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At least 145 records · Page 8Linked to original sources

PubMed2003

Maize-targeted mutagenesis: A knockout resource for maize.

We describe an efficient system for site-selected transposon mutagenesis in maize. A total of 43,776 F1 plants were generated by using Robertson's Mutator (Mu) pollen parents and self-pollinated to establish a library of transposon-mutagenized seed. The frequency of new seed mutants was between 10-4 and 10-5 per F1 plant. As a service to the maize community, maize-targeted mutagenesis selects insertions in genes of interest from this library by using the PCR. Pedigree, knockout, sequence, phenotype, and other information is stored in a powerful interactive database (maize-targeted mutagenesis database) that enables analysis of the entire population and the handling of knockout requests. By inhibiting Mu activity in most F1 plants, we sought to reduce somatic insertions that may cause false positives selected from pooled tissue. By monitoring the remaining Mu activity in the F2, however, we demonstrate that seed phenotypes depend on it, and false positives occur in lines that appear to lack it. We conclude that more than half of all mutations arising in this population are suppressed on losing Mu activity. These results have implications for epigenetic models of inbreeding and for functional genomics.

Base Sequence↗

PubMed1994

Insertional inactivation of the gene for the meningococcal lactoferrin binding protein.

The lactoferrin binding protein (LBP) of Neisseria meningitidis (the putative meningococcal receptor for human lactoferrin, LF), has been previously characterized as an outer-membrane protein of approximately 105 kDa. Using N-terminal amino acid sequence to generate an oligonucleotide probe, a clone from a lambda gt11 phage library was isolated. This clone was subjected to shuttle mutagenesis, in which an erythromycin mini-transposon was used to interrupt the LBP coding sequence. This insertion mutation was introduced into the meningococcus. A N. meningitidis strain that carried this transposon insertion no longer produced the 105 kDa protein. The absence of this protein was correlated with the inability to bind LF or to use LF as an iron source. The LBP mutant was able to grow with other Fe sources and demonstrated no other visible membrane protein alterations. These data confirm the suggestion that LBP is the meningococcal receptor.

Amino Acid Sequence↗

PubMed2006

High-throughput transposon mutagenesis of Corynebacterium glutamicum and construction of a single-gene disruptant mutant library.

A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) was developed. Each of the two minitransposons based on IS31831 (ISL3 family) and Tn5 (IS4 family) was integrated into the Corynebacterium glutamicum R genome. By using BLAST and Perl, transposon insertion locations were automatically identified based on the sequences of TAIL-PCR products of mutant cells. Insertion locations of 18,000 mutants were analyzed, and a comprehensive insertion library covering nearly 80% of the 2,990 open reading frames of C. glutamicum R was generated. Eight thousand of the mutants, exhibiting disruption in 2,330 genes, survived on complex medium under normal laboratory conditions, indicating that the genes were not essential for cell survival. Of the 2,330 genes, 30 exhibited high similarity to essential genes of Escherichia coli or Bacillus subtilis. This approach could be useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms.

Chromosome Mapping↗

PubMed2003

Biocontrol traits of Pseudomonas spp. are regulated by phase variation.

Of 214 Pseudomonas strains isolated from maize rhizosphere, 46 turned out to be antagonistic, of which 43 displayed clear colony phase variation. The latter strains formed both opaque and translucent colonies, designated as phase I and phase II, respectively. It appeared that important biocontrol traits, such as motility and the production of antifungal metabolites, proteases, lipases, chitinases, and biosurfactants, are correlated with phase I morphology and are absent in bacteria with phase II morphology. From a Tn5luxAB transposon library of Pseudomonas sp. strain PCL1171 phase I cells, two mutants exhibiting stable expression of phase II had insertions in gacS. A third mutant, which showed an increased colony phase variation frequency was mutated in mutS. Inoculation of wheat seeds with PCL1171 bacteria of phase I morphology resulted in efficient suppression of take-all disease, whereas disease suppression was absent with phase II bacteria. Neither the gacS nor the mutS mutant was able to suppress take-all, but biocontrol activity was restored after genetic complementation of these mutants. Furthermore, in a number of cases, complementation by gacS of wild-type phase II sectors to phase I phenotype could be shown. A PCL1171 phase I mutant defective in antagonistic activity appeared to have a mutation in a gene encoding a lipopeptide synthetase homologue and had lost its biocontrol activity, suggesting that biocontrol by strain PCL1171 is dependent on the production of a lipopeptide. Our results show that colony phase variation plays a regulatory role in biocontrol by Pseudomonas bacteria by influencing the expression of major biocontrol traits and that the gacS and mutS genes play a role in the colony phase variation process. Therefore phase variation not only plays a role in escaping animal defense but it also appears to play a much broader and vital role in the ecology of bacteria producing exoenzymes, antibiotics, and other secondary metabolites.

Genetic Variation↗

PubMed2004

Identification of Neisseria meningitidis genetic loci involved in the modulation of phase variation frequencies.

It has been proposed that increased phase variation frequencies in Neisseria meningitidis augment transmissibility and invasiveness. A Himar1 mariner transposon mutant library was constructed in serogroup A N. meningitidis and screened for clones with increased phase variation frequencies. Insertions increasing the frequency of slippage events within mononucleotide repeat tracts were identified in three known phase variation-modulating genes (mutS, mutL, and uvrD), as well as six additional loci (pilP, fbpA, fbpB, NMA1233, and two intergenic regions). The implications of these insertion mutations are discussed.

Bacterial Proteins↗

PubMed1998

Libraries of green fluorescent protein fusions generated by transposition in vitro.

Two artificial transposons have been constructed that carry a gene encoding Green Fluorescent Protein and can be used for generating libraries of GFP fusions in a gene of interest. One such element, AT2GFP, can be used to generate GFP insertions in frame with the amino acid sequence of the protein of interest, with a stop codon at the end of the GFP coding sequence; AT2GFP also contains a selectable marker that confers trimethoprim resistance in bacteria. The second element, GS, can be used to generate tribrid GFP fusions because there is no stop codon in the GFP transposon, and the resulting fusion proteins contain the entire amino acid sequence encoded by the gene. The GS element consists of a gfp open reading frame and a supF amber suppressor tRNA gene; the supF portion of the GS transposon can be utilized as a selectable marker in bacteria. Its sequence contains a fortuitous open reading frame, and thus it can be translated continuously with the gfp amino acid sequence. As a target for GFP insertions, we used a plasmid carrying the native Ty1 retrotransposon of the yeast Sacharomyces cerevisiae. The resulting multiple GFP fusions to Ty1 capsid protein Gag and Ty1 integrase were useful in determining the cellular localization of these proteins. Libraries of GFP fusions generated by transposition in vitro represent a novel and potentially powerful method to study the cell distribution and cellular localization signals of proteins.

Animals↗

PubMed2005

A histidine kinase sensor protein gene is necessary for induction of low pH tolerance in Sinorhizobium sp. strain BL3.

The aim of this investigation was to identify and isolate genes involved in acid tolerance from Sinorhizobium sp. strain BL3. It was hypothesized that acid tolerance of strain BL3 could be enhanced by high level expression of certain genes involved in acid tolerance, following insertion of these genes in a multiple copy plasmid. A cosmid clone library of BL3 was introduced into BL3, and the transconjugant colonies were selected at low pH. A single cosmid containing genes for acid tolerance was isolated from 40 different colonies. By transposon-insertion mutagenesis, subcloning and DNA sequencing, a gene involved in acid tolerance, actX, was identified in a 4.4-kb fragment of this cosmid. The actX mutant of BL3 showed increased acid sensitivity and was complemented by the 4.4-kb subcloned fragment. Phaseolus lathyroides seedlings inoculated with recombinant strains containing multiple copies of actX showed increased symbiotic performance at low pH. By constructing an actX::gus fusion, it was shown that actX was induced at low pH. actX encodes a putative histidine kinase sensor protein of a two-component regulatory system. The method of gene identification used in this study for isolation of actX may be applied for the isolation of other genes involved in tolerance to adverse environmental factors.

Bacterial Proteins↗

PubMed1995

A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis.

Yersinia enterocolitica serotype O:3 strain 6471/76-c (YeO3-c) was sensitive to bacteriophage phi R1-37 when grown at 37 degrees C but not when grown at 22 degrees C because of steric hindrance by abundant lipopolysaccharide (LPS) O-side chain (O-antigen) expressed at 22 degrees C. The transposon library of YeO3-c was grown at 37 degrees C and screened for phage phi R1-37-resistant transposon insertion mutants. Three types of mutant were isolated: (i) phage receptor mutants expressing O-antigen (LPS-smooth), (ii) phage receptor mutants not expressing O-antigen (LPS-rough), and (iii) LPS-smooth mutants with the phage receptor constitutively sterically blocked. Mutant type (i) was characterized in detail; the transposon insertion inactivates an operon, named the trs operon. The main findings based on this mutant are: (i) the frs operon is involved in the biosynthesis of the LPS outer core in YeO3-c; the nucleotide sequence of the trs operon revealed eight novel genes showing similarly to known polysaccharide biosynthetic genes of various Gram-negative bacteria as well as to capsule biosynthesis genes of Staphylococcus aureus; (ii) the biosynthesis of the core of YeO3-c involves at least two genetic loci; (iii) the trs operon is required for the biosynthesis of the bacteriophage phi R1-37 receptor structures; (iv) the homopolymeric O-antigen of YeO3-c is ligated to the inner core in Y. enterocolitica O:3; (v) the trs operon is located between the adk-hemH and galE-gsk gene pairs in the Y. enterocolitica chromosome; and (vi) the phage phi R1-37 receptor is present in many but not in all Y. enterocolitica serotypes. The results also allow us to speculate that the trs operon is a relic of the ancestral rfb region of Y. enterocolitica O:3 carrying genes indispensable for the completion of the core polysaccharide biosynthesis.

Amino Acid Sequence↗

PubMed1990

Cloning and expression in Escherichia coli of LKP pilus genes from a nontypeable Haemophilus influenzae strain.

Nontypeable Haemophilus influenzae HF0295, isolated by aspiration from the middle ear of a patient with otitis media, expresses long, thick, and hemagglutinating pili of a single serotype (LKP1) on its surface. An intact pilus vaccine consisting of the LKP1 serotype protected chinchillas against experimental otitis media (C. C. Brinton, Jr., M. J. Carter, D. B. Derber, S. Kar, J. A. Kramarik, A. C. C. To, S. C. M. To, and S. W. Wood, Pediatr. Infect. Dis. J. 8:554-561, 1989; R. B. Karasic, D. J. Beste, S. C. M. To, W. J. Doyle, S. W. Wood, M. J. Carter, A. C. C. To, K. Tanpowpong, C. D. Bluestone, and C. C. Brinton, Jr., Pediatr. Infect. Dis. J. 8:562-565, 1989). The genes encoding LKP1 pili were cloned from a genomic library of the clinical strain as a 12.5-kilobase insert on a plasmid vector and inserted into Escherichia coli K-12. Transposon mutagenesis and deletion constructs mapped the pilus-coding region within a 7-kilobase region of insert DNA. The recombinant bacteria were found by electron microscopy to express pili morphologically similar to LKP1 pili. Purified pilus rods from the recombinant and its parental strain were composed of a single detectable protein with an apparent molecular weight of 27,500. Antibodies raised against LKP1 pili purified from H. influenzae immunologically reacted with pili from the recombinant bacteria. Pili from both strains also adhered to human erythrocytes and buccal cells with the same specificity.

Bacterial Adhesion↗

PubMed2004

TE-based mutagenesis systems in plants: a gene family approach.

Insertions in specific genes belonging to large and homogeneous gene families often do not cause a visible phenotype due to genetic redundancy. Therefore, several single-insertion mutants may have to be combined into double or even triple mutants in order to obtain a loss-of-function phenotype. It is therefore most useful to shift from single-gene insertion selection toward selection at the gene family level. Here, we present an alternative screening methodology that is highly suited for the functional analysis of gene families. Labeled primers designed from conserved regions present in the gene family and a transposon derived primer are combined in an optimized polymerase chain reaction (PCR) to screen a three-dimensionally pooled insertion library in a single step. PCR products are sized by polyacrylamide gel electrophoresis (PAGE), and putative insertion fragments are isolated from the gel, reamplified, and sequenced directly. Because the identification of insertions is sequence-based, insertions into highly homologous genes can easily be distinguished. Taking advantage of the presence of conserved domains in gene families, this approach allows simultaneous screening for insertion events in different family members, and it has the additional advantage of identifying yet unknown family members through their corresponding insertion mutant.

DNA Transposable Elements↗

PubMed1996

Characterization of genes required for pilus expression in Pseudomonas syringae pathovar phaseolicola.

Nonpiliated, phage phi 6-resistant mutants of Pseudomonas syringae pv. phaseolicola were generated by Tn5 transposon mutagenesis. A P. syringae pv. phaseolicola LR700 cosmid library was screened with Tn5-containing EcoRI fragments cloned from nonpiliated mutants. The cosmid clone pVK253 complemented the nonpiliated mutant strain HB2.5. A 3.8-kb sequenced region spanning the Tn5 insertion site contained four open reading frames. The transposon-inactivated gene, designated pilP, is 525 bp long, potentially encoding a 19.1-kDa protein precursor that contains a typical membrane lipoprotein leader sequence. Generation of single mutations in each of the three remaining complete open reading frames by marker exchange also resulted in a nonpiliated phenotype. Expression of this gene region by the T7 expression system in Escherichia coli resulted in four polypeptides of approximately 39, 26, 23, and 18 kDa, in agreement with the sizes of the open reading frames. The three genes upstream of pilP were designated pilM (39 kDa), pilN (23 kDa), and pilO (26 kDa). The processing of the PilP precursor into its mature form was shown to be inhibited by globomycin, a specific inhibitor of signal peptidase II. The gene region identified shows a high degree of homology to a gene region reported to be required for Pseudomonas aeruginosa type IV pilus production.

Amino Acid Sequence↗

PubMed2003

Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis.

Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

Actinomycetales Infections↗

PubMed1990

Transposon tagging of genes for cell-cell interactions in Myxococcus xanthus.

The prokaryote Myxococcus xanthus is a model for cell interactions important in multicellular behavior. We used the transposon TnphoA to specifically identify genes for cell-surface factors involved in cell interactions. From a library of 10,700 insertions of TnphoA, we isolated 36 that produced alkaline phosphatase activity. Three TnphoA insertions tagged cell motility genes, called cgl, which control the adventurous movement of cells. The products of the tagged cgl genes could function in trans upon other cells and were localized primarily in the cell envelope and extracellular space, consistent with TnphoA tagging genes for extracellular factors controlling motility.

Alkaline Phosphatase↗

PubMed2006

Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition.

Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous fungi that infect immunocompromised and immunocompetent people. We developed an insertional mutagenesis strategy for these species based on in vitro transposition and we tested the method by disrupting the URA5 gene in a strain of C. neoformans and the CAP10 gene in three strains of C. gattii. We targeted plasmid DNA containing the URA5 gene or plasmid DNA containing the CAP10 gene from genomic libraries from the shotgun sequencing project for the C. gatti strain WM276. In the latter case, the availability of the end sequences of the clones from the assembled genomic sequence allows rapid selection of target genes for disruption. Modified transposons containing the nourseothricin (NAT) or neomycin (Neo) resistance cassettes were randomly inserted into the target DNA by in vitro transposition. The disrupted genes were used for biolistic transformation and homologous integration was subsequently confirmed by PCR and Southern blot analysis. These results demonstrate that the emerging genomic resources, combined with in vitro transposition into plasmid DNAs from shotgun sequencing libraries or cloned PCR products, will facilitate high-throughput genetic analysis in Cryptococcus species.

Biolistics↗

PubMed2000

Pentapeptide scanning mutagenesis: encouraging old proteins to execute unusual tricks.

Pentapeptide scanning mutagenesis is a facile transposon-based procedure for the random insertion of a variable five amino acid cassette into a target protein. The analysis of a library of proteins harbouring pentapeptide insertions can provide invaluable information on the essential and inessential regions of a target protein, as well as revealing surprising aspects of target protein function and activity.

Amino Acid Sequence↗

PubMed2001

Analysis of functional domains of the Enterococcus faecalis pheromone-induced surface protein aggregation substance.

Pheromone-inducible aggregation substance (AS) proteins of Enterococcus faecalis are essential for high-efficiency conjugation of the sex pheromone plasmids and also serve as virulence factors during host infection. A number of different functions have been attributed to AS in addition to bacterial cell aggregation, including adhesion to host cells, adhesion to fibrin, increased cell surface hydrophobicity, resistance to killing by polymorphonuclear leukocytes and macrophages, and increased vegetation size in an experimental endocarditis model. Relatively little information is available regarding the structure-activity relationship of AS. To identify functional domains, a library of 23 nonpolar 31-amino-acid insertions was constructed in Asc10, the AS encoded by the plasmid pCF10, using the transposons TnlacZ/in and TnphoA/in. Analysis of these insertions revealed a domain necessary for donor-recipient aggregation that extends further into the amino terminus of the protein than previously reported. In addition, insertions in the C terminus of the protein also reduced aggregation. As expected, the ability to aggregate correlates with efficient plasmid transfer. The results also indicated that an increase in cell surface hydrophobicity resulting from AS expression is not sufficient to mediate bacterial aggregation.

Bacterial Adhesion↗

PubMed2006

Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway.

BACKGROUND: Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. RESULTS: A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in C. glutamicum. The cg0910 gene, renamed hisN, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes. CONCLUSION: The absence of regional and sequence preferences of IS6100-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain C.glutamicum ATCC 13032. The identification of the hisN gene encoding histidinol-phosphate phosphatase in C. glutamicum closed the last gap in histidine synthesis in the Actinobacteria. The system might be a valuable genetic tool also in other bacteria due to the broad host-spectrum of IS6100.

Actinobacteria↗

PubMed2002

Efficient gene identification and targeted gene disruption in the wheat blotch fungus Mycosphaerella graminicola using TAGKO.

TAGKO ( transposon- arrayed gene knock out) is a highly efficient method for gene discovery and gene function assignment in the rice blast fungus Magnaporthe grisea. Here, we report the application of genome-wide TAGKO to the wheat blotch fungus Mycosphaerella graminicola, including the successful development of electroporation-based transformation for this fungus. A M. graminicola genomic cosmid library was constructed and a pool of 250 cosmid clones was mutagenized by in vitro transposition. Sequence analysis identified 5,110 unique insertion events in the M. graminicola genome. Eleven transposon-tagged cosmid clones (TAGKO clones) were chosen and transformed into the wild-type strain by electroporation. Ten TAGKO clones out of 11 produced gene-specific mutants at a targeting frequency of 15-28%, significantly higher than that of conventional gene-disruption constructs. The remaining clone failed to produce viable mutants, thereby providing indirect evidence for the identification of an essential gene.

Ascomycota↗