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Results for “Transposon insertion library”

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PubMed2001

Identification of lipopolysaccharide O antigen synthesis genes required for attachment of the S-layer of Caulobacter crescentus.

The outer surface of Caulobacter crescentus consists of a two-dimensional crystalline protein lattice layer (S-layer). A fraction of the LPS has an O antigen polymer attached to the core to form a 'smooth' LPS (S-LPS), which is required for attachment of the protein S-layer to the outer-membrane surface. A method to screen for strains defective in LPS production, based on loss of S-layer attachment, was developed and applied to libraries of transposon-generated mutants. Eighteen distinct insertions were found with transposon interruptions in genes affecting S-LPS production, 12 of which were located near the S-layer subunit protein gene, rsaA, and its transporter genes. Sequence adjacent to transposon insertion points was determined and used to search a C. crescentus genome database. Twelve ORFs likely to be involved in S-LPS synthesis were identified. Seven of the predicted ORFs were linked to rsaA. Six of the putative genes had identity with proteins involved in synthesis of sugar residues, including five predicted to make perosamine. The remaining six ORFs were similar to glycosyltransferases involved in forming linkages between sugar residues in the O antigen, while one may be a transcription repressor. Other chemical and preliminary proton NMR studies of the S-LPS O antigen indicate that it contains an N-acetylated 4,6-dideoxy-4-aminohexose, but is not assembled as a simple, uniform homopolymer, consisting of several different linkages between sugar residues. The ORFs described here include homologues of all the enzymes involved in the synthesis of N-acetylperosamine, a 4,6-dideoxy-4-aminohexose. Overall, the data are consistent with the hypothesis that the O antigen of C. crescentus S-LPS consists primarily of N-acetylperosamine residues polymerized with multiple anomeric linkages.

Acetyltransferases↗

PubMed2004

Identification of candidate gammaherpesvirus 68 genes required for virus replication by signature-tagged transposon mutagenesis.

Current methods for determining the role of a given gene product in the gammaherpesvirus 68 (gammaHV68) life cycle require generation of a specific mutation by either homologous recombination in mammalian cells or bacterial artificial chromosome-mediated mutagenesis in Escherichia coli. The mutant virus is then compared to wild-type virus, and the role of the gene in the viral life cycle is deduced from its phenotype. This process is both time-consuming and labor intensive. Here we present the use of random, transposon-mediated signature-tagged mutagenesis for the identification of candidate viral genes involved in virus replication. Pools of viral mutants, each containing a random insertion of a transposon, were generated with a transposon donor library in which each transposon contains a unique sequence identifier. These pools were transfected into mammalian cells, and the ability of each mutant to replicate was assessed by comparing the presence of virus in the output pool to that present in the input pool of viral genomes. With this approach we could rapidly screen up to 96 individual mutants simultaneously. The location of the transposon insertion was determined by sequencing individual clones with a common primer specific for the transposon end. Here we present the characterization of 53 distinct viral mutants that correspond to insertions in 29 open reading frames within the gammaHV68 genome. To confirm the results of the signature-tagged mutagenesis screen, we quantitated the ability of each mutant to replicate compared to wild-type gammaHV68. From these analyses we identified 16 gammaHV68 open reading frames that, when disrupted by transposon insertions, score as essential for virus replication, and six other open reading frames whose disruption led to significant attenuation of virus replication. In addition, transposon insertion in five other gammaHV68 open reading frames did not affect virus replication. Notably, all but one of the candidate essential replication genes identified in this screen have been shown to be essential for the replication of at least one other herpesvirus.

Animals↗

PubMed1992

NotI genomic cleavage map of Escherichia coli K-12 strain MG1655.

Several approaches were used to construct a complete NotI restriction enzyme cleavage map of the genome of Escherichia coli MG1655. The approaches included use of transposable element insertions that created auxotrophic mutations and introduced a NotI site into the genome, hybridization of NotI fragments to the ordered lambda library constructed by Kohara et al. (BioTechniques 10:474-477, 1991), Southern blotting of NotI digests with cloned genes as probes, and analysis of the known E. coli DNA sequence for NotI sites. In all, 22 NotI cleavage sites were mapped along with 26 transposon insertions. These sites were localized to clones in the lambda library and, when possible, sequenced genes. The map was compared with that of strain EMG2, a wild-type E. coli K-12 strain, and several differences were found, including a region of about 600 kb with an altered restriction pattern and an additional fragment in MG1655. Comparison of MG1655 with other strains revealed minor differences but indicated that this map was representative of that for many commonly used E. coli K-12 strains.

Base Sequence↗

PubMed1999

The sae locus of Staphylococcus aureus encodes a two-component regulatory system.

Sae is a regulatory locus that activates the production of several exoproteins in Staphylococcus aureus. A 3.4-kb fragment of a S. aureus genomic library, screened with a probe adjacent to the transposon insertion of a sae::Tn551 mutant, was cloned into a bifunctional vector. This fragment was shown to carry the sae locus by restoration of exoprotein production in sae mutants. The sae locus was mapped to the SmaI-D fragment of the staphylococcal chromosome by pulse-field electrophoresis. Sequence analysis of the cloned fragment revealed the presence of two genes, designated saeR and saeS, encoding a response regulator and a histidine protein kinase, respectively, with high homology to other bacterial two-component regulatory systems.

Amino Acid Sequence↗

PubMed2005

Genetic analysis of cytomegalovirus by shuttle mutagenesis.

The genomes of Herpesviridae family members are among the largest of all viruses and therefore present a formidable challenge in understanding the roles of every gene in replication or pathogenesis. For example, murine cytomegalovirus (MCMV) has a genome of 230 kb that encodes more than 170 genes, many of which have unknown functions. Many techniques for the genetic analysis of a herpesvirus have been developed over the past two decades. One such procedure involves the use of a shuttle mutagenesis system, and it has successfully generated a pool of MCMV mutants that contained an engineered Tn3-type transposon inserted within their genome. The process of shuttle mutagenesis involves the construction of a genomic fragment library, transposon mutagenesis of the library, and generation of virus mutants through homologous recombination. This chapter details the methodologies required for implementing a Tn3-based shuttle mutagenesis system for construction of a mutant virus library.

Cytomegalovirus↗

PubMed2001

Gene discovery and gene function assignment in filamentous fungi.

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.

Alleles↗

PubMed2004

Establishment of a functional genomics platform for Leifsonia xyli subsp. xyli.

Leifsonia xyli subsp. xyli, the causal agent of ratoon stunting disease in sugarcane, is a xylem-limited, nutritionally fastidious, slow growing, gram-positive coryneform bacterium. Because of the difficulties in growing this bacterium in pure culture, little is known about the molecular mechanisms of pathogenesis. Currently, the genome sequence of L. xyli subsp. xyli is being completed by the Agronomical and Environmental Genomes group from the Organization for Nucleotide Sequencing and Analysis in Brazil. To complement this work, we produced 712 Lxx::Tn4431 transposon mutants and sequenced flanking regions from 383 of these, using a rapid polymerase chain reaction-based approach. Tn4431 insertions appeared to be widespread throughout the L. xyli subsp. xyli genome; however, there were regions that had significantly higher concentrations of insertions. The Tn4431 mutant library was screened for individuals unable to colonize sugarcane, and one noncolonizing mutant was found. The mutant contained a transposon insertion disrupting two open reading frames (ORF), one of which had homology to an integral membrane protein from Mycobacterium leprae. Sequencing of the surrounding regions revealed two operons, pro and cyd, both of which are believed to play roles in disease. Complementation studies were carried out using the noncolonizing Lxx::Tn4431 mutant. The noncolonizing mutant was transformed with a cosmid containing 40 kbp of wild-type sequence, which included the two ORF disrupted in the mutant, and several transformants were subsequently able to colonize sugarcane. However, analysis of each of these transformants, before and after colonization, suggests that they have all undergone various recombinant events, obscuring the roles of these ORF in L. xyli subsp. xyli pathogenesis.

Actinomycetales↗

PubMed1996

Optimized BlaM-transposon shuttle mutagenesis of Helicobacter pylori allows the identification of novel genetic loci involved in bacterial virulence.

Helicobacter pylori is an important etiologic agent of gastroduodenal disease in humans. In this report, we describe a general genetic approach for the identification of genes encoding exported proteins in H. pylori. The novel TnMax9 mini-blaM transposon was used for insertion mutagenesis of a H. pylori gene library established in Escherichia coli. A total of 192 E. coli clones expressing active beta-lactamase fusion proteins (BlaM+) were obtained, indicating that the corresponding target plasmids carry H. pylori genes encoding putative extracytoplasmic proteins. Natural transformation of H. pylori P1 or P12 using the 192 mutant plasmids resulted in 135 distinct H. pylori mutant strains (70%). Screening of the H. pylori collection of mutant strains allowed the identification of mutant strains impaired in motility, in natural transformation competence and in adherence to gastric epithelial cell lines. Motility mutants could be grouped into distinct classes: (i) mutant strains lacking the major flagellin subunit FlaA and intact flagella (class I); (ii) mutant strains with apparently normal flagella, but reduced motility (class II), and (iii) mutant strains with obviously normal flagella, but completely abolished motility (class III). Two independent mutations that exhibited defects in natural competence for genetic transformation mapped to different genetic loci. In addition, two independent mutant strains were isolated by their failure to bind to the human gastric carcinoma cell line KatoIII. Both mutant strains carried a transposon in the same gene, 0.8 kb apart, and showed decreased autoagglutination when compared to the wild-type strain.

Amino Acid Sequence↗

PubMed2003

Maize genome sequencing by methylation filtration.

Gene enrichment strategies offer an alternative to sequencing large and repetitive genomes such as that of maize. We report the generation and analysis of nearly 100,000 undermethylated (or methylation filtration) maize sequences. Comparison with the rice genome reveals that methylation filtration results in a more comprehensive representation of maize genes than those that result from expressed sequence tags or transposon insertion sites sequences. About 7% of the repetitive DNA is unmethylated and thus selected in our libraries, but potentially active transposons and unmethylated organelle genomes can be identified. Reverse transcription polymerase chain reaction can be used to finish the maize transcriptome.

Algorithms↗

PubMed1994

Reassessment of the number of auxiliary genes essential for expression of high-level methicillin resistance in Staphylococcus aureus.

A new transposon library constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL yielded 70 independent insertional mutants with reduced levels of antibiotic resistance. Restriction analysis with HindIII, EcoRV, EcoRI, and PstI and then Southern hybridization with probes for the transposon and for the femA-femB gene demonstrated that 41 of the 70 Tn551 mutants carried distinct and novel, as yet undescribed insertion sites, all of which were outside of the mecA gene and were also outside the already-characterized auxiliary genes femA, femB, femC, and femD. All previously described Tn551 mutations of this type were in genes located either on SmaI fragment A or SmaI fragment I. In contrast, inserts of the new library were located in 7 of the 16 SmaI chromosomal fragments, fragments A, B, C, D, E, F, and I. In all of the mutants, expression of methicillin resistance became heterogeneous, and the MIC for the majority of cells was reduced (1.5 to 200 micrograms ml-1) from the homogeneous methicillin MIC (1,600 micrograms ml-1) of the parental cells. Although identification of the exact number of genes inactivated through the new set of transposon inserts will require cloning and sequencing, a rough estimate of this number from mapping data suggests a minimum of at least 10 to 12 new genetic determinants, all of which are needed together with femA, femB, femC, and femD for the optimal expression of methicillin resistance.

Evaluation Studies as Topic↗

PubMed1998

Characterization of the haemolytic activity of Streptococcus equi.

The haemolytic activity of Streptococcus equi, the cause of equine strangles, was characterized. Production of haemolysin in Todd Hewitt broth was dependent on an equine serum supplement and the logarithmic phase of growth after which activity declined sharply. RNA core also induced haemolysin production from cells harvested at the end of the logarithmic phase of growth. Haemolysis was not affected by cholesterol, was only slightly increased in reducing conditions and was completely inactivated by trypan blue, identifying the haemolytic activity as streptolysin S-like (SLS-like). Purification by hydroxyapatite and Sephacryl column chromatography yielded proteins of molecular weights of approximately 6000 and 17 000-22 000 Da with a 64-fold increase in specific activity. Low molecular weight proteins from the RNA core were still present in the purified toxin. Two non-haemolytic mutants were derived by conjugation with an Enterococcus faecalis-carrying transposon Tn916. Southern blots of HindIII digests of DNA revealed that one of the mutants contained three transposon insertions and the other just one. A lambda phage library of S. equi contained plaques whose haemolytic activity was enhanced by reducing conditions and inhibited by cholesterol, suggesting a streptolysin O-like (SLO-like) activity. However, haemolysin in culture sonicates of host E. coli in which the lambda phage insert was subcloned into plasmid (pUC18), was not affected by these conditions. Seven isolates of S. equi in medium without SLS-like inducers showed no SLO-like activity and no evidence for an SLO-like toxin could be found by immunoblotting with pneumolysin antiserum and monoclonal antibodies or by polymerase chain reaction with primers derived from sequences conserved between the SLO genes of Lancefield group A, C and G streptococci. S. equi does not appear to possess a streptolysin O but does make a streptolysin S-like toxin whose production can be interrupted at just one genetic locus.

Antibodies, Bacterial↗

PubMed2006

Selection of novel vesicular stomatitis virus glycoprotein variants from a peptide insertion library for enhanced purification of retroviral and lentiviral vectors.

The introduction of new features or functions that are not present in an original protein is a significant challenge in protein engineering. For example, modifications to vesicular stomatitis virus glycoprotein (VSV-G), which is commonly used to pseudotype retroviral and lentiviral vectors for gene delivery, have been hindered by a lack of structural knowledge of the protein. We have developed a transposon-based approach that randomly incorporates designed polypeptides throughout a protein to generate saturated insertion libraries and a subsequent high-throughput selection process in mammalian cells that enables the identification of optimal insertion sites for a novel designed functionality. This method was applied to VSV-G in order to construct a comprehensive library of mutants whose combined members have a His6 tag inserted at likely every site in the original protein sequence. Selecting the library via iterative retroviral infections of mammalian cells led to the identification of several VSV-G-His6 variants that were able to package high-titer viral vectors and could be purified by Ni-nitrilotriacetic acid affinity chromatography. Column purification of vectors reduced protein and DNA impurities more than 5,000-fold and 14,000-fold, respectively, from the viral supernatant. This substantially improved purity elicited a weaker immune response in the brain, without altering the infectivity or tropism from wild-type VSV-G-pseudotyped vectors. This work applies a powerful new tool for protein engineering to construct novel viral envelope variants that can greatly improve the safety and use of retroviral and lentiviral vectors for clinical gene therapy. Furthermore, this approach of library generation and selection can readily be extended to other challenges in protein engineering.

Animals↗

PubMed1999

A novel approach for the construction of a Campylobacter mutant library.

Given the lack of functional transposons for use in Campylobacter spp., an alternative method of insertional mutagenesis using natural transformation was developed. High efficiencies of transformation were only obtained with species-specific DNA. This feature was a key element in the construction of mutant libraries of this bacterium. A chromosomal library of Campylobacter jejuni 81116 DNA was made in shuttle vector pUOA18. Next, a kanamycin-resistance (KmR) cassette was ligated into the inserts of the plasmids. C. jejuni 81116 was then transformed with the resulting products to allow homologous recombination between genomic fragments present in the shuttle vector and the chromosome. Transformants were pooled and chromosomal DNA from these transformants was used to retransform C. jejuni 81116. This resulted in transformants containing the KmR cassette in the chromosome but lacking the vector. In order to evaluate this approach for the construction of a mutant bank, the KmR insertional mutants were screened for loss of motility. Partial characterization of 11 non-motile mutants indicated that the inserted genes are involved in motility. Four mutants had the KmR cassette inserted in genes involved in flagella biosynthesis, namely flaA/B, neuB and flgK, and produced incomplete or no flagella. Four mutants had the KmR cassette inserted in genes possibly involved in flagella motor function: pflA, fliM and orf1 downstream of the fliN gene. Three mutants had the KmR cassette inserted in genes that are homologous to genes encoding hypothetical proteins of Helicobacter pylori.

Blotting, Southern↗

PubMed2006

Identification of potential virulence determinants by Himar1 transposition of infectious Borrelia burgdorferi B31.

Lyme disease Borrelia organisms are highly invasive spirochetes that alternate between vertebrate and arthropod hosts and that establish chronic infections and elicit inflammatory reactions in mammals. Although progress has been made in the targeted mutagenesis of individual genes in infectious Borrelia burgdorferi, the roles of the vast majority of gene products in pathogenesis remain unresolved. In this study, we examined the feasibility of using transposon mutagenesis to identify infectivity-related factors in B. burgdorferi. The transformable, infectious strain 5A18 NP1 was transformed with the spirochete-adapted Himar1 transposon delivery vector pMarGent to create a small library of 33 insertion mutants. Single mouse inoculations followed by culture of four tissue sites and serology were used to screen the mutants for infectivity phenotypes. Mutants that appeared attenuated (culture positive at some sites) or noninfectious (negative at all sites) and contained the virulence-associated plasmids lp25 and lp28-1 were examined in more extensive animal studies. Three of these mutants (including those with insertions in the putative fliG-1-encoded flagellar motor switch protein and the guaB-encoded IMP dehydrogenase) were noninfectious, whereas four clones appeared to exhibit reduced infectivity. Serological reactivity in VlsE enzyme-linked immunosorbent assays correlated with the assignment of mutants to the noninfectious or attenuated-infectivity groups. The results of this study indicate that random transposon mutagenesis of infectious B. burgdorferi is feasible and will be of value in studying the pathogenesis of Lyme disease Borrelia.

Animals↗

PubMed1993

A physical and genetic map of the Stigmatella aurantiaca DW4/3.1 chromosome.

A physical map of the myxobacterium Stigmatella aurantiaca DW4/3.1 chromosome was constructed by pulsed-field gel (PFG) long-range mapping. One-and two-dimensional pulsed-field gel analyses were used together with reciprocal double-restriction, cross-hybridization and hybridization fingerprint analysis. These PFG results were confirmed by Smith-Birnstiel analysis, by Southern hybridization using linking clones and clones of a lambda genomic library for the determination of adjacent restriction fragments and by transposon insertion mapping using defined genomic sequences for hybridization. It was thus possible to construct a circular restriction map of the single 9.35 Mbp chromosome of S. aurantiaca based on the endonucleases Asel and Spel. Genetic loci as well as the replication origin were located on the physical map by Southern hybridization using heterologous (derived from Myxococcus xanthus, Escherichia coli and Streptomyces lividans) and homologous probes that are mainly involved in development and cell motility.

Bacteriophage lambda↗

PubMed1997

Applications of transposition mutagenesis in antibiotic producing streptomycetes.

Several transposons have been developed from the streptomycete insertion sequence IS493. They have broad host specificity in Streptomyces species and insert relatively randomly into a consensus target sequence of gNCaNTgNNy. Collectively, they have specialized features that facilitate the following: cloning of DNA flanking insertions; physical mapping of insertions; construction of highly stable mutants; and efficient construction of mutant libraries. All of the transposons can be introduced into streptomycetes by conjugation from E. coli, and can be delivered by curing the temperature sensitive delivery plasmid. Tn5099 was used to physically map genes involved in daptomycin and red pigment production in Streptomyces roseosporus, and to clone daptomycin biosynthetic genes. Tn5099 was also used in Streptomyces fradiae to identify and clone a neutral genomic site for the insertion of a second copy of the tylF gene. Recombinants containing two copies of the tylF gene carried out the normally rate limiting conversion of macrocin to tylosin very efficiently, thus causing substantial increases in tylosin yield.

Anti-Bacterial Agents↗

PubMed1999

Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa.

Bacteria communicate with each other to coordinate expression of specific genes in a cell density-dependent fashion, a phenomenon called quorum sensing and response. Although we know that quorum sensing via acyl-homoserine lactone (HSL) signals controls expression of several virulence genes in the human pathogen Pseudomonas aeruginosa, the number and types of genes controlled by quorum sensing have not been studied systematically. We have constructed a library of random insertions in the chromosome of a P. aeruginosa acyl-HSL synthesis mutant by using a transposon containing a promoterless lacZ. This library was screened for acyl-HSL induction of lacZ. Thirty-nine quorum sensing-regulated genes were identified. The genes were organized into classes depending on the pattern of regulation. About half of the genes appear to be in seven operons, some seem organized in large patches on the genome. Many of the quorum sensing-regulated genes code for putative virulence factors or production of secondary metabolites. Many of the genes identified showed a high level of induction by acyl-HSL signaling.

4-Butyrolactone↗

PubMed2026

Dual roles of genes required for intrinsic resistance to clarithromycin in evasion of killing by serum complement in Haemophilus influenzae.

Macrolide antibiotics are commonly prescribed to treat Haemophilus influenzae respiratory tract infections. Studies have primarily focused on emerging H. influenzae strains with acquired macrolide resistance, while the bacterium's intrinsic resistance to antibiotics has been underexamined. Here, we used a genome-wide approach of transposon insertion-site sequencing to screen an H. influenzae mutant library grown in sub-inhibitory doses of the macrolide antibiotic clarithromycin (CLR) to identify 33 genes involved in intrinsic CLR resistance. Almost half of these genes are also needed for survival in the mouse lung. We focused on candidate genes necessary for both intrinsic macrolide resistance and lung survival. Two of these genes affect the outer-membrane composition of H. influenzae, orfH and omp26. Deletions of these genes in Rd and nontypeable H. influenzae clinical isolates, Hi375 and NT127, conferred sensitivity to CLR and polymyxin B and increased membrane permeability to ethidium bromide (EtBr). The omp26 mutant was sensitive to killing by human serum. Deletions of orfH or omp26 in an acrR mutant strain overexpressing a multidrug efflux pump abrogated resistance of the acrR mutant to CLR and restored permeability to EtBr. Thus, deletion of these genes not only mitigates the effects of an acquired resistance mechanism but also remarkably overrides it. Complementation of these deletion mutations restored CLR resistance and decreased permeability to EtBr. Our results indicate that the subset of genes with dual roles in intrinsic resistance and host lung survival may provide potential novel combination antimicrobial therapeutic targets.

Animals↗