used to scan Borrelia plasmid genomes for IS605 transposable element specific stem loop sequences. Two variant stem loop motifs are found in the left and right flanking regions of the transposase gene. Both motifs appear to have dispersed in plasmid genomes and are found “free-standing” and phylogenetically conserved without the associated IS605 transposase gene or the adjacent flanking sequence. Importantly, IS605 specific stem loop sequences are also found at the 3′ ends of lipoprotein genes (PFam12 and PFam60), however the left and right sequences appear to develop their own evolutionary patterns. The lipoprotein gene-linked left stem loop sequences maintain the IS605 stem loop motif in orthologs but only at the RNA level. These show mutations whereby variants fold into phylogenetically conserved RNA-type stem loops that contain the wobble non-Watson-Crick G-U base-pairing. The right flanking sequence is associated with the family lipoprotein-1 genes. A comparison of homologs shows that the IS605 stem loop motif rapidly dissipates, but a more elaborate secondary structure appears to develop in its place.

Conclusions/Significance

Stem loop sequences specific to the transposable element IS605 are present in plasmid regions devoid of a transposase gene and significantly, are found linked to lipoprotein genes in Borrelia plasmids. These sequences are evolutionarily conserved and/or structurally developed in an RNA format. The findings show that IS605 stem loop sequences are multifaceted and are selectively conserved during evolution when the transposable element dissipates.     

Site-specific deletions in the recombinant plasmid pSC101 containing the redB-ori region of phage lambda

Large deletions occur in the hybrid plasmid formed by pSC101 and the EcoRI fragment f2 of phage lambda (redB-ori region) under well defined growth conditions (Bernardi and Bernardi, 1980). We have sequenced the novel joints of the four deletions so obtained and shown that they have one endpoint in pSC101, identical in all four cases, the other endpoint being located in four different lambda sequences. Furthermore, the nucleotide sequences of the novel joints show homologies between the conserved pSC101 sequence and the lambda sequences both conserved and deleted. The presence of an IS-type element in pSC101 is postulated; however, this element is unrelated to the 200 bp element already described in pSC101 (Ravetch et al., 1976).     

A survey of bacterial insertion sequences using IScan

Bacterial insertion sequences (ISs) are the simplest kinds of bacterial mobile DNA. Evolutionary studies need consistent IS annotation across many different genomes. We have developed an open-source software package, IScan, to identify bacterial ISs and their sequence elements—inverted and target direct repeats—in multiple genomes using multiple flexible search parameters. We applied IScan to 438 completely sequenced bacterial genomes and 20 IS families. The resulting data show that ISs within a genome are extremely similar, with a mean synonymous divergence of K_s = 0.033. Our analysis substantially extends previously available information, and suggests that most ISs have entered bacterial genomes recently. By implication, their population persistence may depend on horizontal transfer. We also used IScan's ability to analyze the statistical significance of sequence similarity among many IS inverted repeats. Although the inverted repeats of insertion sequences are evolutionarily highly flexible parts of ISs, we show that this ability can be used to enrich a dataset for ISs that are likely to be functional. Applied to the thousands of genomes that will soon be available, IScan could be used for many purposes, such as mapping the evolutionary history and horizontal transfer patterns of different ISs.     

The partition (par) locus of pSC101 is an enhancer of plasmid incompatibility

The incompatibitity that pSC101-derived plasmids express toward each other is mediated by directly repeated sequences (iterons) located near the plasmid's replication origin. We report here that the pSC101 par locus, which stabilizes plasmid inheritance in dividing cell populations and alters DNA superheliclty, can function as a cis-acting enhancer of incompatibility, which we show is determined jointly by the copy number of the plasmid and the number of iterons per copy. A single synthetic 32 bp iteron sequence carried by the pUC19 plasmid confers strong pSC101-specific incompatibility in the absence of any other pSC101 sites but requires the par locus to express strong incompatibility when carried by a lower-copy-number plasmid. We propose a model by which the par locus can enchance the apparently antagonistic processes of incompatibility and pSC101 DNA replication while concurrently facilitating plasmid distribution during cell division.     

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF

Summary We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.     

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.     

Construction and BamHL analysis of chimeric plasmids containing EcoRL DNA fragments of the F sex factor.

The construction of seven chimeric plasmids (pRS series) carrying EcoRI endonuclease-generated segments of the F sex factor cloned onto the vector pSC101 is described. BamHI endonuclease analysis of these seven plasmids, the six previously described pRS plasmids (Skurray, R. A., Nagaishi, H., and Clark, A. J. (1976) Proc. Nat. Acad. Sci. USA73, 64–68) and F plasmid DNA has enabled a partial BamHI map of F to be constructed; the orientation of insertion of F DNA segments into the pSC101 vector was also established for nine of the pRS plasmids. Results indicate that in the absence of their normal promoter, F cistrons cloned into the EcoRI site of pSC101 are expressed regardless of orientation of insertion although there is a preferred orientation for high levels of expression.     

Activation and Inactivation of Pseudomonas stutzeri Methylbenzene Catabolism Pathways Mediated by a Transposable Element

The arrangement of the genes involved in o-xylene, m-xylene, and p-xylene catabolism was investigated in three Pseudomonas stutzeri strains: the wild-type strain OX1, which is able to grow on o-xylene but not on the meta and para isomers; the mutant M1, which grows on m-xylene and p-xylene but is unable to utilize the ortho isomer; and the revertant R1, which can utilize all the three isomers of xylene. A 3-kb insertion sequence (IS) termed ISPs1, which inactivates the m-xylene and p-xylene catabolic pathway in P. stutzeri OX1 and the o-xylene catabolic genes in P. stutzeri M1, was detected. No IS was detected in the corresponding catabolic regions of the P. stutzeri R1 genome. ISPs1 is present in several copies in the genomes of the three strains. It is flanked by 24-bp imperfect inverted repeats, causes the direct duplication of 8 bp in the target DNA, and seems to be related to the ISL3 family.     

Regions associated with the stable maintenance of plasmid pSC101 and its tetracycline resistance

Summary Two regions tentatively called unsA and unsR were identified on pSC101. One, unsA, corresponds to less than 650 bp of the N-terminal in the tetracycline resistance structural gene and seems to inhibit stable maintenance of pSC101. The other, unsR, is defined within the 1 kb XhoI-EcoRI region located upstream of the tetracycline resistance structural gene and is a regulatory gene clearly distinct from tetR (Unger et al. 1984); it serves as a suppressor of the unsA function.     

Novel one-step cloning vector with a transposable element: application to the Myxococcus xanthus genome.

A new strategy was developed for rapid cloning of genes with a transposon mutation library. We constructed a transposon designated TnV that was derived from Tn5 and consists of the gene coding for neomycin phosphotransferase II as well as the replication origin of an Escherichia coli plasmid, pSC101, flanked by Tn5 inverted repeats (IS50L and IS50R). TnV can transpose to many different sites of DNA in E. coli and Myxococcus xanthus and confers kanamycin resistance (Kmr) to the cells. From the Kmr cells, one-step cloning of a gene which is mutated as a result of TnV insertion can be achieved as follows. Chromosomal DNA isolated from TnV-mutagenized cells is digested with an appropriate restriction enzyme, ligated, and transformed into E. coli cells with selection for Kmr. The plasmids isolated contain TnV in the target gene. The plasmid DNA can then be used as a probe for characterization of the gene and screening of clones from a genomic library. We used this vector to clone DNA fragments containing genes involved in the development of M. xanthus.     

A family of IS1031 elements in the genome of Acetobacter xylinum: nucleotide sequences and strain distribution

An insertion sequence (here called IS 1031A) from Acetobacter xylinum ATCC 23769 has recently been isolated. This study describes the complete nucleotide sequence of IS 1031A as well as the sequences of two novel iso-IS 1031 elements, IS1031C and IS1031D, from A. xylinum ATCC 23769. The three ISs are all exactly 930 bp long, have imperfect terminal inverted repeats of 24 bp for IS1031A and 21 bp for IS1031C and IS1031D, are flanked by three base pair direct repeats, and contain an open reading frame encoding a putative basic protein of 278 amino acids. Because of nucleotide substitutions, IS1031C and IS1031D differ from IS 1031A by 12.9% while IS1031C differs from IS1031D by only 0.6%. Hybridization analyses of total DNA from nine A. xylinum strains showed that all strains contained IS 1031-like elements varying in copy number from three to at least 16. None of three Acetobacter aceti strains examined contained IS1031-like elements. Taken together, the results suggest that A. xylinum contains a family of IS 1031 elements with considerably diversified nucleotide sequences.     

Identification by deletion analysis of an inducible protein required for plasmid pSC101-mediated tetracycline resistance.

Plasmids containing small deletions within a tetracycline (Tc) resistance gene(s) of plasmid pHA121 were isolated. Plasmid pHA121 was formed by ligating the EcoRI-digested Tc resistance plasmid pSC101 and similarly digested mini-ColE1 plasmid pHA105. The DNA deletion plasmids were constructed by digesting plasmid pHA121 DNA with the restriction endonucleases BamH1 and Sal1 and, in addition, λ exonuclease. Two plasmids, designated pJT131 and pJT133, had small deletions of approximately 0.64 to 0.8 kb and a comparison of the radioactive polypeptides synthesized in plasmid-containing minicells revealed that a 34-kdal polypeptide was not specified by either pJT131 or pJT133. We conclude that the 34-kdal polypeptide is required for the expression of Tc resistance and that its structural gene probably maps in the deleted region of pSC101 DNA.     

ISMapper: identifying transposase insertion sites in bacterial genomes from short read sequence data

Background

Insertion sequences (IS) are small transposable elements, commonly found in bacterial genomes. Identifying the location of IS in bacterial genomes can be useful for a variety of purposes including epidemiological tracking and predicting antibiotic resistance. However IS are commonly present in multiple copies in a single genome, which complicates genome assembly and the identification of IS insertion sites. Here we present ISMapper, a mapping-based tool for identification of the site and orientation of IS insertions in bacterial genomes, directly from paired-end short read data.

Results

ISMapper was validated using three types of short read data: (i) simulated reads from a variety of species, (ii) Illumina reads from 5 isolates for which finished genome sequences were available for comparison, and (iii) Illumina reads from 7 Acinetobacter baumannii isolates for which predicted IS locations were tested using PCR. A total of 20 genomes, including 13 species and 32 distinct IS, were used for validation. ISMapper correctly identified 97 % of known IS insertions in the analysis of simulated reads, and 98 % in real Illumina reads. Subsampling of real Illumina reads to lower depths indicated ISMapper was able to correctly detect insertions for average genome-wide read depths >20x, although read depths >50x were required to obtain confident calls that were highly-supported by evidence from reads. All ISAba1 insertions identified by ISMapper in the A. baumannii genomes were confirmed by PCR. In each A. baumannii genome, ISMapper successfully identified an IS insertion upstream of the ampC beta-lactamase that could explain phenotypic resistance to third-generation cephalosporins. The utility of ISMapper was further demonstrated by profiling genome-wide IS6110 insertions in 138 publicly available Mycobacterium tuberculosis genomes, revealing lineage-specific insertions and multiple insertion hotspots.

Conclusions

ISMapper provides a rapid and robust method for identifying IS insertion sites directly from short read data, with a high degree of accuracy demonstrated across a wide range of bacteria.     

Analysis of the promoter-distal region of the tra operon of the F sex factor of Escherichia coli K-12 encoded by EcoRI restriction fragments f17, f19, and f2.

The promoter-distal region of the tra operon of the F sex factor Escherichia coli K-12 was analyzed, using the chimeric plasmid pRS31, which contains the F EcoRI restriction fragments f17, f19, and f2 cloned into the EcoRI site of pSC101. A series of deletion plasmids of pRS31, extending increasing distances from a site in f17 through f19 and ending in f2, were isolated. These plasmids were examined by heteroduplex analysis with the parent DNA, and a restriction map of this region of DNA was constructed. A series of Tn5 insertion derivatives of pRS31 were also isolated and mapped, using both heteroduplex analysis and restriction mapping. Both the insertion and deletion mutants were tested in minicells for the synthesis of radioactively labeled proteins. This allowed the identification of the individual gene products and mapping of the genes. The result is a saturated physical map of this region of DNA from fragment f17 through to the IS3 insertion sequence near the promoter-distal end of f2.     

New cosmid vectors developed for eukaryotic DNA cloning

A series of ColE1 and pSC101 cosmid vectors have been constructed suitable for cloning large stretches of DNA. All contain a single BamHI site allowing cloning of Sau3A, MboI, BglII, BclI , and BamHI-generated fragments. These vectors have the following characteristics: (i) they are relatively small (1.7-3.4 kb); (ii) the BamHI cloning site is flanked by restriction enzyme sites enabling direct cloning of unfractionated insert DNA without generating multiple insert or vector ligation products [ Ish - Horowitz and Burke, Nucl . Acids Res. 9 (1981) 2989-2998]; (iii) two vectors ( pHSG272 and pHSG274 ) contain a hybrid Tn5 KmR/ G418R gene which is selectable in both prokaryotic and eukaryotic cells, making them suitable for transferring DNA into eukaryotic cells, and (iv) the different prokaryotic selectable markers available in the other vectors described facilitate cosmid rescue of the transferred DNA sequences from the eukaryotic cell: CmR, ApR, KmR, ( pHSG429 ), CmR, ( pHSG439 ), colicin E1 immunity ( pHSG250 ), (v) the cosmid pHSG272 was used successfully to construct a shuttle vector based on the BPVI replicon [ Matthias et al., EMBO J. 2 (1983) 1487-1492].