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Results for “transposon insertion sequencing”

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PubMed2004

MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719.

MexAB-OprM is a multidrug efflux system that contributes to intrinsic and acquired multidrug resistance in Pseudomonas aeruginosa, the latter as a result of mutational hyperexpression of the mexAB-oprM operon. While efflux gene hyperexpression typically results from mutations in the linked mexR repressor gene, it also occurs independently of mexR mutations in so-called nalC mutants that demonstrate more modest mexAB-oprM expression and, thus, more modest multidrug resistance than do mexR strains. Using a transposon insertion mutagenesis approach, nalC mutant strains were selected and the disrupted gene, PA3721, identified. Amplification and sequencing of this gene from previously isolated spontaneous nalC mutants revealed the presence of mutations in all instances and as such, PA3721 has been renamed nalC. PA3721 (nalC) encodes a probable repressor of the TetR/AcrR family and occurs upstream of an apparent two-gene operon, PA3720-PA3719, whose expression was negatively regulated by PA3721. Thus, PA3720-PA3719 was hyperexpressed in transposon insertion and spontaneous nalC mutants. The loss of PA3719 but not of PA3720 expression in a spontaneous nalC mutant reduced MexAB-OprM expression to wild-type levels and compromised multidrug resistance, an indication that hyperexpression of PA3719 only was necessary for the nalC phenotype. Introduction of PA3719 into wild-type P. aeruginosa on a multicopy plasmid was, in fact, sufficient to promote elevated MexAB-OprM expression and multidrug resistance characteristic of a nalC strain. Thus, the nalC (PA3721) mutation serves only to enhance PA3720-PA3719 expression, with expression of PA3719 (encodes a 53 amino acid protein of predicted pI 10.4) directly or indirectly impacting MexAB-OprM expression. Intriguingly, nalC strains produce markedly elevated levels of stable MexR protein suggesting that PA3720-PA3719 hyperexpression somehow modulates MexR repressor activity. The deduced products of PA3720-PA3719 show no homology to sequences presently in the GenBank databases, however, and as such provide no clues as to how this might occur.

Bacterial Outer Membrane Proteins↗

PubMed2004

A faster way to make GFP-based biosensors: two new transposons for creating multicolored libraries of fluorescent fusion proteins.

BACKGROUND: There are now several ways to generate fluorescent fusion proteins by randomly inserting DNA encoding the Green Fluorescent Protein (GFP) into another protein's coding sequence. These approaches can be used to map regions in a protein that are permissive for GFP insertion or to create novel biosensors. While remarkably useful, the current insertional strategies have two major limitations: (1) they only produce one kind, or color, of fluorescent fusion protein and (2) one half of all GFP insertions within the target coding sequence are in the wrong orientation. RESULTS: We have overcome these limitations by incorporating two different fluorescent proteins coding sequences in a single transposon, either in tandem or antiparallel. Our initial tests targeted two mammalian integral membrane proteins: the voltage sensitive motor, Prestin, and an ER ligand gated Ca2+ channel (IP3R). CONCLUSIONS: These new designs increase the efficiency of random fusion protein generation in one of two ways: (1) by creating two different fusion proteins from each insertion or (2) by being independent of orientation.

Biosensing Techniques↗

PubMed1993

comF, a Bacillus subtilis late competence locus, encodes a protein similar to ATP-dependent RNA/DNA helicases.

We have sequenced and genetically characterized comF, a Bacillus subtilis competence locus, previously identified by Tn917 transposon insertion mutagenesis. Expression of the locus, in which three open reading frames (ORFs) were found, is driven by a single sigma A-like promoter in front of comFORF1 and is dependent on early regulatory competence genes and only expressed in competence medium. The predicted amino acid sequences of two of the ORFs showed similarities to known proteins in the GenBank and SwissProt databases: ComFORF1 is similar to an extensive family of ATP-dependent RNA/DNA helicases with closer similarity to the DEAD protein subfamily and to the PriA protein in Escherichia coli. The latter is a DNA translocase/helicase required for primosome assembly at the replication fork of phage phi X174. ComFORF3 is 22% identical to Com101, a protein required for genetic competence in Haemophilus influenzae, a naturally competent Gram-negative bacterium. In-frame comFORF1 deletions were 1000-fold deficient in transformability compared to the wild-type, whereas disruptions of the other two ORFs were only five- to 10-fold lower. These observations allow us to hypothesize that the ComFORF1 late gene product plays an essential role during the binding and uptake events involved in Bacillus subtilis transformation.

Adenosine Triphosphatases↗

PubMed1987

Polymorphisms on the right arm of yeast chromosome III associated with Ty transposition and recombination events.

The region of Saccharomyces cerevisiae chromosome III centromere-distal to the PGK gene is the site of frequent chromosome polymorphisms. We have sequenced this region from fragments of chromosome III isolated from three different yeast strains, GRF88, CN31C and CF4-16B. The sequence analysis demonstrates that these polymorphisms are associated with the presence of Ty and delta elements and defines a region of the chromosome which is a hot-spot for transposition events (the RAHS). The three strains can be arranged into a logical evolutionary series in which successive transposition and recombination events insert Ty elements and fuse them with consequent deletions of chromosome and of transposon sequences. The influence of such events on yeast genome evolution is discussed.

Base Sequence↗

PubMed1997

Circularized Ac/Ds transposons: formation, structure and fate.

The maize Ac/Ds transposable elements are thought to transpose via a cut-and-paste mechanism, but the intermediates formed during transposition are still unknown. In this work we present evidence that circular Ac molecules are formed in plants containing actively transposing elements. In these circles, transposon ends are joined head-to-head. The sequence at the ends' junction is variable, containing small deletions or insertions. Circles containing deleted Ac ends are probably unable to successfully reintegrate. To test the ability of circles with intact transposon ends to integrate into the genome, an artificial Ds circle was constructed by cloning the joined ends of Ac into a plasmid carrying a plant selectable marker. When such a circular Ds was introduced into tobacco protoplasts in the presence of Ac-transposase, no efficient transposase-mediated integration was observed. Although a circular transposition intermediate cannot be ruled out, the findings of circles with deleted transposon ends and the absence of transposase-mediated integration of the circular Ds suggest that some of the joined-ends-carrying elements are not transposition intermediates, but rather abortive excision products. The formation of Ac circles might account for the previously described phenomenon of Ac-loss. The origin of Ac circles and the implications for models of Ac transposition are discussed.

Chromosomes↗

PubMed2003

Identification of an activator protein required for the induction of fruA, a gene essential for fruiting body development in Myxococcus xanthus.

Myxococcus xanthus exhibits social behavior and multicellular development. FruA is an essential transcription factor for fruiting body development in M. xanthus. In the present study, the upstream promoter region was found to be necessary for the induction of fruA expression during development. A cis-acting element required for the induction was identified and was located between nucleotides -154 and -107 with respect to the transcription initiation site. In addition, it was found that two binding sites exist within this element of the fruA promoter. By using DNA affinity column chromatography containing the cis-acting element, a fruA promoter-binding protein was purified. The purified protein was shown by N-terminal sequence analysis to be identical to MrpC, a protein identified previously by transposon insertion mutagenesis as an essential locus for fruiting body development [Sun, H. & Shi, W. (2001) J. Bacteriol. 183, 4786-4795]. Furthermore, fruA mRNA was not detectable in the mrpC::km strain, demonstrating that MrpC is essential for fruA expression. Moreover, mutational analysis of the binding sites for MrpC in the fruA promoter indicates that binding of MrpC activates transcription of fruA in vivo. This report provides evidence for a direct molecular interaction involved in temporally regulated gene expression in M. xanthus.

Amino Acid Sequence↗

PubMedDate not supplied

DNA sequence at the end of IS1 required for transposition.

The insertion sequence IS1 belongs to a class of bacterial transposable genetic elements that can form compound transposons in which two copies of IS1 flank an otherwise non-transposable segment of DNA. IS1 differs from other known elements of this class (such as IS10, IS50 and IS903) in several respects. It is one of the smallest known insertion elements, exhibits a relatively complex array of open reading frames, is present in the chromosomes of various Enterobacteria, in some cases in many copies, and its insertion can result in the duplication of either 8 or 9 base pairs (bp) in the target DNA. Furthermore, although, like other members of the compound class, it seems to undergo direct transposition, IS1 also promotes replicon fusion (co-integrate formation) at a relatively high frequency. Like all other elements studied to date, the integrity of the extremities of IS1 are essential for efficient transposition. We have constructed a test system to determine the minimal DNA sequences at the extremities of IS1 required for transposition. Sequential deletions of the end sequences reveal that 21-25 bp of an isolated extremity are sufficient for transposition. A specific sequence 13-23 bp from the ends, defining the edge of the minimal sequence, is implicated as an essential site. The sites, symmetrically arrayed at both ends of IS1, correspond to the apparent consensus sequence of the known binding sites for the Escherichia coli DNA-binding protein (called integration host factor or IHF) which is required for the site-specific recombination that leads to integration of bacteriophage lambda into the bacterial genome. The sites at the ends of IS1 may thus bind a host protein, such as JHF or a related protein, that is involved in regulating the transposition apparatus.

Base Sequence↗

PubMed1997

Structural and genetic analysis of a mutant of Rhodobacter sphaeroides WS8 deficient in hook length control.

Motility in the photosynthetic bacterium Rhodobacter sphaeroides is achieved by the unidirectional rotation of a single subpolar flagellum. In this study, transposon mutagenesis was used to obtain nonmotile flagellar mutants from this bacterium. We report here the isolation and characterization of a mutant that shows a polyhook phenotype. Morphological characterization of the mutant was done by electron microscopy. Polyhooks were obtained by shearing and were used to purify the hook protein monomer (FlgE). The apparent molecular mass of the hook protein was 50 kDa. N-terminal amino acid sequencing and comparisons with the hook proteins of other flagellated bacteria indicated that the Rhodobacter hook protein has consensus sequences common to axial flagellar components. A 25-kb fragment from an R. sphaeroides WS8 cosmid library restored wild-type flagellation and motility to the mutant. Using DNA adjacent to the inserted transposon as a probe, we identified a 4.6-kb SalI restriction fragment that contained the gene responsible for the polyhook phenotype. Nucleotide sequence analysis of this region revealed an open reading frame with a deduced amino acid sequence that was 23.4% identical to that of FliK of Salmonella typhimurium, the polypeptide responsible for hook length control in that enteric bacterium. The relevance of a gene homologous to fliK in the uniflagellated bacterium R. sphaeroides is discussed.

Amino Acid Sequence↗

PubMed1991

Genetic analysis, nucleotide sequence, and products of two Pseudomonas denitrificans cob genes encoding nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase and cobalamin (5'-phosphate) synthase.

Tn5 Sp(r) transposons have been inserted into the 8-kb Pseudomonas denitrificans DNA fragment from complementation group D, which carries cob genes. Genetic analysis and the nucleotide sequence revealed that only two cob genes (cobU and cobV) were found on this cob genomic locus. Nicotinate-nucleotide: dimethylbenzimidazole phosphoribosyltransferase (EC 2.4.2.21) was assayed and purified to homogeneity from a P. denitrificans strain in which cobU and cobV were amplified. The purified enzyme was identified as the cobU gene product on the basis of identical molecular weights and N-terminal sequences. Cobalamin (5'-phosphate) synthase activity was increased when cobV was amplified in P. denitrificans. The partially purified enzyme catalyzed not only the synthesis of cobalamin 5'-phosphate from GDP-cobinamide and alpha-ribazole 5'-phosphate but also the one-step synthesis of cobalamin from GDP-cobinamide and alpha-ribazole. Biochemical data provided evidence that cobV encodes cobalamin (5'-phosphate) synthase.

Amino Acid Sequence↗

PubMed2004

Identification of two new genes involved in twitching motility in Pseudomonas aeruginosa.

Mu transposition complexes were used for transposon mutagenesis of Pseudomonas aeruginosa strain PA68. Mu DNA transposition complexes were assembled with MuA transposase and an artificial mini-Mu transposon in vitro, and introduced into Pseudomonas aeruginosa by electroporation. Eight mutants deficient in twitching motility were isolated. Southern blotting confirmed that the insertions had occurred as single events. DNA sequencing of the region flanking the insertion in the twitching-motility mutants revealed that the mini-Mu transposon had inserted into six different genes, PAO171, PA1822, PAO413, PA4959, PA4551 and PA5040. Four of these have previously been proven to be needed for twitching motility, whereas the PA1822 and PA0171 genes have not previously been shown to be required for twitching motility. The twitching-motility defect in the PA1822 mutant was partially complemented by providing the PA1822 gene in trans, and the defect in the PA0171 mutant was fully complemented when PA0171 was provided. A PA0171 mutant and a PA1822 mutant were constructed by gene replacement in the P. aeruginosa PAO1 strain. These mutants were deficient in twitching motility, showing that both the PA1822 and the PA0171 gene are involved in twitching motility.

Base Sequence↗

PubMed1991

Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point.

From examination of published DNA sequences of genes found inserted at a specific site in integrons, all genes are shown to be associated, at their 3' ends, with a short imperfect inverted repeat sequence, a 59-base element or relative of this element. The similarity of the arrangement of gene inserts in the integron and in the Tn7 transposon family is described. A refined consensus for the 59-base element is reported. Members of this family are highly diverged and the relationship of a group of longer elements to the 59-base elements is demonstrated. The ability of 59-base elements of different length and sequence to act as sites for recombination catalysed by the integron-encoded DNA integrase is demonstrated, confirming that elements of this family have a common function. The ability of elements located between gene pairs to act as recombination sites has also been demonstrated. The recombination cross-over point has been localized to the GTT triplet which is conserved in the core sites, GTTRRRY, found at the 3' end of 59-base elements. Recombination at the core site found in inverse orientation at the 5' end of the 59-base elements was not detected, and the sequences responsible for orientation of the recombination event appear to reside within the 59-base element. A model for site-specific insertion of genes into integrons and Tn7-like transposons is proposed. Circular units consisting of a gene associated with a 59-base element are inserted into an ancestral element which contains neither a gene nor a 59-base element.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1982

Molecular cloning of sequences from a Drosophila RNA polymerase II locus by P element transposon tagging.

We have identified a lethal mutation in the D. melanogaster RNA polymerase II locus, RpIIC4, caused by insertion of a transposable element associated with the phenomenon of hybrid dysgenesis (P element). Using previously cloned P element sequences as a hybridization probe we have isolated a hybrid lambda phage clone carrying a 10 kb genomic DNA fragment containing a 1.3 kb P element insert and flanking sequences from the RpII locus. The non-P sequences in this clone (lambda DmRpII-1) hybridize to polytene chromosome band region 10C, the cytogenetic location of RpIIC4, and revertants which lose the lethal RNA polymerase II mutation also lose P element sequences from the locus. We have generated several additional P element insertions into the locus and shown that they can occur at two or more different sites. These experiments illustrate that mutagenesis by P element insertion and use of cloned P DNA to retrieve the DNA sequences into which insertion has occurred may be a general method for cloning genetically defined loci in Drosophila.

Alleles↗

PubMed1995

Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants.

Establishment of loss-of-function phenotypes is often a key step in determining the biological function of a gene. We describe a procedure to obtain mutant petunia plants in which a specific gene with known sequence is inactivated by the transposable element dTph1. Leaves are collected from batches of 1000 plants with highly active dTph1 elements, pooled according to a three-dimensional matrix, and screened by PCR using a transposon- and a gene-specific primer. In this way individual plants with a dTph1 insertion can be identified by analysis of about 30 PCRs. We found insertion alleles for various genes at a frequency of about 1 in 1000 plants. The plant population can be preserved by selfing all the plants, so that it can be screened for insertions in many genes over a prolonged period.

Alleles↗

PubMed1986

Molecular cloning and characterization of sequences from the regulatory cluster of the Pseudomonas plasmid alk system.

Alkane oxidation functions encoded by the Pseudomonas plasmid CAM-OCT are positively regulated by one or more products of a locus designated alkR. To characterize this locus in greater detail, molecular cloning and restriction mapping of sequences covering the alkR region have been carried out in Escherichia coli, followed by mobilization to Pseudomonas recipients for analysis of genetic content. Inserts from Pseudomonas (CAM-OCT) strains were cloned into vectors pLAFR1, the pLAFR1::Tn7S derivative pXJS5403, and the transposon vector Tn3 delta 596. This has made it possible to: (1) construct a detailed restriction map of cloned fragments and the alkR region of CAM-OCT; (2) map insertion sites of the transposon Tn7S into alkR cistrons; and (3) analyze the ability of cloned sequences to complement or effect marker rescue of alkR nitrosoguanidine- and Tn7S-induced mutations. In addition, transcription of an alkB'-lacZ transcription fusion in the presence of a cloned 18.5 kb EcoRI alkR fragment and an inducer of the alk system confirmed that our cloned sequences contain functional alkR cistrons. The complementation/marker rescue results indicate that alkR is a complex locus and that the products of at least three cistrons are required for the complete AlkR+ phenotype. One of these cistrons is identified by mutations which alter a component of the inducer recognition system.

Alkanes↗

PubMed1980

A new filamentous phage cloning vector: fd-tet.

We have constructed a hybrid chromosome composed of the genome of wild-type fd (a filamentous, male-specific bacteriophage) and a segment of transposon Tn10 coding for tetracycline resistance but not including the Tn10 insertion sequences. The hybrid phage infects male E. coli, thereby transducing the infected cells to tetracycline resistance. The phage DNA can also be propagated in F- cells after transfection. This new phage, fd-tet, may be used as a cloning vector to produce large quantities of cloned DNA in single-stranded form. Its usefulness has been demonstrated by cloning of a fragment from bacteriophage lambda. Some unexpected sequence alterations have been identified in lambda cloning experiments.

Coliphages↗

PubMed2003

NaCl-sensitive mutant of Staphylococcus aureus has a Tn917-lacZ insertion in its ars operon.

Staphylococcus aureus is a Gram-positive bacterium that is extremely halotolerant. To investigate the molecular mechanisms by which S. aureus can cope with osmotic stress, Tn917-lacZ-induced NaCl-sensitive mutants were isolated. An NaCl-sensitive mutant showed a longer lag period, slower growth rate, and lower final culture turbidity than the parent strain in liquid medium containing 1.5 M NaCl. Electron microscopic observation of the NaCl-sensitive mutant under NaCl stress conditions revealed large, pseudo-multicellular cells. Addition of exogenous osmoprotectants, such as glycine betaine, choline, L-proline, and proline betaine, did not relieve the NaCl sensitivity of the mutant. The region flanking the transposon insertion site in the NaCl-sensitive S. aureus chromosome was sequenced. The mutated gene was 99% identical to arsR, the arsenic operon regulatory protein present on the pI258 plasmid of S. aureus. The ars operon from pI258 was subcloned into the shuttle vector pLI50 and transferred into the NaCl-sensitive mutant. The ars operon in trans restored NaCl tolerance in the mutant, suggesting that NaCl sensitivity is due to the mutation in arsR.

Bacterial Proteins↗

PubMed1999

Cloning and sequence analysis of the lipase and lipase chaperone-encoding genes from Acinetobacter calcoaceticus RAG-1, and redefinition of a proteobacterial lipase family and an analogous lipase chaperone family.

The genes encoding the lipase (LipA) and lipase chaperone (LipB) from Acinetobacter calcoaceticus RAG-1 were cloned and sequenced. The genes were isolated from a genomic DNA library by complementation of a lipase-deficient transposon mutant of the same strain. Transposon insertion in this mutant and three others was mapped to a single site in the chaperone gene. The deduced amino acid (aa) sequences for the lipase and its chaperone were found to encode mature proteins of 313 aa (32.5kDa) and 347 aa (38.6kDa), respectively. The lipase contained a putative leader sequence, as well as the conserved Ser, His, and Asp residues which are known to function as the catalytic triad in other lipases. A possible trans-membrane hydrophobic helix was identified in the N-terminal region of the chaperone. Phylogenetic comparisons showed that LipA, together with the lipases of A. calcoaceticus BD413, Vibrio cholerae El Tor, and Proteus vulgaris K80, were members of a previously described family of Pseudomonas and Burkholderia lipases. This new family, which we redefine as the Group I Proteobacterial lipases, was subdivided into four subfamilies on the basis of overall sequence homology and conservation of residues which are unique to the subfamilies. LipB, moreover, was found to be a member of an analogous family of lipase chaperones. We propose that the lipases produced by P. fluorescens and Serratia marcescens, which comprise a second sequence family, be referred to as the Group II Proteobacterial lipases. Evidence is provided to support the hypothesis that both the Group I and Group II families have evolved from a combination of common descent and lateral gene transfer.

Acinetobacter calcoaceticus↗

PubMed2005

A resource of 5,814 dissociation transposon-tagged and sequence-indexed lines of Arabidopsis transposed from start loci on chromosome 5.

We report here the generation of an additional collection of Dissociation (Ds) transposon-tagged, sequence-indexed lines of Arabidopsis thaliana. Our RIKEN Ds insertion collection now totals 17,668 lines. Our collection has preferential insertions in chromosomes 1 and 5, because Ds was transposed from start loci on those chromosomes (11,854 and 5,814 lines, respectively). We describe here features of the latter 5,814 lines. The former 11,854 lines have been described previously. We have created a searchable database of the insertion sites and mutated genes (http://rarge.gsc.riken.jp/), and are depositing these lines in the RIKEN BioResource Center (http://www.brc.riken.go.jp/lab/epd/Eng/). Our collection of these mutants will contribute to progress in functional genomics of plants.

Arabidopsis↗