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PubMed1991

Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA.

The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots). Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites. We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences. Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 bp away from this hotspot. Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam- mutation did not affect Tn5 insertion relative to an I end sequence in target DNA. These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.

Base Sequence↗
PubMed1993

PilS and PilR, a two-component transcriptional regulatory system controlling expression of type 4 fimbriae in Pseudomonas aeruginosa.

Transposon mutagenesis was used to identify genes necessary for the expression of Pseudomonas aeruginosa type 4 fimbriae. In a library of 12,700 mutants, 147 were observed to have lost the spreading colony morphology associated with the presence of functional fimbriae. Of these, 28 had also acquired resistance to the fimbrial-specific bacteriophage PO4. The mutations conferring this phage resistance were found to have occurred at at least six different loci, including the three that had been previously shown to be required for fimbrial biosynthesis or function: the structural subunit (pilA) and adjacent genes (pilB,C,D), the twitching motility gene (pilT), and the sigma 54 RNA polymerase initiation factor gene (rpoN). One novel group of phage-resistant mutants was identified in which the transposon had inserted near sequences that cross-hybridized to an oligonucleotide probe designed against conserved domains in regulators of RpoN-dependent promoters. These mutants had no detectable transcription of pilA and did not produce fimbriae. A probe derived from inverse polymerase chain reaction was used to isolate the corresponding wild-type sequences from a P. aeruginosa PAO cosmid reference library, and two adjacent genes affected by transposon insertions, pilS and pilR, were located and sequenced. These genes were shown to be capable of complementing the corresponding mutants, both at the level of restoring the phenotypes associated with functional fimbriae and by the restoration of pilA transcription. The pilSR operon was physically mapped to Spel fragment 5 (corresponding to about 72-75/0 min on the genetic map), and shown to be located approximately 25 kb from pilA-D. PilS and PilR clearly belong to the family of two-component transcriptional regulatory systems which have been described in many bacterial species. PilS is predicted to be a sensor protein which when stimulated by the appropriate environmental signals activates PilR through kinase activity. PilR then activates transcription of pilA, probably by interacting with RNA polymerase containing RpoN. The identification of pilS and pilR makes possible a more thorough examination of the signal transduction systems controlling expression of virulence factors in P. aeruginosa.

Amino Acid Sequence↗
PubMed2000

Rapid amplification and cloning of Tn5 flanking fragments by inverse PCR.

A simple approach is described to efficiently amplify DNA sequences flanking transposon Tn5 insertions. The method involves: (i) digestion with a restriction enzyme that cuts within Tn5; (ii) self-ligation under conditions favouring the production of monomeric circles; (iii) four parallel PCR reactions using primers designed to amplify left or right flanking sequences, and to distinguish target amplicons from non-specific products. This reveals the number of Tn5 insertions and the size of flanking genomic restriction fragments, without Southern blot analysis. The amplified product contains restriction sites that facilitate cohesive-end cloning. This rapid method is demonstrated using Tn5 and Tn5-Mob tagged DNA sequences involved in albicidin biosynthesis in Xanthomonas albilineans. It is generally applicable for efficient recovery of DNA sequences flanking transposon Tn5 derivatives in insertional mutagenesis studies.

Anti-Bacterial Agents↗
PubMed1997

The anthrax toxin activator gene atxA is associated with CO2-enhanced non-toxin gene expression in Bacillus anthracis.

The Bacillus anthracis toxin genes, cya, lef, and pag, can be viewed as a regulon, in which transcription of all three genes is activated in trans by the same regulatory gene, atxA, in response to the same signal, CO2. In atxA+ strains, toxin gene expression is increased 5- to 20-fold in cells grown in 5% CO2 relative to cells grown in air. CO2-enhanced toxin gene transcription is not observed in atx4-null mutants. Here, we used two independent techniques to obtain evidence for additional CO2-induced atxA-regulated genes. First, total protein preparations from atxA4+ and atxA isolates grown in 5% CO2 and in air were examined by two-dimensional electrophoresis. Comparison of the resulting protein patterns indicated that synthesis of non-toxin proteins is influenced by growth in elevated CO2 and the toxin gene regulator, atxA. Second, we generated random transcriptional lacZ fusions in B. anthracis with transposon Tn917-LTV3. Transposon-insertion libraries were screened for mutants expressing CO2-enhanced atxA-dependent beta-galactosidase activity. DNA sequence analysis of transposon insertion sites in 17 mutants carrying CO2- and atxA-regulated fusions revealed 10 mutants carrying independent insertions on the 185-kb toxin plasmid pXO1 which did not map to the toxin genes. The tcr-lacZ fusion mutants (tcr for toxin coregulated) were Tox+, indicating that these genes may not be involved in anthrax toxin gene activation. Our data indicate a clear association of atxA with CO2-enhanced gene expression in B. anthracis and provide evidence that atxA regulates genes other than the structural genes for the anthrax toxin proteins.

Antigens, Bacterial↗
PubMed2000

Novel genes involved in the regulation of pathogenicity factor production within the rpf gene cluster of Xanthomonas campestris.

The synthesis of extracellular enzymes and extracellular polysaccharide (EPS) in Xanthomonas campestris pathovar campestris (Xcc) is subject to co-ordinate regulation by a cluster of genes called rpf (for regulation of pathogenicity factors). These genes are located within a 21.9 kb region of the chromosome isolated as the cosmid clone pIJ3020. The genes in the left-hand section of this region of the chromosome have previously been characterized. This paper reports on the genes in the right-hand section and on the phenotypes of mutants with transposon insertions in these genes. Sequence analysis identified eight genes or ORFs with the gene order rpfD-orf1-orf2-orf3-orf4-recJ-rpf E-greA. RecJ and GreA have established functions in recombination and transcriptional elongation, respectively. rpfD encoded a protein with some amino acid sequence relatedness to a hypothetical protein from Caulobacter crescentus and an autolysin response regulator in Bacillus subtilis. The predicted protein products of orf1, 2 and 3 were related to each other and had substantial amino acid sequence relatedness to hypothetical proteins from C. crescentus. Transposon insertions in orf1, 2 and 3 had no effect on the synthesis of extracellular enzymes or EPS. The predicted proteins RpfE and Orf4 showed the highest amino acid sequence relatedness to hypothetical proteins from Bordetella pertussis and Klebsiella pneumoniae, respectively. Transposon insertions in rpfE led to reduced levels of some extracellular enzymes (endoglucanase and protease) and increased levels of others (polygalacturonate lyase). Transposon insertions in orf4 had no effect on polygalacturonate lyase but led to reduced levels of protease and endoglucanase. Levels of EPS were reduced in both rpfE and orf4 mutants. These alterations in the levels of extracellular enzymes, which were relatively modest (between two- and threefold), did not affect the pathogenicity of Xcc on turnip. It is proposed that the gene designation should be rpfI for orf4.

Amino Acid Sequence↗
PubMed2006

Sequence analysis of a novel insertion site of transposon IS10.

A sacB mutant was obtained by transposon IS10 inactivation of a plasmid pXT3sacB carrying the sacB gene. Sequencing of this mutant plasmid DNA (GenBank accession No. AY580883.1) showed that the IS10 flanking the 22 bp inverted repeats were 5'-CTGAGAGATCCCCTCATAATTT-3' and 5'-AAATCATTAGGGGATTCATCAG-3', which were the similar to those published in reports previously. However, the target sequence adjacent to IS10 was 5'-TGCTTGGTT-3' instead of the previously reported 5'-NGCTNAGCN-3'. To our knowledge, this is the first report on the novel insertion site of IS10. In addition, Southern blot hybridization confirmed that the mobile IS10 originated from the chromosomal DNA of the host strain Escherichia coli DH5alpha and that there were two copies in the DH5alpha genome.

Base Sequence↗
PubMed2003

Nucleotide sequence, functional characterization and evolution of pFKN, a virulence plasmid in Pseudomonas syringae pathovar maculicola.

Pseudomonas syringae pv. maculicola strain M6 (Psm M6) carries the avrRpm1 gene, encoding a type III effector, on a 40 kb plasmid, pFKN. We hypothesized that this plasmid might carry additional genes required for pathogenesis on plants. We report the sequence and features of pFKN. In addition to avrRpm1, pFKN carries an allele of another type III effector, termed avrPphE, and a gene of unknown function (ORF8), expression of which is induced in planta, suggesting a role in the plant-pathogen interaction. The region of pFKN carrying avrRpm1, avrPphE and ORF8 exhibits several features of pathogenicity islands (PAIs). Curing of pFKN (creating Psm M6C) caused a significant reduction in virulence on Arabidopsis leaves. However, complementation studies using Psm M6C demonstrated an obvious virulence function only for avrRpm1. pFKN can integrate and excise from the chromosome of Psm M6 at low frequency via homologous recombination between identical sequence segments located on the chromosome and on pFKN. These segments are part of two nearly identical transposons carrying avrPphE. The avrPphE transposon was also detected in other strains of P. s. pv. maculicola and in P. s. tomato strain DC3000. The avrPphE transposon was found inserted at different loci in different strains. The analysis of sequences surrounding the avrPphE transposon insertion site in the chromosome of Psm M6 indicates that pFKN integrates into a PAI that encodes type III effectors. The integration of pFKN into this chromosomal region may therefore be seen as an evolutionary process determining the formation of a new PAI in the chromosome of Psm M6.

Arabidopsis↗
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↗
PubMed1991

Identification of two fructose transport and phosphorylation pathways in Xanthomonas campestris pv. campestris.

Fructose was shown to be phosphorylated by a specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) in Xanthomonas campestris pv. campestris. Transposon mutagenesis of X. campestris was performed and two mutants affected in growth on fructose were isolated. Both mutants were deficient in PTS activity. Comparison of the rate of uptake and phosphorylation of fructose in the wild-type and in the mutant strains revealed the presence of a second fructose permeation and phosphorylation pathway in this bacterium: an unidentified permease coupled to an ATP-dependent fructokinase. One of the two mutants was also deficient in fructokinase activity. Chromosomal DNA fragments containing the regions flanking the transposon insertion site were cloned from both mutant strains. Their physical study revealed that the insertion sites were separated by 1.4 kb, allowing the reconstruction of a wild-type DNA fragment which complemented one of the two mutants. The region flanking the transposon insertion site was sequenced in one of the mutants, showing that the transposon had interrupted the gene encoding the fructose EII. The mutant strains also failed to utilize mannose, sucrose and mannitol, suggesting the existence of a branch point between the metabolism of fructose and of these latter carbohydrates.

Amino Acid Sequence↗
PubMed2003

Large-scale analysis of the meningococcus genome by gene disruption: resistance to complement-mediated lysis.

The biologic role of a majority of the Neisseria meningitidis 2100 predicted coding regions is still to be assigned or experimentally confirmed. Determining the phenotypic effect of gene disruption being a fundamental approach to understanding gene function, we used high-density signature-tagged transposon mutagenesis, followed by a large-scale sequencing of the transposon insertion sites, to construct a genome-wide collection of mutants. The sequencing results for the first half of the 4548 mutants composing the library suggested that we have mutations in 80%-90% of N. meningitidis nonessential genes. This was confirmed by a whole-genome identification of the genes required for resistance to complement-mediated lysis, a key to meningococcal virulence. We show that all the genes we identified, including four previously uncharacterized, were important for the synthesis of the polysialic acid capsule or the lipooligosaccharide (LOS), suggesting that these are likely to be the only meningococcal attributes necessary for serum resistance. Our work provides a valuable and lasting resource that may lead to a global map of gene function in N. meningitidis.

Antigens, Bacterial↗
PubMed1991

Transposon mutagenesis in Proteus mirabilis.

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

DNA Transposable Elements↗
PubMed1991

Identification of a new operon involved in Listeria monocytogenes virulence: its first gene encodes a protein homologous to bacterial metalloproteases.

The region flanking the transposon in a Tn1545-induced lecithinase-negative mutant of Listeria monocytogenes EGD was cloned and sequenced. The transposon had inserted in ORF D, the open reading frame previously identified downstream from hlyA, the gene encoding listeriolysin O. The complete sequence of ORF D from strain EGD has been determined as well as that of two other strains: LO28, a clinical isolate; and LM8, an epidemic strain. ORF D is 1,533 bp long and encodes a protein highly homologous to metalloproteases of bacilli, Serratia sp., Legionella pneumophila, and Pseudomonas aeruginosa. It was renamed prtA. Northern RNA blot analysis indicated that prtA is the first gene of a 6-kb operon, suggesting that the lecithinase-negative phenotype of the mutant might be due to a polar effect of the transposon insertion.

Amino Acid Sequence↗
PubMed2003

Cytadherence-deficient mutants of Mycoplasma gallisepticum generated by transposon mutagenesis.

Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gm(r) HA-negative (HA(-)) colonies displaying a stable HA(-) phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA(-) mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA(-) mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.

Adhesins, Bacterial↗
PubMed1991

Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes.

The agropine/mannopine synthesis region of the TR region of the Ri plasmid of Agrobacterium rhizogenes strain A4 was localized on the basis of sequence similarity with probes from Ti plasmids of Agrobacterium tumefaciens and analysis of transposon insertions. The nucleotide sequence of the right part of the TR-DNA of pRiA4, encompassing the three genes involved in mannityl-opine synthesis, was determined and compared to the sequence of the corresponding region of the octopine-type Ti plasmid pTi15955. The organization of this region is strongly conserved between Ri and Ti plasmids, but the similarity is restricted to the coding sequences: no homology was detected in the 5' and 3' flanking sequences. The mas1' and ags proteins are the most conserved, showing more than 68% amino acid conservation, whereas the mas2' proteins are only 59% identical. Significant G/C content and codon usage differences are observed between pTi15955 and pRiA4. An open reading frame strongly similar to that of bacterial repressors is situated immediately to the right of the TR region.

Amino Acid Sequence↗
PubMed1997

The mouse Clc1/myotonia gene: ETn insertion, a variable AATC repeat, and PCR diagnosis of alleles.

Myotonias are muscle diseases in which the function of the muscular chloride channel ClC-1 is impaired. Null alleles of the corresponding Clc1 gene on mouse chromosome (Chr) 6 provide animal models for human myotonias. It was shown that the allele adr (Clc1adr) is due to an insertion of an ETn type transposon that is transcribed and leads to multiple splicing events; the allele mto (Clc1adr-mto) involves a stop codon near the N-terminus. We have determined the genomic organization of the mouse Clc1 gene and the sequence requirements for the transposon insertion in the Clc1adr allele. The mouse Clc1 gene is composed of 23 exons, ranging from 39 to 372 bp, and spans approximately 23 kb of genomic DNA. The exon/intron organization is highly homologous to that of the human CLCN1 gene; the homology of the coding sequence is 97% to rat and 89% to human. In the adr allele the ETn transposon is inserted into intron 12, the largest intron. Whereas the 5' and 3' LTR sequences of the ETn transposon are homologous to those reported for other insertional mutations of the mouse, no consensus motif for an insertion target site could be defined. On the basis of flanking sequences, we provide duplex PCR diagnoses for the adr, adr-mto, and wild-type alleles of Clc1. Close to the 3' end of intron 12, a tetranucleotide repeat (AATC)n was found that is polymorphic between mouse species Mus musculus, M. molossinus, M. castaneus, and M. spretus, and can thus be used for chromosomal mapping studies.

Alleles↗
PubMed2003

Isolation of the Pseudomonas aeruginosa gene affecting uptake of dibenzothiophene in n-tetradecane.

The dsz desulfurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred into the chromosomes of Pseudomonas aeruginosa strain NCIMB9571 using a transposon vector. All of the recombinant strains completely desulfurized 1 mM dibenzothiophene (DBT) in n-tetradecane (n-TD) except one, named strain PARMI. Strain PARMI was unable to desulfurize DBT in n-TD, but was able to desulfurize it in water. The n-alkane utilization ability, the biosurfactant production and the fatty acid composition of cells in strain PARMI were the same level as those of the other recombinants. The transposon insertion area of strain PARMI was analyzed by transposon tagging. We cloned three possible open reading frames (ORFs), designated hcuA, hcuB and hcuC, from the genomic DNA of P. aeruginosa NCIMB9571 using the transposon insertion area of strain PARMI as a DNA probe. Examination of the sequence revealed the transposon was inserted into hcuA. The full length of the hcuABC genes transformed into strain PARMI achieved 87% recovery of the desulfurization activity of DBT in n-TD, but partial hcuABC genes achieved only 0-12%. These results indicate that DBT desulfurization in the oil phase by recombinant P. aeruginosa strain NCIMB9571 requires the full length of the hcuABC gene cluster. The hcuABC gene cluster relates to DBT uptake from the oil phase to inside of the cell, and the uptake ability is independent of the n-alkane utilization ability, the biosurfactant production and the fatty acid composition of cells.

Journal Article↗
PubMed1999

A transposon insertion in the gumG homologue of Xanthomonas oryzae pv. oryzae causes loss of extracellular polysaccharide production and virulence.

Xanthomonas oryzae pv. oryzae causes a serious disease of rice called bacterial leaf blight. It produces copious amounts of extracellular polysaccharide (EPS). An EPS- and virulence-deficient mutant of X. oryzae pv. oryzae was isolated by Tn5 mutagenesis. The mutant allele in this strain was cloned by transposon tagging in the Escherichia coli vector pBluescript and the DNA sequences flanking the transposon insertion site were determined. Computer-based similarity searches in the DNA database using the BLAST algorithm showed these sequences to be 78% identical at the nucleotide level to a gene, gumG, in the gum cluster, which is required for EPS biosynthesis in Xanthomonas campestris pv. campestris. A 36-kb X. oryzae pv. oryzae genomic clone containing the putative EPS biosynthetic gene cluster of X. oryzae pv. oryzae restored both EPS production and virulence proficiency to the gumGXo::Tn5 mutant. The results suggest that EPS is an important virulence factor of X. oryzae pv. oryzae.

Algorithms↗