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Results for “transposon insertion sequencing”

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PubMed1998

Identification of csrR/csrS, a genetic locus that regulates hyaluronic acid capsule synthesis in group A Streptococcus.

The hyaluronic acid capsule of group A Streptococcus (GAS) is an important virulence factor, but little is known about mechanisms that regulate capsule expression. Transposon Tn916 mutagenesis of the poorly encapsulated M-type 3 GAS strain DLS003 produced a transconjugant that exhibited a mucoid colony morphology, reflecting increased hyaluronic acid capsule production. Analysis of chromosomal DNA sequence immediately downstream of the transposon insertion identified two open reading frames, designated csrR and csrS, which exhibited sequence similarity to bacterial two-component regulatory systems. We constructed an in-frame deletion mutation within csrR, which encodes the putative response component. Replacement of the native csrR gene in the DLS003 chromosome with the mutant allele resulted in a sixfold increase in capsule production and a corresponding increase in transcription of the has operon, which contains the essential genes for hyaluronic acid synthesis. Increased capsule production by the csrR mutant strain was associated with enhanced resistance to complement-mediated opsonophagocytic killing in vitro and with a 500-fold increase in virulence in mice. These results establish CsrR as a negative regulator of hyaluronic acid capsule synthesis and suggest that it is part of a two-component regulatory system that influences capsule expression and virulence.

Amino Acid Sequence↗

PubMed2000

Transposome insertional mutagenesis and direct sequencing of microbial genomes.

Preformed transposase-transposon complexes called 'Transposomes' have been electroporated into bacterial cells. The magnesium dependent process of insertion of the transposable element into bacterial chromosomal DNA occurs in vivo. The transposition efficiency of a Transposome containing a kanamycin marker was between 1.0 x 10(4) and 1.0 x 10(7) kanamycin resistant clones per microgram of transposon DNA in three gram-negative enteric bacterial species. Transposon integration sites were examined by direct genome sequencing of chromosomal DNA. Genomic DNA was isolated from transposition clones and directly cycle sequenced with primers specific for the ends of the transposon. The precise location of genome interruption for a transposition clone was identified by homology to known genes or sequences. Mutant phenotypes were rapidly correlated with genomic insertions sites.

Chromosomes, Bacterial↗

PubMed2000

Identification of Pasteurella multocida virulence genes in a septicemic mouse model using signature-tagged mutagenesis.

P. multocida is the causative agent of several economically significant veterinary diseases occurring in numerous species worldwide. Signature-tagged mutagenesis (STM) is a powerful genetic technique used to simultaneously screen multiple transposon mutants of a pathogen for their inability to survive in vivo. We have designed an STM system based on a mini-Tn10 transposon, chemiluminescent detection and semi-quantitative analysis and have identified transposon insertions into genes of Pasteurella multocida that attenuate virulence in a septicemic mouse model. A bank of 96 transposons containing strongly-hybridizing tags was used to create 19 pools of P. multocida transposon mutants containing approximately 70-90 mutants/pool. A total of 62 mutants were attenuated when checked individually, and 25 unique single transposon insertion mutations were identified from this group. The sequence of the disrupted ORF for each attenuated mutant was determined by either cloning or PCR-amplifying and sequencing the flanking regions. The attenuated mutants contained transposon insertions in genes encoding biosynthetic enzymes, virulence factors, regulatory components and unknown functions. This study should contribute to an understanding of the pathogenic mechanisms by which P. multocida and other pathogens in the Pasteurellaceae family cause disease and identify novel live vaccine candidates and new potential antibiotic targets.

Animals↗

PubMed1994

Length of the coupling sequence of Tn916.

The coupling sequences of conjugative transposons are short variable sequences derived from the DNA flanking the transposon insertion site. We show here that for Tn916 the left coupling sequence is 6 bases long. The right-hand end of the transposon can excise with either four or five T's, but integration occurs to restore the five T's at the transposon's right end.

Bacillus subtilis↗

PubMed2004

Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo.

We have used the Tol2 transposable element to design and perform effective enhancer trapping in zebrafish. Modified transposon DNA and transposase RNA were delivered into zebrafish embryos by microinjection to produce heritable insertions in the zebrafish genome. The enhancer trap construct carries the EGFP gene controlled by a partial epithelial promoter from the keratin8 gene. Enhanced green fluorescent protein (EGFP) is used as a marker to select F1 transgenic fish and as a reporter to trap enhancers. We have isolated 28 transgenic lines that were derived from the 37 GFP-positive F0 founders and displayed various specific EGFP expression patterns in addition to basal expression from the modified keratin 8 promoter. Analyses of expression by whole-mount RNA in situ hybridization demonstrated that these patterns could recapitulate the expression of the tagged genes to a variable extent and, therefore, confirmed that our construct worked effectively as an enhancer trap. Transgenic offspring from the 37 F0 EGFP-positive founders have been genetically analyzed up to the F2 generation. Flanking sequences from 65 separate transposon insertion sites were identified by thermal asymmetric interlaced polymerase chain reaction. Injection of the transposase RNA into transgenic embryos induced remobilization of genomic Tol2 copies producing novel insertions including some in the germ line. The approach has great potential for developmental and anatomical studies of teleosts.

Animals↗

PubMed1988

Sequence requirements of Escherichia coli attTn7, a specific site of transposon Tn7 insertion.

Transposon Tn7 transposes at high frequency to a specific site, attTn7, in the Escherichia coli chromosome. We devised a quantitative assay for Tn7 transposition in which Tn7-end derivatives containing the cis-acting transposition sequences of Tn7 transpose from a bacteriophage lambda vector upon infection into cells containing the Tn7-encoded transposition proteins. We used this assay to identify a 68-base-pair DNA segment containing the sequences essential for attTn7 target activity. This segment is positioned asymmetrically with respect to the specific point of Tn7 insertion in attTn7 and lacks obvious homology to the sequences at the ends of Tn7 which participate directly in transposition. We also show that some sequences essential for attTn7 target activity are contained within the protein-coding sequence of a bacterial gene.

Base Sequence↗

PubMed1992

The evolution of insertion sequences within enteric bacteria.

To identify mechanisms that influence the evolution of bacterial transposons, DNA sequence variation was evaluated among homologs of insertion sequences IS1, IS3 and IS30 from natural strains of Escherichia coli and related enteric bacteria. The nucleotide sequences within each class of IS were highly conserved among E. coli strains, over 99.7% similar to a consensus sequence. When compared to the range of nucleotide divergence among chromosomal genes, these data indicate high turnover and rapid movement of the transposons among clonal lineages of E. coli. In addition, length polymorphism among IS appears to be far less frequent than in eukaryotic transposons, indicating that nonfunctional elements comprise a smaller fraction of bacterial transposon populations than found in eukaryotes. IS present in other species of enteric bacteria are substantially divergent from E. coli elements, indicating that IS are mobilized among bacterial species at a reduced rate. However, homologs of IS1 and IS3 from diverse species provide evidence that recombination events and horizontal transfer of IS among species have both played major roles in the evolution of these elements. IS3 elements from E. coli and Shigella show multiple, nested, intragenic recombinations with a distantly related transposon, and IS1 homologs from diverse taxa reveal a mosaic structure indicative of multiple recombination and horizontal transfer events.

Base Sequence↗

PubMed2001

Transposon mutations in the flagella biosynthetic pathway of the solvent-tolerant Pseudomonas putida S12 result in a decreased expression of solvent efflux genes.

Fourteen solvent-sensitive transposon mutants were generated from the solvent-tolerant Pseudomonas putida strain S12 by applying the TnMod-KmO mutagenesis system. These mutants were unable to grow in the presence of octanol and toluene. By cloning the region flanking the transposon insertion point a partial sequence of the interrupted genes was determined. Comparison of the deduced amino acid sequences with a protein database revealed the following interrupted putative gene products: organic solvent efflux proteins SrpA and SrpB, the flagellar structural proteins FlgK, FlaG, FliI, FliC, and FliH, the transcriptional activator FleQ, the alternative RNA polymerase sigma factor RpoN, and the flagellum-specific RNA polymerase sigma factor FliA (RpoF). The transposon mutants, except for the organic solvent efflux mutants, were nonmotile as determined by a swarm assay and the formation of the flagellum was totally impaired. Expression studies with a srp promoter probe showed a decreased expression of the SrpABC efflux pump in the nonmotile mutants.

Bacterial Proteins↗

PubMed1982

ISRm1: A Rhizobium meliloti insertion sequence that transposes preferentially into nitrogen fixation genes.

After transposon Tn5 mutagenesis, a high proportion of Rhizobium meliloti symbiotic mutants do not contain Tn5 insertions in symbiotic genes. Instead, the mutations in these strains are correlated with the presence of an endogenous insertion sequence (ISRm1) in nitrogen fixation (nif) or symbiotic genes which are adjacent to the nif genes. ISRm1 is 1.4 kb and transposes to at least three restriction fragments in the nif region at a frequency between 10(-2) and 10(-3). A nif region restriction fragment containing ISRm1 was cloned from one of the mutant strains unable to fix nitrogen symbiotically (Fix-) and the resulting plasmid was used as a hybridization probe. ISRm1 is present at least ten times in the R. meliloti genome but is not present in any other R. meliloti strains, E. coli strains, or Rhizobium species tested. We demonstrated that the Fix- phenotype correlated with ISRm1 transposition is indeed caused by ISRm1 insertion by conjugating a cloned fragment containing ISRm1 into a wild type Fix+ R. meliloti host and replacing the normal genomic nif fragment with the nif::ISRm1 fragment. The resulting strain was Fix-.

DNA Transposable Elements↗

PubMed1980

Nucleotide sequence of the bacterial transposon Tn1681 encoding a heat-stable (ST) toxin and its identification in enterotoxigenic Escherichia coli strains.

the Escherichia coli heat-stable toxin (ST I) is encoded within a transposon (Tn1681) flanked by inverted repeats of insertion sequence 1 (IS1) [So, M., Heffron, F. & McCarthy, B. J. (1979) Nature (London) 277, 453-456]. By subcloning restriction fragments and by insertion mutagenesis, we located precisely the gene for ST I within the transposon. We determined the complete nucleotide sequence of the central portion of Tn1681 (i.e., that part flanked by IS1) and identified the coding sequence of the toxin. From the nucleotide sequence, we deduced a probable amino acid sequence for ST I. The NH2-terminal portion of the amino acid sequence is extremely hydrophobic and bears a striking resemblance to the signal sequence of the fd phage minor coat protein. By using a subcloned restriction fragment containing the gene for ST I but no IS1 sequences, we determined (i) that the ST toxin with activity assayable in suckling mice (ST I) is genetically distinct from the St toxin assayable in ligated ileal loops (ST II) and (ii) that ST I can be responsible for diarrheal disease in different animals.

Amino Acid Sequence↗

PubMed1987

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.

Base Composition↗

PubMed1992

DNA sequence of IS91 and identification of the transposase gene.

IS91 is a 1,830-bp insertion sequence that inserts specifically at the sequence CAAG or GAAC of the target and does not duplicate any sequence upon insertion (23). By transposon mutagenesis, we have identified open reading frame 426 (ORF426; bp 454 to 1731) as the putative ORF for the transposase. It displays a cysteine-rich, potential metal-binding domain in its N-terminal region. Adjacent to ORF426, there is an ORF (ORF121) which precedes and terminally overlaps ORF426 by one amino acid. Tn1732 insertions in ORF121 do not affect the transposition frequency. IS91 has sequence similarities to IS801 from Pseudomonas syringae. Their putative transposases are 36% identical, including conservation of the cysteine-rich cluster. The information concerning IS801 insertion specificity and target duplication has been reevaluated in the light of our results.

Amino Acid Sequence↗

PubMed2000

Construction and application of mycobacterial reporter transposons.

The transposon Tn5367, which is a derivative of the mycobacterial insertion sequence IS1096, was modified by introducing novel genes to produce reporter transposons which can be used to generate transposon insertion libraries containing mycobacterial gene or operon fusions. A plasmid that is temperature-sensitive for replication in mycobacteria was used to deliver promoterless lacZ or aph reporter genes to Mycobacterium smegmatis as transcriptional (lacZ), or translational ('aph) fusions. Mutants containing lacZ produced varying intensities of blue colour on indicator media. This reporter activity could be used as a quantitative measure of promoter strength. Mutants displaying varying levels of resistance to kanamycin were obtained by transpositional insertion of the 'aph reporter lacking a promoter, ribosome binding site and start codon to form functionally active translational fusions. Finally, inclusion of the R6Kgamma origin within Tn5367 allowed transposon insertions to be rescued in an Escherichia coli host strain permissive for the replication of this origin. This study demonstrates that transcriptional and translational reporter derivatives of Tn5367 are functional, and they supplement the growing range of molecular tools available for the study of mycobacteria.

Amino Acid Sequence↗

PubMed1989

Transposable elements for efficient manipulation of a wide range of gram-negative bacteria: promoter probes and vectors for foreign genes.

We describe here the construction and use of a series of modified transposons based on the insertion sequence IS1. Like their parent, omegon-Km [Fellay et al., Gene 76 (1989) 215-226], these elements permit efficient insertional mutagenesis of a variety of Gram-negative bacteria. The presence of a functional pBR322 origin of replication within the transposable element facilitates subsequent cloning of the mutated gene. The omegon-Km system was previously shown to function in Pseudomonas putida, Rhizobium leguminosarum and Paracoccus denitrificans. The results we present here demonstrate that its use can be extended to Xanthomonas campestris, a plant pathogen, and to the microaeroduric Zymomonas mobilis. Derivative transposons carrying unique restriction sites for ScaI, NdeI, XbaI and XhoI have been constructed, allowing the cloning and introduction of foreign genes. We have also constructed two derivatives which can be used to generate operon fusions upon insertion and are thus useful for isolating and characterising indigenous promoters. One carries a promoterless chloramphenicol acetyl-transferase (CAT)-encoding gene (cat) and the second, the entire promoterless Escherichia coli lac operon. We demonstrate the utility of the cat promoter probe in X. campestris to target conditional promoters inducible by high salt or subject to repression by glucose.

Base Sequence↗

PubMed1987

Precise insertion of antibiotic resistance determinants into Tn21-like transposons: nucleotide sequence of the OXA-1 beta-lactamase gene.

Several plasmid-encoded beta-lactamases are on multiresistance transposable elements. The OXA-1 beta-lactamase gene is part of Tn2603, which is borne on the R plasmid RGN238. We report here the complete nucleotide sequence of the OXA-1 beta-lactamase gene and flanking sequences. The OXA-1 gene shows a greater than 50% sequence divergence from the OXA-2 gene, yet there is significant functional similarity at the peptide level. Analysis of 5' and 3' flanking sequences shows that Tn2603 differs from its probable precursor, Tn21, by a precise 1004-base-pair insertion, containing the OXA-1 structural gene, at the target sequence AAAGTT, which is located between the Tn21 streptomycin/spectinomycin (aadA) promoter and its structural gene. A 5- for 6-base repeat of the target sequence is found at the end of the insertion. The same precise insertion and repeat of the target sequence are found for the OXA-2 gene from R46. The 5' flanking regions of two other genes, the trimethoprim-resistance gene from R388 and the gentamicin resistance (aadB) gene from pDGO100, are greater than 98% homologous to the 5' flanking sequences of the OXA-1, OXA-2, and aadA genes until they diverge at the target sequence. From the available sequence data a recombinational hot spot is defined at the nucleotide level 5' of the aadA gene of Tn21, and a second potential hot spot is proposed 3' of this gene.

Amino Acid Sequence↗

PubMed2004

[Positional analysis of a gene related to salt tolerance in Sinorhizobium meliloti by transposon rescue].

Salt sensitive mutant 042BML-2 was obtained by mutating Sinorhizobium meliloti 042BM with transposon Tn5 carried on the plasmid pRL1063a. By transposon rescue, a 1.179 kb of DNA sequence of S. meliloti flanking the Tn5 insertion site was obtained. The sequence similarity analysis through BLAST analysis in GenBank revealed the transposon was inserted into a functionally unknown gene, which is 6 270 bp in length, of S. meliloti, and the gene was named rtsC. This study indicated that rtsC was associated with salt tolerance in S. meliloti 042BM. Hydrophobicity profile analysis of the putative amino acid sequence showed that two predicted transmembrane domains existed in N-terminal portion of RtsC. The significance of RtsC protein in the salt-tolerance in S. meliloti was discussed.

Amino Acid Sequence↗

PubMed1988

Cloning and transposon insertion mutagenesis of virulence genes of the 100-kilobase plasmid of Salmonella typhimurium.

We have cloned regions of the 100-kilobase (kb) plasmid, pStSR100, of Salmonella typhimurium SR-11 that confer virulence to plasmid-cured S. typhimurium. Cells carrying recombinant plasmids that conferred virulence were selected by inoculating mice orally with recombinant libraries in virulence plasmid-cured S. typhimurium and harvesting isolates that infected spleens. Three plasmids, pYA401, pYA402, and pYA403, constructed with the cosmid vector pCVD305 conferred wild-type levels of virulence to plasmid-cured S. typhimurium and had a common 14-kb DNA insert sequence. Another recombinant plasmid, pYA422, constructed with the vector pACYC184, conferred to plasmid-cured S. typhimurium a wild-type 50% lethal dose (LD50) level, but mice died more slowly than when infected with wild-type S. typhimurium. Furthermore, pYA422 conferred the ability to cause a higher, but not a wild-type, level of splenic infection on plasmid-cured S. typhimurium. pYA422 had a 3.2-kb insert sequence which mapped to the center of the 14-kb common sequence of the cosmid clones. Transposon Tn5 insertion mutations in pYA403 inhibited virulence to various degrees, and when transduced into the native virulence plasmid of S. typhimurium, these Tn5 insertions decreased virulence to degrees similar to those observed when the Tn5 insertions were present in pYA403. vir-22::Tn5 in pStSR100 greatly lowered infection of spleens relative to unmutagenized virulence plasmid, while vir-26::Tn5 and vir-27::Tn5 lowered splenic infection to lesser degrees. At least three proteins were encoded by pYA403 containing 23 kb of insert sequence and subclone pYA420, containing the 14-kb common insert sequence present in all of the cosmid clones. One of these proteins, with an apparent molecular weight of 28,000, was also encoded by pYA422. The Tn5 insertion that most attenuated virulence, vir-22::Tn5, inhibited synthesis of the 28,000-molecular-weight protein. The vir-22::Tn5 insertion was complemented by recombinant plasmids encoding only the 28,000-molecular-weight protein, suggesting a role of this protein in virulence. However, recombinant plasmids, exemplified by pYA422, that encoded only the 28,000-molecular-weight protein did not confer full virulence.

Animals↗

PubMed1996

Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium.

Cytadherence and subsequent parasitism of host cells by the human pathogens, Mycoplasma pneumoniae and Mycoplasma genitalium, are mediated by adhesins and adherence-related accessory proteins. In this report we demonstrate the use of transposon Tn4001 to generate Tn-induced transformants displaying cytadherence-deficient characteristics. Mycoplasma pneumoniae Tn-generated transformant, designated 8R, lacked the high-molecular weight adherence-accessory proteins HMW1/4 and was deficient in hemadsorption and cytadherence capabilities. In transformant 8R, Tn4001 was not localized in or near the hmw1 gene or in the upstream adhesin (p30/hmw3) locus, suggesting an alternate site associated with the regulation of hmw1 gene expression. Sequence analysis identified the transposon insertion site at the crl locus previously reported, although the protein characteristics of transformant 8R differed from the earlier described transformants. The M. genitalium Tn-transformant, designated G26, was also defective in hemadsorption and cytadherence. However, transformant G26 synthesized adhesins P140 and P32 suggesting that Tn4001 transposed into a new gene or site previously unlinked to cytadherence, namely ORF MG032. This study demonstrates the utility of Tn4001 mutagenesis for both M. pneumoniae and M. genitalium which, in the latter case, has special relevance in light of the recent complete characterization of its continuous total genomic sequence.

Adhesins, Bacterial↗