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PubMed1994

Genetic rearrangement associated with in vivo mucoid conversion of Pseudomonas aeruginosa PAO is due to insertion elements.

The conversion of Pseudomonas aeruginosa PAO to the mucoid phenotype has been reported for a chronic pulmonary infection model in rats (D. E. Woods, P. A. Sokol, L. E. Bryan, D. G. Storey, S. J. Mattingly, H. J. Vogel, and H. Ceri, J. Infect. Dis. 163:143-149, 1991). This conversion was associated with a genetic rearrangement upstream of the exotoxin A gene. To characterize the genetic rearrangement, the region upstream of the toxA gene was cloned from PAO, PAO-muc (a mucoid strain), and PAO-rev (a nonmucoid revertant strain). The nucleotide sequence of a 4.8-kb fragment from PAO-muc was determined. A+T-rich regions of approximately 2 kb (IS-PA-4) and 0.4 kb (IS-PA-5) were identified in this fragment. DNA probes constructed internal to these regions hybridized to PAO-muc but not to PAO or PAO-rev, suggesting that PAO-muc contains an insertion element. Sequence analysis of the nonmucoid clones indicated that a 2,561-bp fragment corresponding to IS-PA-4 and a 992-bp fragment corresponding to IS-PA-5 were not present in PAO or PAO-rev. Both nonmucoid clones, however, contained in the same location as IS-PA-4, a 1,313-bp region which was not present in PAO-muc. DNA probes complementary to this sequence, designated IS-PA-6, did not hybridize with PAO-muc, indicating that this sequence had been replaced upon conversion to the mucoid phenotype. Between IS-PA-4 and IS-PA-5 there was a 500-bp sequence which was 94% identical to the 500-bp sequence downstream of IS-PA-6. These insertion elements had some DNA sequence similarity to plasmid and transposon sequences, suggesting that they may be of plasmid origin. IS-PA-4 and IS-PA-5 were shown also to be present in two mucoid isolates from cystic fibrosis patients. The insertions occurred in the same location upstream of the toxA gene, suggesting that this type of genetic recombination may also be associated with mucoid conversion in some P. aeruginosa clinical isolates.

ADP Ribose Transferases↗
PubMed1995

Sequence of a transposon identified as Tn1000 (gamma delta).

We report the complete sequence of a transposon found in a cosmid clone of a human DNA sequence. The transposon is identified as the Escherichia coli transposon Tn1000 (also known as gamma delta) on the basis of the identity of the restriction map of the new sequence with that previously recorded for Tn1000 and homology between parts of the new sequence and that of published fragments of Tn1000 sequence. The transposon, which comprises 5,981 nucleotides including two 35 bp inverted terminal repeat sequences (ITRs), contains three open reading frames. The sequence of the resolvase coding region (tnpR) is identical to that published by others. A second reading frame can be identified as the tnpA gene, coding for the transposase, on the grounds of its strong homology with the corresponding gene from transposon Tn3. The third reading frame has the potential to code for a protein of unknown function containing 698 amino acids.

Amino Acid Sequence↗
PubMed2025

Genome-wide analysis of Enterococcus faecalis genes that facilitate interspecies competition with Lactobacillus crispatus.

Enterococci are opportunistic pathogens notorious for causing a variety of infections. While both Enterococcus faecalis and Lactobacillus crispatus are commensal residents of the vaginal tract, the molecular mechanisms that enable E. faecalis to take advantage of a vaginal biome with lower counts of lactobacilli to colonize the vaginal tract and induce aerobic vaginitis remain unknown. Here, we show that L. crispatus eradicates E. faecalis in a contact-independent manner. Using transposon sequencing to identify E. faecalis OG1RF transposon (Tn) mutants that are either under-represented or over-represented when co-cultured with L. crispatus, we found that Tn mutants with disruption in the dltABCD operon, that encodes the proteins responsible for the D-alanylation of teichoic acids, and OG1RF_11697 encoding for an uncharacterized hypothetical protein are more susceptible to killing by L. crispatus. Inversely, Tn mutants with disruption in ldh1, which encodes for L-lactate dehydrogenase, are more resistant to L. crispatus killing. Using the Galleria mellonella infection model, we show that co-injection of L. crispatus with E. faecalis OG1RF enhances larvae survival while this L. crispatus-mediated protection was lost in larvae co-infected with either L. crispatus and E. faecalisΔldh1 or Δldh1Δldh2 strains. Last, using RNA sequencing to identify E. faecalis genes that are differently expressed in the presence of L. crispatus, we found major changes in the expression of genes associated with glycerophospholipid metabolism, central metabolism, and general stress responses. The findings in this study provide insights into how E. faecalis mitigate assaults by L. crispatus.IMPORTANCEEnterococcus faecalis is an opportunistic pathogen notorious for causing a multitude of infections. As vaginal commensals, E. faecalis must interact with Lactobacillus crispatus, but how E. faecalis overcomes or mitigate assaults by L. crispatus killing remains unknown. We show that L. crispatus eradicates E. faecalis temporally in a contact-independent manner. Using high-throughput molecular approaches, we identified genetic determinants that enable E. faecalis to compete with L. crispatus. This study represents an important first step for the identification of adaptive genetic traits required for enterococci to tolerate assaults by lactobacilli.

Enterococcus faecalis↗
PubMed2002

Random sequencing of Paramecium somatic DNA.

We report a random survey of 1 to 2% of the somatic genome of the free-living ciliate Paramecium tetraurelia by single-run sequencing of the ends of plasmid inserts. As in all ciliates, the germ line genome of Paramecium (100 to 200 Mb) is reproducibly rearranged at each sexual cycle to produce a somatic genome of expressed or potentially expressed genes, stripped of repeated sequences, transposons, and AT-rich unique sequence elements limited to the germ line. We found the somatic genome to be compact (>68% coding, estimated from the sequence of several complete library inserts) and to feature uniformly small introns (18 to 35 nucleotides). This facilitated gene discovery: 722 open reading frames (ORFs) were identified by similarity with known proteins, and 119 novel ORFs were tentatively identified by internal comparison of the data set. We determined the phylogenetic position of Paramecium with respect to eukaryotes whose genomes have been sequenced by the distance matrix neighbor-joining method by using random combined protein data from the project. The unrooted tree obtained is very robust and in excellent agreement with accepted topology, providing strong support for the quality and consistency of the data set. Our study demonstrates that a random survey of the somatic genome of Paramecium is a good strategy for gene discovery in this organism.

Amino Acid Sequence↗
PubMed1982

Insertion sites and the terminal nucleotide sequences of the Tn4 transposon.

The nucleotide sequences at the ends of the Tn4 transposon (mercury spectinomycin and sulfonamide resistance) have been determined. They are inverted repeated sequences of 38 nucleotides with three mismatched base pairs. These sequences are strongly homologous with the terminal sequences of Tn501 (mercury resistance) but less so with those of Tn3 (ampicillin resistance). The Tn4 transposon generates pentanucleotide members (Tn3, Tn1000, Tn501, Tn551, IS2) with the exception of Tn1721 and bacteriophage Mu. Among the three Tn4 insertion sites examined here, two of them occurred near a nonanucleotide sequence in perfect homology with part of the terminal inverted-repeat sequence of Tn4 and the third insertion occurred near a sequence of partial homology to one end of Tn4. All three insertions were in the same orientation such that IRb is proximal to its homologous sequence on the recipient DNA.

Base Composition↗
PubMed1999

Multiple causes of size variation in the diploid megabase chromosomes of African tyrpanosomes.

The chromosomes of many protozoans are polymorphic in size, but African trypanosomes contain diploid homologues which are exceptionally size-polymorphic. We present the first complete analysis of the structure of a Trypanosoma brucei megabase chromosome which reveals the concentration of repetitive sequence, non-random distribution of transposon-like elements, and a hemizygous variant surface glycoprotein gene expression site. Subsequent comparative analyses of size-polymorphic homologues show that the repetitive regions are highly polymorphic, as demonstrated in studies on the chromosomes of other protozoan parasites. We show that a large number of the transposon-like elements are located in these regions. However, although we have shown elsewhere that synteny is maintained in coding regions, homologous chromosomes may vary along their entire length. Thus, the variable chromosomal location of variant surface glycoprotein expression gene sites, the expansion and contraction of repetitive DNA, the number of putative transposons, sequence polymorphism at chromosome ends, and expansion and contraction within or between coding regions all contribute to huge chromosomal size polymorphisms in T brucei.

Animals↗
PubMed1991

Transposon mutagenesis in Proteus mirabilis.

A technique of transposon mutagenesis involving the use of Tn5 on a suicide plasmid was developed for Proteus mirabilis. Analysis of the resulting exconjugants indicated that Tn5 transposed in P. mirabilis at a frequency of ca. 4.5 x 10(-6) per recipient cell. The resulting mutants were stable and retained the transposon-encoded antibiotic resistance when incubated for several generations under nonselective conditions. The frequency of auxotrophic mutants in the population, as well as DNA-DNA hybridizaiton to transposon sequences, confirmed that the insertion of the transposon was random and the Proteus chromosome did not contain significant insertional hot spots of transposition. Approximately 35% of the mutants analyzed possessed plasmid-acquired ampicillin resistance, although no extrachromosomal plasmid DNA was found. In these mutants, insertion of the Tn5 element and a part or all of the plasmid had occurred. Application of this technique to the study of swarmer cell differentiation in P. mirabilis is discussed.

DNA Transposable Elements↗
PubMed2000

Identifying tagged transposon insertion sites in yeast by direct genomic sequencing.

Tagged transposons are powerful tools for large-scale studies of gene expression, protein localization, and gene disruption in Saccharomyces cerevisiae. The current techniques used to identify transposon insertion sites in the yeast genome require a DNA amplification step that can be time-consuming and problematic. We show that the DNA amplification step can be bypassed. Insertion sites can be identified rapidly and reliably by direct genomic sequencing using a transposon-specific primer, BigDye-labelled terminators, and an automated sequencer. Direct genomic sequencing can also save time on the genetic analysis phase of transposon-based projects.

Base Sequence↗
PubMed1999

An alternative inverse PCR (IPCR) method to amplify DNA sequences flanking Tn5 transposon insertions.

We have developed an alternative method to amplify DNA sequences flanking Tn5 transposon insertions. This method relies on the identical sequences of inverted terminal repeats, located at the 5' and 3' ends of Tn5, to determine the location and orientation of a transposon insertion within a restriction endonuclease fragment. From this information, PCR primers can be designed to selectively amplify by inverse PCR the DNA flanking one side of the transposon. This method avoids the problem of amplifying or cloning long sequences flanking Tn5. To demonstrate the applicability of this method, we generated Tn5 transposon mutants of Pseudomonas abietaniphila BKME-9 which no longer grew on dehydroabietic acid (DhA). The flanking sequence of one of the mutant (strain BKME-941) which accumulated 7-oxoDhA, was amplified.

Abietanes↗
PubMed2005

Genomic approaches reveal unexpected genetic divergence within Ciona intestinalis.

The invertebrate chordate Ciona intestinalis is a widely used model organism in biological research. Individuals from waters ranging from arctic to temperate are morphologically almost indistinguishable. However, we found significant differences in whole genomic DNA sequence between northern European and Pacific C. intestinalis. Intronic and transposon sequences often appear unrelated between these geographic origins and amino acid substitutions in protein coding sequences indicate a divergence time in excess of 20 MYA. This finding suggests the existence of two cryptic species within the present C. intestinalis species. We found five marker loci which distinguish the two genetic forms by PCR. This analysis revealed that specimens from Naples, Italy, have the Pacific-type genome, perhaps due to human-mediated marine transport of species. Despite major genomic divergence, the two forms could be hybridized in the laboratory.

Animals↗
PubMed2005

piggyBac internal sequences are necessary for efficient transformation of target genomes.

A previously reported piggyBac minimal sequence cartridge, which is capable of efficient transposition in embryo interplasmid transposition assays, failed to produce transformants at a significant frequency in Drosophila melanogaster compared with full-length or less extensive internal deletion constructs. We have re-examined the importance of these internal domain (ID) sequences for germline transformation using a PCR strategy that effectively adds increasing lengths of ID sequences to each terminus. A series of these piggyBac ID synthetic deletion plasmids containing the 3xP3-ECFP marker gene are compared for germline transformation of D. melanogaster. Our analyses identify a minimal sequence configuration that is sufficient for movement of piggyBac vectored sequences from plasmids into the insect genome. Southern hybridizations confirm the presence of the piggyBac transposon sequences, and insertion site analyses confirm these integrations target TTAA sites. The results verify that ID sequences adjacent to the 5' and 3' terminal repeat domains are crucial for effective germline transformation with piggyBac even though they are not required for excision or interplasmid transposition. Using this information we reconstructed an inverted repeat cartridge, ITR1.1k, and a minimal piggyBac transposon vector, pXL-BacII-ECFP, each of which contains these identified ID sequences in addition to the terminal repeat configuration previously described as essential for mobility. We confirm in independent experiments that these new minimal constructs yield transformation frequencies similar to the control piggyBac vector. Sequencing analyses of our constructs verify the position and the source of a point mutation within the 3' internal repeat sequence of our vectors that has no apparent effect on transformation efficiency.

Animals↗
PubMed2005

The post-transcriptional gene silencing machinery functions independently of DNA methylation to repress a LINE1-like retrotransposon in Neurospora crassa.

Post-transcriptional gene silencing (PTGS) involving small interfering RNA (siRNA)-directed degradation of RNA transcripts and transcriptional silencing via DNA methylation have each been proposed as mechanisms of genome defence against invading nucleic acids, such as transposons and viruses. Furthermore, recent data from plants indicates that many transposons are silenced via a combination of the two mechanisms, and siRNAs can direct methylation of transposon sequences. We investigated the contribution of DNA methylation and the PTGS pathway to transposon control in the filamentous fungus Neurospora crassa. We found that repression of the LINE1-like transposon, Tad, requires the Argonaute protein QDE2 and Dicer, each of which are required for transgene-induced PTGS (quelling) in N.crassa. Interestingly, unlike quelling, the RNA-dependent RNA polymerase QDE1 and the RecQ DNA helicase QDE3 were not required for Tad control, suggesting the existence of specialized silencing pathways for diverse kinds of repetitive elements. In contrast, Tad elements were not significantly methylated and the DIM2 DNA methyltransferase, responsible for all known DNA methylation in Neurospora, had no effect on Tad control. Thus, an RNAi-related transposon silencing mechanism operates during the vegetative phase of N.crassa that is independent of DNA methylation, highlighting a major difference between this organism and other methylation-proficient species.

DNA Methylation↗
PubMed1992

Construction and application of plasmid- and transposon-based promoter-probe vectors for Streptomyces spp. that employ a Vibrio harveyi luciferase reporter cassette.

Several versatile promoter-probe vectors have been constructed for Streptomyces strains which utilize the production of blue-green light as a measure of transcription activity. Three plasmid vectors (two high and one low copy number) and two transposons are described. The multicopy plasmids pRS1106 and pRS1108 contain a transcription terminator and multiple-cloning polylinker upstream of promoterless luciferase (lux) and neomycin resistance reporter genes. Plasmid pHI90 is similar in structure to the pRS vectors except that its single copy number is an advantage for regulation studies or situations in which overexpression is otherwise toxic to the cell. The two transposons carry a promoterless lux cassette cloned such that transposition into a target DNA and fusion to the target's transcription unit occur simultaneously. Tn5351 was created by inserting the luciferase genes near the right end of the viomycin resistance transposon Tn4563. Tn5353 carries the luciferase genes near the right end of a neomycin resistance transposon derived from Tn4556. The size of Tn5353 was minimized by deleting nonessential transposon sequences, making this element small enough to be cloned into phi C31 bacteriophages for efficient transposon delivery to target cells of Streptomyces strains. The two Tnlux transposons have been used to generate Streptomyces coelicolor morphological mutants and to monitor transcription from chromosomal promoters during development.

Chromosomes, Bacterial↗
PubMed1985

The nucleotide sequence of the tnpA gene completes the sequence of the Pseudomonas transposon Tn501.

The nucleotide sequence of the gene (tnpA) which codes for the transposase of transposon Tn501 has been determined. It contains an open reading frame for a polypeptide of Mr = 111,500, which terminates within the inverted repeat sequence of the transposon. The reading frame would be transcribed in the same direction as the mercury-resistance genes and the tnpR gene. The amino acid sequence predicted from this reading frame shows 32% identity with that of the transposase of the related transposon Tn3. The C-terminal regions of these two polypeptides show slightly greater homology than the N-terminal regions when conservative amino acid substitutions are considered. With this sequence determination, the nucleotide sequence of Tn501 is fully defined. The main features of the sequence are briefly presented.

Amino Acid Sequence↗
PubMed1991

Kinetic and structural analysis of a cleaved donor intermediate and a strand transfer intermediate in Tn10 transposition.

Tn10 transposes by a nonreplicative "cut and paste" mechanism. We describe here two protein-DNA complexes that are reaction intermediates in the Tn10 transposition process: a cleaved donor complex whose DNA component consists of transposon sequences cleanly excised from flanking donor DNA, and a strand transfer complex whose DNA component contains transposon termini specifically joined to a target site. The kinetic behavior of the first species suggests that it is an early intermediate in the transposition reaction. These two Tn10 complexes are closely analogous to complexes identified in the pathway for replicative "cointegrate" formation by bacteriophage Mu and thus represent intermediates that may be common to both nonreplicative and replicative transposition. These and other results suggest that the Tn10 and Mu reactions are fundamentally very similar despite their very different biological outcomes. The critical difference between the two reactions is the fate of the DNA strand that is not joined to target DNA.

Bacterial Proteins↗
PubMed2004

Counterselection and co-delivery of transposon and transposase functions for Sleeping Beauty-mediated transposition in cultured mammalian cells.

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.

3T3 Cells↗
PubMed2003

Characterization of a plasmid-encoded adhesin of an avian pathogenic Escherichia coli (APEC) strain isolated from a case of swollen head syndrome (SHS).

An avian pathogenic Escherichia coli (APEC) strain designated SHS4, isolated from a chicken with clinical signs of swollen head syndrome (SHS), adhered to but did not invade Hep-2 and tracheal epithelial cells. The PCR amplified fimA, csgA and tsh gene sequences. It produced Ia, Ib, E1, E3, K, and B colicins, but not colicin V and aerobactin. It harboured two plasmids of 60 and 98MDa and was resistant to streptomycin and tetracycline. Conjugation with a nalidixic acid (Na) resistant K-12 recipient strain (MS101) showed that the 98MDa plasmid did not transfer, whereas transfer of the 60MDa plasmid resulted in concomitant transfer of adhesion to Hep-2 and tracheal epithelial cells, production of the colicins Ia, E1, E3, and K, and the tsh-related DNA sequence. Transposon (TnphoA) mutagenesis of strain TR4 gave rise to strain Mut23, which lost its adhesive capacities, but was still able to express the same colicins as did strain TR4. PCR was able to amplify the tsh-related DNA sequence in this strain and a molecular probe based on transposon TnphoA indicated that the transposon was inserted in the 60MDa plasmid. Based on these results, we suggest that the 60MDa plasmid have adhesion genes, which may be responsible for the initial colonization of the upper respiratory tract of chickens.

Adhesins, Escherichia coli↗