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Results for “transposon insertion sequencing”

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PubMed1997

Insertion mutagenesis as a tool in the modification of protein function. Extended substrate specificity conferred by pentapeptide insertions in the omega-loop of TEM-1 beta-lactamase.

The TEM-1 beta-lactamase enzyme efficiently hydrolyzes beta-lactam antibiotics such as ampicillin but cleaves third generation cephalosporin antibiotics poorly. Variant beta-lactamases that conferred elevated levels of resistance to the cephalosporin ceftazidime were identified in a set of beta-lactamase derivatives previously generated by pentapeptide scanning mutagenesis in which a variable 5-amino acid cassette was introduced randomly in the target protein. This mutagenesis procedure was also modified to allow the direct selection of variant beta-lactamases with pentapeptide insertions that conferred extended substrate specificities. All insertions associated with enhanced resistance to ceftazidime were targetted to the 19-amino acid Omega-loop region, which forms part of the catalytic pocket of the beta-lactamase enzyme. However, pentapeptide insertions in the C- and N-terminal halves of this region had different effects on the ability of the enzyme to hydrolyze ampicillin in vivo. Larger insertions that increased the length of the Omega-loop by up to 2-fold also retained catalytic activity toward ampicillin and/or ceftazidime in vivo. In accord with previous substitution mutation studies, these results emphasize the extreme flexibility of the Omega-loop with regards the primary structure requirements for ceftazidime hydrolysis by beta-lactamase. The potential of pentapeptide scanning mutagenesis in mimicking evolution events that result from the insertion and excision of transposons in nature is discussed.

Amino Acid Sequence↗

PubMed1991

A directed DNA sequencing strategy based upon Tn3 transposon mutagenesis: application to the ADE1 locus on Saccharomyces cerevisiae chromosome I.

We have developed a directed DNA sequencing strategy based upon the Escherichia coli transposon Tn3. This transposon displays little sequence specificity for transposition and is thus well suited to this task. Both mini-Tn3 transposons and sequencing vectors bearing the phage f1 single stranded origin of replication have been constructed. Upon mutagenesis of a target sequence, a population is produced in which each clone has two f1 origins of replication, one of which is at a variable position depending upon the transposon insertion site. When helper phage is added to the mutagenised population, the two f1 origins present on each clone are nicked, dividing the packaged strand into two segments, each of which is packaged into a separate phage particle. One of these segments contains no resistance markers and is lost, whilst the other is recovered as a deleted clone with a single chimeric f1 origin. A unidirectionally, variably-deleted set of sequencing clones is produced, and appropriately sized clones are sequenced using a primer complimentary to the transposon end. In addition to being inexpensive, the method does not require the same degree of technical expertise needed for many in vitro, enzymatically based methods. The strategy has been used to determine 2.6 kilobases of nucleotide sequence in the Saccharomyces cerevisiae ADE 1 locus.

Amino Acid Sequence↗

PubMed1998

Characterization of the genes for the biosynthesis of the compatible solute ectoine in the moderately halophilic bacterium Halomonas elongata DSM 3043.

The ectoine synthesis genes of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been precisely located in a 2.8-kb EcoRI region of a cosmid clone previously isolated (CANOVAS, D., VARGAS, C., IGLESIAS-GUERRA, F., CSONKA, L. N., RHODES, D., VENTOSA, A., NIETO, J. J.: Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes. J. Biol. Chem. 272, 25794-25801, 1997). This region was sequenced and three open reading frames were found corresponding to the genes ectA (encoding the diaminobutyric acid acetyl transferase), ectB (encoding the diaminobutyric acid aminotransferase) and ectC (encoding the ectoine synthase). These three genes were able to restore the salt tolerance of two H. elongata mutants defective in the synthesis of ectoine (strains CHR62 and CHR63). However, the H. elongata ectoine synthesis genes did not confer to Escherichia coli the ability to synthesize ectoine. Transposon insertion in the salt-sensitive mutant strain CHR63 was exactly mapped within the ectC gene. Moreover, sequences homologous to the H. elongata ect region have been found in a number of moderately halophilic bacteria belonging to the genera Halomonas and Chromohalobacter.

Acetyltransferases↗

PubMed2005

Genetic and physiological characterization of 23S rRNA and ftsJ mutants of Borrelia burgdorferi isolated by mariner transposition.

Borrelia burgdorferi contains one 16S rRNA gene and two tandem sets of 23S and 5S rRNA genes located in a single chromosomal region. This unusual rRNA gene organization has been speculated to be involved in the slow growth of this organism. Because we were repeatedly unable to isolate a 23S ribosomal mutant in B. burgdorferi by allelic exchange, we developed a transposition mutagenesis system for this bacterium. To this end, Himar1 transposase is expressed in B. burgdorferi from a resident plasmid containing an erythromycin resistance marker, and this strain is then electroporated with suicide plasmids containing mariner transposons and kanamycin resistance genes expressible in B. burgdorferi. This system permitted us to generate hundreds of erythromycin/kanamycin-resistant B. burgdorferi clones with each of three suicide plasmids. DNA sequencing of several kanamycin-resistant clones generated with one of the suicide plasmids showed stable and random insertion of the transposon into the B. burgdorferi chromosomal and plasmid genome. One mutant was inactivated in rrlA (23S rRNA), another in ftsJ (rrmJ). rrlA disruption had no effect on growth rate under a wide range of culture conditions, but disruption of ftsJ interfered significantly with growth rate and bacterial morphology. These data show it is possible to isolate random and stable B. burgdorferi transposition mutants for physiological analysis of this pathogenic spirochete.

Bacterial Proteins↗

PubMed1999

Signalling by the fungus Pythium ultimum represses expression of two ribosomal RNA operons with key roles in the rhizosphere ecology of Pseudomonas fluorescens F113.

Pseudomonas fluorescens F113 produces antifungal metabolites that protect the roots of sugarbeet from the fungus Pythium ultimum. The phytopathogen, in turn, has the ability to downregulate the expression of genes fundamental to the rhizosphere competence of the bacterial strain. This paper describes the characterization of two of these genes, which were isolated by screening a mini-Tn5::lacZ mutant bank for differential expression of beta-galactosidase in the presence of P. ultimum. In order to identify the genes affected in reporter mutants SF3 and SF5, the transposons and flanking regions were cloned. Sequence analysis of the regions flanking the transposons in SF3 revealed that mini-Tn5::lacZ had inserted into a tRNA(Ile) gene, which maps within a ribosomal RNA (rrn) operon. In SF5, the transposon inserted between the promoter of a second rrn operon and a gene encoding a 16S rRNA. Southern blot analysis demonstrated that there are five rrn operons in P. fluorescens F113 and that the transposons in SF3 and SF5 had inserted into two different operons. Further characterization of these mutants suggests that their reduced rhizosphere competence is not the result of reduced viability in the short term but may be accounted for partly by reduced growth rates under conditions that support rapid growth. Analysis of lacZ expression in the reporter mutants indicate that the marked rrn operons are regulated differently, suggesting different physiological roles.

Base Sequence↗

PubMed1997

DNA replication promotes high-frequency homologous recombination during Autographa californica multiple nuclear polyhedrosis virus infection.

The relative ease with which foreign genes can be incorporated into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) indicates that a highly efficient recombinational process exists within infected cells. However, it is unclear whether this is due to marker transfer mediated by host cell enzymes or recombination events promoted by AcMNPV itself. To address the latter possibility, a pair of inverted repeat IS50 elements derived from the bacterial transposon Tn5 was inserted into the polyhedrin gene locus of the AcMNPV genome. Inversion of Tn5 sequences arising from recombination between its IS50 repeats could be readily detected in this virus, indicating that AcMNPV DNA undergoes high-frequency recombination during infection. To further characterize this process, a transient recombination assay was developed and used to identify the cis- and trans-acting requirements for Tn5 inversion in AcMNPV. A transfected Tn5-containing plasmid was found to undergo the same sequence inversion events seen in the viral genome, but only if it also contained a putative AcMNPV origin of replication (homologous region 2) in cis and was replicated by AcMNPV gene products supplied in trans. Taken together, these results indicated that recombination events which occur in infected cells were strictly dependent upon AcMNPV-mediated DNA replication. Direct support for this hypothesis was provided by the observation that the minimal set of AcMNPV genes that was essential for plasmid DNA replication also promoted recombination events leading to Tn5 inversion in the absence of any other viral function. Finally, using a panel of deletion mutants of the IS50 elements in Tn5, sequence inversion was shown to be the result of homologous rather than site-specific recombination, since it occurred independently of a discrete sequence within the transposon. These results demonstrate that the AcMNPV DNA replication machinery exhibits a strong propensity to promote homologous recombination events during infection and is likely to play a role in the high frequency of marker transfer observed in this virus.

Animals↗

PubMed1992

A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy.

A new procedure is described to recombine plasmid-borne lacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flanking lacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncated lacZ and aadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carrying lacZ and aadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy in Pseudomonas putida the regulatory elements controlling expression of the XylS-activated Pm promoter of the TOL catabolic plasmid pWWO. We show here that the Pm promoter undergoes a XylS-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.

Blotting, Southern↗

PubMed1984

Multiple IS10 rearrangements in Escherichia coli.

We have investigated the occurrence of multiple transposon-promoted chromosomal rearrangements in Escherichia coli K12 strains containing transposon Tn10. We show that a single Tn10 element, with its two closely spaced insertion sequence (IS10) elements, frequently gives rise to complex rearrangements that can be accounted for as the sum of two "classical" IS10 events. Using a strain containing differentially marked Tn10 elements at widely separated locations, we have investigated the possibility that IS10-promoted rearrangements occur in cell-wide "bursts", as expected if cells could occasionally undergo brief periods when all IS10 transposition events were activated, interspersed with longer periods of relative quiescence. We find no evidence for strong (greater than 60-fold), periodic cell-wide activation under our experimental conditions. The sensitivity of this experiment has been evaluated using an expression for the accumulation of double mutations in populations with heterogeneous, fluctuating mutation rates (see Appendix). We discuss several mechanisms by which two closely linked IS10 elements could undergo coupled double events without cell-wide activation: local activation of small chromosomal regions, periodic bursts of synthesis of cis-acting transposase protein, and/or a propensity for elements that have actually engaged in one rearrangement event to initiate a second successive event immediately thereafter. We favor the last possibility.

Base Sequence↗

PubMed2004

Transposon-independent increase of transcription by the Sleeping Beauty transposase.

When a plasmid encoding the Sleeping Beauty (SB) transposase (pCMV-SB) was cointroduced with luciferase expression plasmid DNA into mouse skeletal muscle at a molar ratio of 4:1, luciferase gene expression was 5 times higher than the expression without pCMV-SB on day 28. This enhancement was not dependent on the presence of transposon (Tn) sequence in luciferase expression plasmid. Southern blot analysis failed to detect luciferase gene insertion into the host genome. Then, expression, a luciferase expression plasmid without Tn, was cointroduced into HeLa cells with or without pCMV-SB. With pCMV-SB, the mRNA amount and the luciferase activity were 1.5 times and 2 times higher, respectively, than without pCMV-SB, even though the cells with pCMV-SB had a smaller copy number of luciferase plasmids than the cells without pCMV-SB. These results suggest that SB transposase enhances the transcription of an exogenous gene regardless of the presence of the Tn sequence.

Animals↗

PubMed1999

A corrinoid-dependent catabolic pathway for growth of a Methylobacterium strain with chloromethane.

Methylobacterium sp. strain CM4, an aerobic methylotrophic alpha-proteobacterium, is able to grow with chloromethane as a carbon and energy source. Mutants of this strain that still grew with methanol, methylamine, or formate, but were unable to grow with chloromethane, were previously obtained by miniTn5 mutagenesis. The transposon insertion sites in six of these mutants mapped to two distinct DNA fragments. The sequences of these fragments, which extended over more than 17 kb, were determined. Sequence analysis, mutant properties, and measurements of enzyme activity in cell-free extracts allowed the definition of a multistep pathway for the conversion of chloromethane to formate. The methyl group of chloromethane is first transferred by the protein CmuA (cmu: chloromethane utilization) to a corrinoid protein, from where it is transferred to H4folate by CmuB. Both CmuA and CmuB display sequence similarity to methyltransferases of methanogenic archaea. In its C-terminal part, CmuA is also very similar to corrinoid-binding proteins, indicating that it is a bifunctional protein consisting of two domains that are expressed as separate polypeptides in methyl transfer systems of methanogens. The methyl group derived from chloromethane is then processed by means of pterine-linked intermediates to formate by a pathway that appears to be distinct from those already described in Methylobacterium. Remarkable features of this pathway for the catabolism of chloromethane thus include the involvement of a corrinoid-dependent methyltransferase system for dehalogenation in an aerobe and a set of enzymes specifically involved in funneling the C1 moiety derived from chloromethane into central metabolism.

Amino Acid Sequence↗

PubMedDate not supplied

Molecular epidemiology of antibiotic resistance plasmids of Haemophilus species and Neisseria gonorrhoeae.

Ampicillin resistance in Haemophilus influenzae and Neisseria gonorrhoeae is most commonly due to plasmid-mediated production of the TEM beta-lactamase. The H. influenzae plasmids may have evolved by insertion of various antibiotic resistance transposons into a phenotypically cryptic plasmid found in one of 699 isolates of H. influenzae examined. The small, nonconjugative, beta-lactamase-specifying plasmids of N. gonorrhoeae and Haemophilus species are highly related. Phenotypically cryptic plasmids found in several epidemiologically distinct isolates of Haemophilus parainfluenzae are highly related to the beta-lactamase plasmids but carry no transposon A (TnA) sequences. This evidence strongly favors the hypothesis that the beta-lactamase plasmids evolved by the insertion of TnA (possibly introduced from enteric bacteria) into cryptic plasmids resident in H. parainfluenzae.

Conjugation, Genetic↗

PubMed1987

A Tn10-lacZ-kanR-URA3 gene fusion transposon for insertion mutagenesis and fusion analysis of yeast and bacterial genes.

We describe here a new variant of transposon Tn10 especially adapted for transposon analysis of cloned yeast genes; it can equally well be used for analysis of prokaryotic genes. We have applied this element to analysis of the LEU2, RAD50, and CDC48 genes of Saccharomyces cerevisiae. This transposon, nicknamed mini-Tn10-LUK, contains a lacZ gene without efficient transcription or translation start signals, an intact URA3 gene, and a kanR determinant. The lacZ gene can be activated by appropriate insertion of the element into an actively expressed gene. Other yeast genes can easily be substituted for URA3 in the available constructs. The mini-Tn10-LUK system has several important advantages. Transposition events occur in Escherichia coli at high frequency and into many different sites in yeast DNA. It is easy to obtain enough insertions to sensitively define the functional limits of a gene. Transposon insertions can be obtained in a single step by standard transposon procedures and can be screened immediately for phenotype either in yeast or in E. coli. The LacZ phenotypes of the insertion mutations provide a good circumstantial indication of the orientation of the target gene. Under favorable circumstances, usable lacZ protein fusions are created. Transposon insertion mutations obtained by this method directly facilitate additional genetic, functional, physical and DNA sequence analysis of the gene or region of interest.

Base Sequence↗

PubMed1995

An in vivo transposase-catalyzed single-stranded DNA circularization reaction.

Expression of the bacterial insertion sequence IS911 transposase in vivo leads to excision and circularization of IS911-based transposons. We show here that transposase produces an unusual molecular form generated by single-strand cleavage, transfer, and ligation of one end of the element to the opposite end. When the transposon is carried by a circular plasmid, this results in the formation of a "figure-eight" molecule in which a single strand of the transposon is circularized while the corresponding strand of the vector backbone retains a single-strand interruption at this position. The results show that a 3' end of the transposon is transferred to the opposite target end. Transposase is therefore capable of introducing single-strand cleavages at the ends of the element, an activity similar to that of retroviral integrases with which it shares significant similarities in amino acid sequence. Kinetic studies demonstrate that the figure-eight accumulates earlier than transposon circles after transposase induction and disappears before circles after inhibition of transposase expression, raising the possibility that the figure-eight molecules are precursors to the circles. Therefore, IS911 excision as a circle may not occur by double-strand cleavage leading to its prior separation from the vector backbone in a linear form but could proceed by consecutive circularization of each strand.

Base Sequence↗

PubMed2001

Identification of Mutator insertional mutants of starch-branching enzyme 2a in corn.

Starch-branching enzymes (SBE) break the alpha-1,4 linkage of starch, re-attaching the chain to a glucan chain by an alpha-1,6 bond, altering starch structure. SBEs also facilitate starch accumulation by increasing the number of non-reducing ends on the growing chain. In maize (Zea mays), three isoforms of SBE have been identified. To examine the function of the SBEIIa isoform, a reverse genetics polymerase chain reaction-based screen was used to identify a mutant line segregating for a Mutator transposon within Sbe2a. To locate the insertion within the second exon of Sbe2a, the genomic sequence of Sbe2a containing the promoter and 5' end was isolated and sequenced. Plants homozygous for sbe2a::Mu have undetectable levels of Sbe2a transcripts and SBEIIa in their leaves. Characterization of leaf starch from sbe2a::Mu mutants shows reduced branching similar to yet more extreme than that seen in kernels lacking SBEIIb activity. Characterization of endosperm starch from sbe2a::Mu mutants shows branching that is indistinguishable from wild-type controls. These mutant plants have a visible phenotype resembling accelerated senescence, which was correlated with the Mutator insertion within Sbe2a. This correlation suggests a specific role for SBEIIa in leaves, which may be necessary for normal plant development.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1992

Isolation and analysis of IS6120, a new insertion sequence from Mycobacterium smegmatis.

Insertion sequence IS6120 from Mycobacterium smegmatis was identified by its ability to transpose into different sites in the lambda repressor gene, cl857, carried on an Escherichia coli/mycobacteria shuttle plasmid. IS6120 is a novel 1.5 kb insertion sequence, which has 24-bp imperfect terminal inverted repeats and generates 9-bp duplications of the target DNA following insertion. IS6120 is present in at least three copies in M. smegmatis but was not found in other species, including Mycobacterium tuberculosis. Nucleotide sequence analysis revealed that IS6120 contains two open reading frames, one of which encodes a putative transposase with similarities to those found in IS256 from Staphylococcus aureus, IST2 from Thiobacillus ferrooxidans, and ISRm3 from Rhizobium meliloti. The fact that IS6120 does not recognize a consensus target sequence for insertion and has no homologous sequences in the other strains studied makes IS6120 useful for transposon mutagenesis in mycobacteria.

Amino Acid Sequence↗

PubMed2000

Fitness in soil and rhizosphere of Pseudomonas fluorescens C7R12 compared with a C7R12 mutant affected in pyoverdine synthesis and uptake.

Fluorescent pseudomonads have evolved an efficient strategy of iron uptake based on the synthesis of the siderophore pyoverdine and its relevant outer membrane receptor. The possible implication of pyoverdine synthesis and uptake on the ecological competence of a model strain (Pseudomonas fluorescens C7R12) in soil habitats was evaluated using a pyoverdine minus mutant (PL1) obtained by random insertion of the transposon Tn5. The Tn5 flanking DNA was amplified by inverse PCR and sequenced. The nucleotide sequence was found to show a high level of identity with pvsB, a pyoverdine synthetase. As expected, the mutant PL1 was significantly more susceptible to iron starvation than the wild-type strain despite its ability to produce another unknown siderophore. As with the wild-type strain, the mutant PL1 was able to incorporate the wild-type pyoverdine and five pyoverdines of foreign origin, but at a significantly lower rate despite the similarity of the outer membrane protein patterns of the two strains. The survival kinetics of the wild-type and of the pyoverdine minus mutant, in bulk and rhizosphere soil, were compared under gnotobiotic and non-gnotobiotic conditions. In gnotobiotic model systems, both strains, when inoculated separately, showed a similar survival in soil and rhizosphere, suggesting that iron was not a limiting factor. In contrast, when inoculated together, the bacterial competition was favorable to the pyoverdine producer C7R12. The efficient fitness of PL1 in the presence of the indigenous microflora, even when coinoculated with C7R12, is assumed to be related to its ability to uptake heterologous pyoverdines. Altogether, these results suggest that pyoverdine-mediated iron uptake is involved in the ecological competence of the strain P. fluorescens C7R12.

Journal Article↗

PubMed1994

Genetics of galactose metabolism of Erwinia amylovora and its influence on polysaccharide synthesis and virulence of the fire blight pathogen.

Galactose metabolism mutants of Erwinia amylovora were created by transposon insertions and characterized for their growth properties and interaction with plant tissue. The nucleotide sequence of the galE gene was determined. The gene, which encodes UDP-galactose 4-epimerase, shows homology to the galE genes of Escherichia coli, Neisseria gonorrhoeae, Rhizobium meliloti, and other gram-negative bacteria. Cloned DNA with the galE and with the galT and galK genes did not share borders, as judged by the lack of common fragments in hybridization with chromosomal DNA. These genes are thus located separately on the bacterial chromosome. In contrast to the gal operon of E. coli, the galE gene of E. amylovora is constitutively expressed, independently of the presence of galactose in the medium. The function of the galE gene but not of the galT or galK gene is required for bacterial virulence on pear fruits and seedlings. In the absence of galactose, the galE mutant was deficient in amylovoran synthesis. Subsequently, the galE mutant cells elicited host defense reactions, and they were not stained by fluorescein isothiocyanate-labelled lectin, which efficiently binds to amylovoran capsules of E. amylovora. The mutation affected the side chains of bacterial lipopolysaccharide, but an intact O antigen was not required for virulence. This was shown with another mutant, which could be complemented for virulence but not for side chain synthesis of lipopolysaccharide.

Amino Acid Sequence↗

PubMed1985

[Cis-active function of plasmid R 57 resolving co-integrates formed during transposition of ISI-elements].

The mechanism of pBR322 plasmid mobilization in the cells of Escherichia coli K-12 recA by conjugative factor R57 was studied. It was shown that mobilization of pB322 is achieved by formation of unstable IS1-mediated cointegrates with R57. In the rec+ E. coli strains cointegrates resolved with formation of pBR322:IS1 plasmids. In the recA bacteria the cointegrates dissociated to pBR:IS1, as well as to other insertion derivatives of pBR322. Some of the latter contain Tn9-like sequence, i.e. a transposon flanked by direct repeats of IS1. The subsequent transposition of IS1 from pBR-. IS1 to pBR3.1 plasmid (Aps deletion derivative of RP1) leads to formation of stable cointegrates incapable of dissociating even in the presence of coresident plasmid R57. It is suggested that R57 encodes the cys-acting function providing recA-independent recombination between direct repeats of IS1.

Ampicillin↗