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Results for “transposon insertion sequencing”

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PubMed1991

IS231A from Bacillus thuringiensis is functional in Escherichia coli: transposition and insertion specificity.

A kanamycin resistance gene was introduced within the insertion sequence IS231A from Bacillus thuringiensis, and transposition of the element was demonstrated in Escherichia coli. DNA sequencing at the target sites showed that IS231A transposition results in direct repeats of variable lengths (10, 11, and 12 bp). These target sequences resemble the terminal inverted repeats of the transposon Tn4430, which are the preferred natural insertion sites of IS231 in B. thuringiensis.

Bacillus thuringiensis↗

PubMed1994

Characterization of a repetitive element detected by NheI in the genomes of Salmo species.

The genomes of the two species in the genus Salmo (Atlantic salmon, S. salar; brown trout, S. trutta) contain a 380-bp repetitive element that is flanked by the recognition sequence of the restriction enzyme NheI. These elements, which comprise approximately 1.2% of the salmon genome, do not exist in long tandem arrays as is typical of satellite DNA. A comparison of the sequences of 16 salmon and 7 trout elements revealed that members of this family of repetitive DNA are closely related to one another (over 95% identity). Subfamily structure exists and there is evidence that members of the same subfamilies are found in both Salmo species. A search of the GenBank database indicated that sequences homologous to the NheI repeat are located within a 1424-bp segment inserted immediately downstream of the 5' end of a Tc1 transposon-like sequence isolated from Atlantic salmon (A.R. Radice, B. Bugaj, D.H. Fitch, and S.W. Emmons, unpublished data; GenBank accession No. L12206).

Animals↗

PubMed2003

Genetic characterization of a novel metallo-beta-lactamase gene, blaIMP-13, harboured by a novel Tn5051-type transposon disseminating carbapenemase genes in Europe: report from the SENTRY worldwide antimicrobial surveillance programme.

OBJECTIVE: In 2001, as part of the SENTRY worldwide antimicrobial surveillance programme, Pseudomonas aeruginosa 86-14571A was isolated at a hospital in Rome from a cancer patient with a bloodstream infection. The isolate was resistant to all antibiotics except amikacin, and displayed an imipenem MIC of 64 mg/L that decreased to 8 mg/L in the presence of EDTA. The resistance determinant was investigated. METHODS: The resistant determinant was cloned in Escherichia coli using a shotgun cloning approach. RESULTS: Sequence analysis revealed the presence of a novel IMP-type metallo-beta-lactamase (MBL) gene, blaIMP-13. This encoded a protein displaying most identity to IMP variants: 93% and 92.3% identity, respectively, to IMP-8 and IMP-2 (previously identified in Italy). The protein had 19 amino acid changes from IMP-2 and 17 amino acid changes from IMP-8. The blaIMP-13 gene was found as a gene cassette in the first position of a class 1 integron. A 25 bp inverted repeat sequence IRi was identified 174 bp upstream of the class I integrase, which suggests that the integron is found on a Tn402-like transposon, or defective transposon derivative. This element, in turn, is located in the transposition locus (tnp region) of a Tn21 subfamily transposon that showed most identity to Tn5051, a transposon recently identified from a strain of Pseudomonas putida isolated in New York. Interestingly, the insertion point of the Tn402-like transposon and the sequence of the Tn5051-like genes were identical to those of the genetic element harbouring blaVIM-2 recently identified in Poland. CONCLUSIONS: The resistance determinant of P. aeruginosa 86-14571A is a novel IMP-type MBL carried on a composite transposon responsible for wide geographical dissemination of MBL genes in Europe.

Amino Acid Sequence↗

PubMed1999

Regional insertional mutagenesis of genes on Arabidopsis thaliana chromosome V using the Ac/Ds transposon in combination with a cDNA scanning method.

For regional insertional mutagenesis of Arabidopsis thaliana genes, we combined a cDNA scanning method (Hayashida et al. Gene 1995; 165:155-161) and an Ac/Ds transposon designed for local mutagenesis, and evaluated this approach with two overlapping yeast artificial chromosome (YAC) clones, CIC7E11 and CIC8B11, on A. thaliana chromosome 5. We applied a previously developed novel cDNA selection method using DNA latex particles (cDNA scanning method) to the two YAC clones and constructed two sub-libraries in which cDNAs for genes on each YAC DNA were concentrated. From each sub-library we isolated cDNAs for genes on each YAC DNA, partially sequenced them, and produced expressed sequence tags (ESTs). In total, 113 non-redundant groups of cDNAs were obtained. Forty-four per cent of these EST clones were novel, and 34% had significant homology to functional proteins from various organisms. In parallel, we transposed Ds from a donor Ds-GUS-T-DNA line, Ds4391-20, already mapped to the CIC7E11/8B11 region. We obtained Ds-transposed lines and recovered their Ds-flanking genomic DNAs by thermal asymmetric interlaced (TAIL) polymerase chain reaction (PCR). Dot-blot analysis indicated that 20% of the lines contained transposed Ds in the CIC7E11/8B11 region, suggesting that this Ac/Ds transposon system is effective for regional insertional mutagenesis. To isolate Ds insertion mutants in the genes identified from the CIC7E11/8B11 region, we carried out PCR screening from 800 Ds-containing lines using Ds-specific and gene-specific primers that were designed from the 113 cDNA sequences identified by the cDNA scanning method. We found that 49 lines contain Ds insertion mutations, and that five lines contain Ds mutations in genes that are mapped to the sequenced CIC7E11/8B11 genomic DNA region. These results indicate that combining the cDNA scanning method and the Ac/Ds transposon gives a powerful tool for regional insertional mutagenesis not only in Arabidopsis but also in other plants or crops whose genomes are not sequenced.

Arabidopsis↗

PubMed2002

Sinorhizobium fredii HH103 has a truncated nolO gene due to a -1 frameshift mutation that is conserved among other geographically distant S. fredii strains.

Strain SVQ121 is a mutant derivative of Sinorhizobium fredii HH103 carrying a transposon Tn5-lacZ insertion into the nolO-coding region. Sequence analysis of the wild-type gene revealed that it is homologous to that of Rhizobium sp. NGR234, which is involved in the 3 (or 4)-O-carbamoylation of the nonreducing terminus of Nod factors. Downstream of nolO, as in Rhizobium sp. NGR234, the noeI gene responsible for methylation of the fucose moiety of Nod factors was found. SVQ121 Nod factors showed lower levels of methylation into the fucosyl residue than those of HH103-suggesting a polar effect of the transposon insertion into nolO over the noel gene. A noeI HH103 mutant was constructed. This mutant, SVQ503, produced Nod factors devoid of methyl groups, confirming that the S. fredii noeI gene is functional. Neither the nolO nor the noeI mutation affected the ability of HH103 to nodulate several host plants, but both mutations reduced competitiveness to nodulate soybean. The Nod factors produced by strain HH103, like those of other S. fredii isolates, lack carbamoyl residues. By using specific polymerase chain reaction primers, we sequenced the nolO gene of S. fredii strains USDA192, USDA193, USDA257, and 042B(s). All the analyzed strains showed the same -1 frameshift mutation that is present in the HH103 nolO-coding region. From these results, it is concluded that, regardless of their geographical origin, S. fredii strains carry the nolO-coding region but that it is truncated by the same base-pair deletion.

Amino Acid Sequence↗

PubMed1982

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.

Base Sequence↗

PubMed1993

Target-selected gene inactivation in Caenorhabditis elegans by using a frozen transposon insertion mutant bank.

To understand how genotype determines the phenotype of the animal Caenorhabditis elegans, one ideally needs to know the complete sequence of the genome and the contribution of genes to phenotype, which requires an efficient strategy for reverse genetics. We here report that the Tc1 transposon induces frequent deletions of flanking DNA, apparently resulting from Tc1 excision followed by imprecise DNA repair. We use this to inactivate genes in two steps. (i) We established a frozen library of 5000 nematode lines mutagenized by Tc1 insertion, from which insertion mutants of genes of interest can be recovered. Their address within the library is determined by PCR. (ii) Animals are then screened, again by PCR, to detect derivatives in which Tc1 and 1000-2000 base pairs of flanking DNA are deleted, and thus a gene of interest is inactivated. We have thus far isolated Tc1 insertions in 16 different genes and obtained deletion derivatives of 6 of those.

Alleles↗

PubMed1991

Purification, characterization and nucleotide sequence of the periplasmic C4-dicarboxylate-binding protein (DctP) from Rhodobacter capsulatus.

A periplasmic binding protein essential for high-affinity transport of the C4-dicarboxylates malate, succinate and fumarate across the cytoplasmic membrane of the purple photosynthetic bacterium Rhodobacter capsulatus has been purified to homogeneity and some of its ligand-binding properties characterized. The protein was not produced in a Tn5 insertion mutant unable to transport C4-dicarboxylates under aerobic conditions in the dark. Wild-type DNA corresponding to the location of the transposon insertion site was subcloned and a 1.5 kb section sequenced. A complete open reading frame of 999 bp was identified that encoded a 333-residue protein (DctP) with a molecular weight of 36,128 with a 26-residue amino-terminal signal peptide. The identify of this protein with the purified dicarboxylate-binding protein and the position of the predicted signal peptide cleavage site was confirmed by N-terminal sequencing. No significant homology with other proteins was detected in database searches. A GC-rich region of dyad symmetry was located 7 bp downstream of the dctP translational stop codon. This structure may be of significance in regulating the relative abundance of DctP and other dct gene products which comprise the high-affinity dicarboxylate transport system in this bacterium.

Amino Acid Sequence↗

PubMed2001

Population dynamics of an Ac-like transposable element in self- and cross-pollinating arabidopsis.

Theoretical models predict that the mating system should be an important factor driving the dynamics of transposable elements in natural populations due to differences in selective pressure on both element and host. We used a PCR-based approach to examine the abundance and levels of insertion polymorphism of Ac-III, a recently identified Ac-like transposon family, in natural populations of the selfing plant Arabidopsis thaliana and its close outcrossing relative, Arabidopsis lyrata. Although several insertions appeared to be ancient and shared between species, there is strong evidence for recent activity of this element family in both species. Sequences of the regions flanking insertions indicate that all Ac-III transposons segregating in natural populations are in noncoding regions and provide no evidence for local transposition events. Transposon display analysis suggests the presence of slightly higher numbers of insertion sites per individual but fewer total polymorphic insertions in the self-pollinating A. thaliana than A. lyrata. Element insertions appear to be segregating at significantly lower frequencies in A. lyrata than A. thaliana, which is consistent with a reduction in transposition rate, reduction in effective population size, or reduced efficacy of natural selection against element insertions in selfing populations.

Arabidopsis↗

PubMed1998

Complete sequence of the IncPbeta plasmid R751: implications for evolution and organisation of the IncP backbone.

The broad host range IncP plasmids are of particular interest because of their ability to promote gene spread between diverse bacterial species. To facilitate study of these plasmids we have compiled the complete sequence of the IncPbeta plasmid R751. Comparison with the sequence of the IncPalpha plasmids confirms the conservation of the IncP backbone of replication, conjugative transfer and stable inheritance functions between the two branches of this family. As in the IncPalpha genome the DNA of this backbone appears to have been enriched for the GCCG/CGGC motifs characteristic of the genome of organisms with a high G+C content, such as P. aeruginosa, suggesting that IncPbeta plasmids have been subjected during their evolution to similar mutational and selective forces as IncPalpha plasmids and may have evolved in pseudomonad hosts. The IncP genome is consistently interrupted by insertion of phenotypic markers and/or transposable elements between oriV and trfA and between the tra and trb operons. The R751 genome reveals a family of repeated sequences in these regions which may form the basis of a hot spot for insertion of foreign DNA. Sequence analysis of the cryptic transposon Tn4321 revealed that it is not a member of the Tn21 family as we had proposed previously from an inspection of its ends. Rather it is a composite transposon defined by inverted repeats of a 1347 bp IS element belonging to a recently discovered family which is distributed throughout the prokaryotes. The central unique region of Tn4321 encodes two predicted proteins, one of which is a regulatory protein while the other is presumably responsible for an as yet unidentified phenotype. The most striking feature of the IncPalpha plasmids, the global regulation of replication and transfer by the KorA and KorB proteins encoded in the central control operon, is conserved between the two plasmids although there appear to be significant differences in the specificity of repressor-operator interactions. The importance of these global regulatory circuits is emphasised by the observation that the operator sequences for KorB are highly conserved even in contexts where the surrounding region, either a protein coding or intergenic sequence, has diverged considerably. There appears to be no equivalent of the parABCDE region which in the IncPalpha plasmids provides multimer resolution, lethality to plasmid-free segregants and active partitioning functions. However, we found that the continuous sector from co-ordinate 0 to 9100 bp, encoding the co-regulated klc and kle operons as well as the central control region, could confer a high degree of segregational stability on a low copy number test vector. Thus R751 appears to exhibit very clearly what was first revealed by study of the IncPalpha plasmids, namely a fully functional co-ordinately regulated set of replication, transfer and stable inheritance functions.

Amino Acid Sequence↗

PubMed1994

IS231 and other Bacillus thuringiensis transposable elements: a review.

Bacillus thuringiensis is an entomopathogenic bacterium whose toxicity is due to the presence in the sporangia of delta-endotoxin crystals active against agricultural pests and vectors of human and animal diseases. Most of the genes coding for these toxin proteins are plasmid-borne and are generally structurally associated with insertion sequences (IS231, IS232, IS240, ISBT1 and ISBT2) and transposons (Tn4430 and Tn5401). Several of these mobile elements have been shown to be active and are believed to participate in the crystal gene mobility, thereby contributing to the variation of bacterial toxicity. Structural analysis of the iso-IS231 elements indicates that they are related to IS1151 from Clostridium perfringens and distantly related to IS4 and IS186 from Escherichia coli. Like the other IS4 family members, they contain a conserved transposase-integrase motif found in other IS families and retroviruses. Moreover, functional data gathered from IS231A in Escherichia coli indicate a non-replicative mode of transposition, with a marked preference for specific targets. Similar results were also obtained in Bacillus subtilis and B. thuringiensis, and a working model for DNA-protein interactions at the target site is proposed.

Amino Acid Sequence↗

PubMed2003

Effect of inactivation of poly(hydroxyalkanoates) depolymerase gene on the properties of poly(hydroxyalkanoates) in Pseudomonas resinovorans.

The phaZ gene of Pseudomonas resinovorans codes for a poly(hydroxyalkanoates) (PHA) depolymerase. Two phaZ mutants of Pseudomonas resinovorans NRRL B-2649, FOAC001 and FOAC002, were constructed by an in vitro transposition procedure followed by chromosomal integration via homologous recombination. A detailed mapping of the transposon insertion sites and an analysis of the resultant sequences showed that putative fusion polypeptides PhaZ(FOAC001) (239 amino-acid residues) and PhaZ(FOAC002) (85 amino-acid residues) could result from the mutant phaZ genes of FOAC001 and FOAC002, respectively. In vivo PHA degradation data indicated that PhaZ(FOAC001) might still retain a partial PHA depolymerization activity, while PhaZ(FOAC002) is completely devoid of this function. The cell yields and PHA contents of B-2649, FOAC001, and FOAC002 were similar when the cells were grown either under a limiting nitrogen-source (low-N) condition for up to 5 days or in excess N-source (high-N) for 3 days. A dramatic decrease in PHA content was observed in the PhaZ-active B-2649 and FOAC001 cells during prolonged cell growth (5 days) in high-N medium or in response to a shift-up in nitrogen-source. The repeat-unit compositions of the PHAs from FOAC001 and FOAC002 contained slightly lower amounts of beta-hydroxyoctanoate and higher beta-hydroxytetradecenoate than that of the wild-type B-2649 when grown under a high-N condition. While the molecular masses of the PHAs from FOAC001 and FOAC002 did not vary under any conditions used in this study, those of the wild-type B-2649 were markedly increased in cells either grown for 5 days under a high-N condition or subjected to a nitrogen-source shift-up. These phaZ mutants thus provide a valuable system to study the influence of PHA depolymerase on the accumulation and properties of medium-chain-length PHA.

Carboxylic Ester Hydrolases↗

PubMed2004

Transposon Tn7 protein TnsD binding to Escherichia coli attTn7 DNA and its eukaryotic orthologs.

Transposon Tn7 inserts itself into the attTn7 target DNA sequence at the 3' end of the Escherichia coli glmS gene with high specificity and efficiency. This site in the E. coli genome displays amino acid conservation and nucleotide similarity with orthologous sequences in Archaebacteria and eukaryotes. On the basis of the high degree of nucleotide similarity, particularly with eukaryotes, we examined the interactions of a set of 20-bp duplex DNA sequences with the Tn7 protein TnsD. The protein was overexpressed in the IPTG-inducible vector pET14b-TnsD in E. coli BL21(DE3)-RIL-Codon-Plus, and purified by nickel chelation and ion exchange chromatography. Changes in the conformation of DNA duplexes upon interaction with TnsD were monitored by circular dichroism (CD) spectroscopy. TnsD binding to and dissociation from immobilized DNA duplexes were monitored by total internal reflectance (TIR). CD and TIR results were analyzed to calculate k(on), k(off), and K(D) values. The 20-bp DNA duplex corresponding to attTn7 at the 3' end of E. coli glmS displayed strong affinity for TnsD protein, with K(D) approximately 20 nM. Eukaryotic orthologs of attTn7 from yeast and mammalian GFPT1 displayed lower affinity, with K(D) approximately 500 nM. Mutant DNA sequences with a single central mismatch did not display any detectable interaction with TnsD. The physical studies validate our biological observation of Tn7 transposition into a plasmid containing the mammalian attTn7 ortholog sequence [Cleaver, S. H., and Wickstrom, E. (2000) Gene 254, 37-44], and suggest that 1-2 amino acid substitutions in TnsD might be sufficient to permit binding to mammalian orthologs that is as strong as wild-type TnsD binding to attTn7.

Animals↗

PubMed1984

Proteus mirabilis chromosome mobilization by plasmid D: a physical characterization.

Plasmid D, a hybrid of plasmids P-lac and R1 drd19, mediates polarized chromosome mobilization from one origin in Proteus mirabilis strain PM5006, while the parental plasmids neither individually nor combined mobilize this chromosome. To elucidate its acquired mobilizing ability plasmid D was characterized physically in relation to P-lac and R1 drd19. Restriction patterns of these plasmids were compared and it was shown that D consists of P-lac and only the r-determinant (r-det) of R1 drd19. A mechanism for the formation of plasmid D, via transduction of the r-det and subsequent transposon-like integration into P-lac, involving insertion sequence IS1, was suggested. Evidence for aberration in plasmid D DNA as a result of r-det integration into P-lac was attributed to IS1 elements which flank the r-det. Recombination regions of parental plasmid DNA were located on HindIII fragments alpha and beta of plasmid D and were subsequently inserted in vitro into IncP-1 plasmid RP4 that fails to mobilize the P. mirabilis chromosome. RP4::HindIII alpha plasmids did not mobilize the latter chromosome, but rendered the Proteus host lac+. RP4::HindIII beta plasmids pMC1 and pMC17, containing the fragment in opposite orientations, mobilized the P. mirabilis chromsome. For pMC17, mobilization was indistinguishable from that of plasmid D, i.e. having the same orientation and the same single origin. However, mobilization promoted by pMC1 was from two distinctly different origins, different from that of pMC17. This apparently deviates from known examples where inversion of homologous DNA inserted into plasmids leads to mobilization from the same origin but in reverse direction.

Chromosome Mapping↗

PubMed1994

Transposon-mediated mobilization of chromosomally located catabolic operons of the CAM plasmid by TOL plasmid transposon Tn4652 and CAM plasmid transposon Tn3614.

The CAM (camphor degradation) plasmid is integrated into the chromosome of Pseudomonas putida PaW-line strains and is not self-transferable as a plasmid via conjugation. Our results show that the mobilization of chromosomally located CAM and the integration of cam-operons into the chromosome of the new Cam+ transconjugants is a recA-independent process mediated by transposons Tn4652 (17 kbp) and Tn3614 (7.2 kbp). Transposon Tn3614 is apparently identical to the left-hand and the right-hand sequences of the TOL plasmid pWW0 transposon Tn4654. The insertion of Tn401 inside the left-hand terminal IR of Tn4652 completely inhibited the mobilization of CAM. According to our data transposons Tn4652 and Tn3614 together with CAM plasmid catabolic operons are integrated into the chromosome. We propose that in pseudomonads the transposons Tn4652 and Tn3614 play a key role in the evolution and spread of new catabolic plasmids in nature.

Chromosomes, Bacterial↗

PubMed2003

Mutation in tcpR gene (Vc0832) of Vibrio cholerae O1 causes loss of tolerance to high osmolarity and affects colonization and virulence in infant mice.

Vibrio cholerae, the agent of cholera, multiplies and colonizes human intestinal tract where it survives high osmolarity due to bile and other sodium salts. In this work, by TnphoA mutagenesis, a mutant of V. cholerae O1 which could not grow and form colonies on LB agar containing 400 mM NaCl has been characterized. The mutant, designated CD83, adhered normally to freshly isolated rabbit intestinal discs, colonized poorly the gut of infant mice and was avirulent in the same model, whereas the parent strain CD81 would colonize the gut and cause death of mice. Attenuation of virulence of CD83 was not attributable to its inability to produce cholera toxin, as no difference was found in the cholera toxin produced in vitro by the mutant and parent strains. Molecular cloning and sequencing of the mutated gene revealed that insertion of transposon occurred in tcpR gene (Vc0832) of V. cholerae. Complementation of the mutant with wild-type tcpR gene resulted in restoration of the ability to survive at high salt concentration (400 mM NaCl), and to colonize the gut and restore virulence. The results suggest that tcpR plays a role in survival of V. cholerae in the small intestine of host as the osmolarity in the intestinal lumen is thought to be equivalent to 300 mM NaCl or higher.

Animals↗

PubMed2000

Identification of a two-component signal transduction system involved in fimbriation of Porphyromonas gingivalis.

Porphyromonas gingivalis, a periodontopathogen, is an oral anaerobic gram-negative bacterium with numerous fimbriae on the cell surface. Fimbriae have been considered to be an important virulence factor in this organism. We analyzed the genomic DNA of transposon-induced, fimbria-deficient mutants derived from ATCC 33277 and found that seven independent mutants had transposon insertions within the same restriction fragment. Cloning and sequencing of the disrupted region from one of the mutants revealed two adjacent open reading frames (ORFs) which seemed to encode a two-component signal transduction system. We also found that six of the mutants had insertions in a gene, fimS, a homologue of the genes encoding sensor kinase, and that the insertion in the remaining one disrupted the gene immediately downstream, fimR, a homologue of the response regulator genes in other bacteria. These findings suggest that this two-component regulatory system is involved in fimbriation of P. gingivalis.

Amino Acid Sequence↗

PubMed2001

Functional analysis of the Bacillus subtilis cysK and cysJI genes.

The function of the Bacillus subtilis cysK and cysJI (previously designated yvgQR) genes, expected to be involved in the assimilatory sulfate reduction pathway, was investigated. A B. subtilis mutant with a deletion in the cysJI genes was unable to use sulfate or sulfite as sulfur source, which confirmed that these genes encode sulfite reductase. A mutant with a transposon insertion in the cysK gene, whose deduced protein sequence showed similarity to cysteine synthases, grew poorly on sulfate and butanesulfonate. A strain in which cysK and yrhA, a cysK paralog, were inactivated was unable to grow with sulfate. Whereas expression of the cysJI genes was induced by sulfate, expression of cysK was repressed both by sulfate and by cysteine.

Amino Acid Sequence↗