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Results for “transposon insertion sequencing”

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PubMed1994

Transposon-mediated mobilization of chromosomally located catabolic operons of the CAM plasmid by TOL plasmid transposon Tn4652 and CAM plasmid transposon Tn3614.

The CAM (camphor degradation) plasmid is integrated into the chromosome of Pseudomonas putida PaW-line strains and is not self-transferable as a plasmid via conjugation. Our results show that the mobilization of chromosomally located CAM and the integration of cam-operons into the chromosome of the new Cam+ transconjugants is a recA-independent process mediated by transposons Tn4652 (17 kbp) and Tn3614 (7.2 kbp). Transposon Tn3614 is apparently identical to the left-hand and the right-hand sequences of the TOL plasmid pWW0 transposon Tn4654. The insertion of Tn401 inside the left-hand terminal IR of Tn4652 completely inhibited the mobilization of CAM. According to our data transposons Tn4652 and Tn3614 together with CAM plasmid catabolic operons are integrated into the chromosome. We propose that in pseudomonads the transposons Tn4652 and Tn3614 play a key role in the evolution and spread of new catabolic plasmids in nature.

Chromosomes, Bacterial↗

PubMed2003

Mutation in tcpR gene (Vc0832) of Vibrio cholerae O1 causes loss of tolerance to high osmolarity and affects colonization and virulence in infant mice.

Vibrio cholerae, the agent of cholera, multiplies and colonizes human intestinal tract where it survives high osmolarity due to bile and other sodium salts. In this work, by TnphoA mutagenesis, a mutant of V. cholerae O1 which could not grow and form colonies on LB agar containing 400 mM NaCl has been characterized. The mutant, designated CD83, adhered normally to freshly isolated rabbit intestinal discs, colonized poorly the gut of infant mice and was avirulent in the same model, whereas the parent strain CD81 would colonize the gut and cause death of mice. Attenuation of virulence of CD83 was not attributable to its inability to produce cholera toxin, as no difference was found in the cholera toxin produced in vitro by the mutant and parent strains. Molecular cloning and sequencing of the mutated gene revealed that insertion of transposon occurred in tcpR gene (Vc0832) of V. cholerae. Complementation of the mutant with wild-type tcpR gene resulted in restoration of the ability to survive at high salt concentration (400 mM NaCl), and to colonize the gut and restore virulence. The results suggest that tcpR plays a role in survival of V. cholerae in the small intestine of host as the osmolarity in the intestinal lumen is thought to be equivalent to 300 mM NaCl or higher.

Animals↗

PubMed2000

Identification of a two-component signal transduction system involved in fimbriation of Porphyromonas gingivalis.

Porphyromonas gingivalis, a periodontopathogen, is an oral anaerobic gram-negative bacterium with numerous fimbriae on the cell surface. Fimbriae have been considered to be an important virulence factor in this organism. We analyzed the genomic DNA of transposon-induced, fimbria-deficient mutants derived from ATCC 33277 and found that seven independent mutants had transposon insertions within the same restriction fragment. Cloning and sequencing of the disrupted region from one of the mutants revealed two adjacent open reading frames (ORFs) which seemed to encode a two-component signal transduction system. We also found that six of the mutants had insertions in a gene, fimS, a homologue of the genes encoding sensor kinase, and that the insertion in the remaining one disrupted the gene immediately downstream, fimR, a homologue of the response regulator genes in other bacteria. These findings suggest that this two-component regulatory system is involved in fimbriation of P. gingivalis.

Amino Acid Sequence↗

PubMed2001

Functional analysis of the Bacillus subtilis cysK and cysJI genes.

The function of the Bacillus subtilis cysK and cysJI (previously designated yvgQR) genes, expected to be involved in the assimilatory sulfate reduction pathway, was investigated. A B. subtilis mutant with a deletion in the cysJI genes was unable to use sulfate or sulfite as sulfur source, which confirmed that these genes encode sulfite reductase. A mutant with a transposon insertion in the cysK gene, whose deduced protein sequence showed similarity to cysteine synthases, grew poorly on sulfate and butanesulfonate. A strain in which cysK and yrhA, a cysK paralog, were inactivated was unable to grow with sulfate. Whereas expression of the cysJI genes was induced by sulfate, expression of cysK was repressed both by sulfate and by cysteine.

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↗

PubMed2002

Role of the htrA gene in Klebsiella pneumoniae virulence.

We recently described the use of mini-Tn5 to generate complement-sensitive mutants derived from a complement-resistant Klebsiella pneumoniae clinical isolate deficient in the lipopolysaccharide O side chain. One mutant with a reduced capacity to survive in nonimmune human sera carried the transposon inserted in the htrA gene. We cloned and sequenced the gene and predicted from the deduced amino acid sequence that the putative HtrA homolog contains structural features similar to those of previously described HtrA proteins. To investigate the biological functions and the role of the htrA gene in the virulence of K. pneumoniae, we constructed an isogenic mutant by insertion-duplication mutagenesis. Characterization of the mutant showed that it had greater sensitivity to temperature (50 degrees C) and oxidative stress (H(2)O(2)) than the parent strain. Furthermore, the htrA mutant produced less capsule, bound more molecules of complement component C3, and was more sensitive to complement and whole-blood killing than was the parent strain. Finally, disruption of the htrA gene in a virulent K. pneumoniae strain caused a reduction of its virulence in a mice model. Our results indicate that the htrA gene plays an important role in the virulence of K. pneumoniae.

Animals↗

PubMed1995

AinS and a new family of autoinducer synthesis proteins.

In Vibrio fischeri, the autoinducer N-3-oxohexanoyl-L-homoserine lactone (AI-1) governs the cell density-dependent induction of the luminescence operon via the LuxR transcriptional activator. The synthesis of AI-1 from bacterial metabolic intermediates is dependent on luxI. Recently, we found a second V. fischeri autoinducer molecule, N-octanoyl-L-homoserine lactone (AI-2), that in E. coli also activates the luminescence operon via LuxR. A locus independent of luxI was identified as being required for AI-2 synthesis. This 2.7-kb ain (autoinducer) locus was characterized by transposon insertion mutagenesis, deletion and complementation analysis, and DNA sequencing. A single 1,185-bp gene, ainS, was found to be the sole exogenous gene necessary for the synthesis of AI-2 in Escherichia coli. In addition, a V. fischeri ainS mutant produced AI-1 but not AI-2, confirming that in its native species ainS is specific for the synthesis of AI-2. ainS is predicted to encode a 45,580-Da protein which exhibits no similarity to LuxI or to any of the LuxI homologs responsible for the synthesis of N-acyl-L-homoserine lactones in a variety of other bacteria. The existence of two different and unrelated autoinducer synthesis genes suggests the occurrence of convergent evolution in the synthesis of homoserine lactone signaling molecules. The C-terminal half of AinS shows homology to a putative protein in Vibrio harveyi, LuxM, which is required for the synthesis of a V. harveyi bioluminescence autoinducer. Together, AinS and LuxM define a new family of autoinducer synthesis proteins. Furthermore, the predicted product of another gene, ainR, encoded immediately downstream of ainS, shows homology to LuxN, which is similarly encoded downstream of luxM in V. harveyi and proposed to have sensor/regulator functions in the bioluminescence response to the V. harveyi auto inducer. This similarity presents the possibility that AI-2, besides interacting with LuxR, also interacts with AinR under presently unknown conditions.

4-Butyrolactone↗

PubMed2004

H-NS represses Salmonella enterica serovar Typhimurium dsbA expression during exponential growth.

Disulfide bond formation catalyzed by disulfide oxidoreductases occurs in the periplasm and plays a major role in the proper folding and integrity of many proteins. In this study, we were interested in elucidating factors that influence the regulation of dsbA, a gene coding for the primary disulfide oxidoreductase found in Salmonella enterica serovar Typhimurium. Strains with mutations created by transposon mutagenesis were screened for strains with altered expression of dsbA. A mutant (NLM2173) was found where maximal expression of a dsbA::lacZ transcriptional fusion occurred in the exponential growth phase in contrast to that observed in the wild type where maximal expression occurs in stationary phase. Sequence analysis of NLM2173 demonstrated that the transposon had inserted upstream of the gene encoding H-NS. Western immunoblot analysis using H-NS and StpA antibodies showed decreased amounts of H-NS protein in NLM2173, and this reduction in H-NS correlated with an increase of StpA protein. Northern blot analysis with a dsbA-specific probe showed an increase in dsbA transcript during exponential phase of growth. Direct binding of H-NS to the dsbA promoter region was verified using purified H-NS in electrophoretic mobility shift assays. Thus, a reduction in H-NS protein is correlated with a derepression of dsbA in NLM2173, suggesting that H-NS normally plays a role in suppressing the expression of dsbA during exponential phase growth.

Bacterial Proteins↗

PubMed2000

Fast screening procedures for random transposon libraries of cloned herpesvirus genomes: mutational analysis of human cytomegalovirus envelope glycoprotein genes.

We have cloned the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli. Here, we have subjected the HCMV BAC to random transposon (Tn) mutagenesis using a Tn1721-derived insertion sequence and have provided the conditions for excision of the BAC cassette. We report on a fast and efficient screening procedure for a Tn insertion library. Bacterial clones containing randomly mutated full-length HCMV genomes were transferred into 96-well microtiter plates. A PCR screening method based on two Tn primers and one primer specific for the desired genomic position of the Tn insertion was established. Within three consecutive rounds of PCR a Tn insertion of interest can be assigned to a specific bacterial clone. We applied this method to retrieve mutants of HCMV envelope glycoprotein genes. To determine the infectivities of the mutant HCMV genomes, the DNA of the identified BACs was transfected into permissive fibroblasts. In contrast to BACs with mutations in the genes coding for gB, gH, gL, and gM, which did not yield infectious virus, BACs with disruptions of open reading frame UL4 (gp48) or UL74 (gO) were viable, although gO-deficient viruses showed a severe growth deficit. Thus, gO (UL74), a component of the glycoprotein complex III, is dispensable for viral growth. We conclude that our approach of PCR screening for Tn insertions will greatly facilitate the functional analysis of herpesvirus genomes.

Cytomegalovirus↗

PubMedDate not supplied

[Virulence factors of Salmonella: from molecular genetics to diagnostic applications].

Salmonella serotype Typhimurium is a facultative intracellular pathogen that causes a systemic infection in naturally, or experimentally, infected mice. After oral contamination, Typhimurium colonizes the ileal mucosa and Peyer's patches and invades draining mesenteric lymph nodes. From these primary sites of infection, bacteria dissiminate to the reticuloendothelial system and proliferate rapidly in spleen and liver. Several virulence factors are encoded by chromosomal genes. The ability of Typhimurium to adhere to and invade epithelial cells has been associated with flagella, pili of type I and mannose-resistant haemagglutinating activity. By comparing the virulence of isogenic strains, it appeared that these traits played a marginal role and were not essential for full virulence expression. It is now clear that other surface structures are important for the invasiness capacity of Typhimurium. To multiply in the reticuloendothelial system, a complete lipopolysaccharide is necessary for the bacteria in resisting serum bactericidal activity and producing tissue damage. Salmonella have evolved a specialized iron-binding ligand, termed enterobactin, to acquire iron necessary for their multiplication. Enterobactin competes with the host iron-binding proteins (transferrin or lactoferrin) to secure the iron required by the bacteria. Though the presence of an enterotoxin in Salmonella is still controversial, there is now substantial evidence to support this concept. Recently, a gene encoding an enterotoxin has been cloned from Typhimurium and expressed in E. coli. Typhimurium strains harbour a 90 kilobases (kb) plasmid which is essential for virulence. This plasmid encodes virulence factors required for replication of Salmonella in liver and spleen. It was postulated that the plasmid allowed Typhimurium to multiply in Kupffer cells and in splenic macrophages. The virulence-associated region of the plasmid restored full virulence to plasmidless strains. Transposon insertion mutagenesis demonstrated the existence of two DNA sequences, designated Vir A and Vir B, which are essential for virulence expression. The Vir A region has been sequenced; it encodes four polypeptides with apparent molecular mass of 27,000, 28,000, 33,000 and 70,000. The Vir B region encodes two polypeptides of 38,000 and 43,000. In an attempt to identify bacterial components contributing to invasion of HeLa cells by Salmonella serovar Typhi, we cloned a 30 kb DNA sequence necessary for entry of bacteria into epithelial cells. However, this sequence is not sufficient for conferring an invasive phenotype to E. coli strains. From this DNA fragment, a short segment of 487 bp was subcloned, sequenced and used as probe to detect Salmonella.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacterial Adhesion↗

PubMed1998

Tn4351-generated non-haemolytic and/or non-pigmented mutants of Porphyromonas gingivalis.

Escherichia coli-Porphyromonas gingivalis plasmid-shuttle vectors R751::* omega 4, pVOH1, and pVAL-1 were used for isolation of non-pigmented, defective protease, or non-haemolytic activity phenotypes in P. gingivalis. Transfer frequencies for R751::* omega 4, pVOH1, and pVAL-1 varied from 1.7 x 10(-9) to 5.3 x 10(-11) depending on the P. gingivalis 381 and ATCC 33277 strains. Two erythromycin-resistant transconjugants were screened from P. gingivalis 381 and eighteen were screened from ATCC 33277. Among isolated transconjugants, two from ATCC 33277 contained only one transposon insertion, while others included both Tn4351 and R751 sequences. The one transconjugant which contained a single insertion of Tn4351, designated NUM-T14, demonstrated defective pigmentation, and defective protease and haemolytic activities. The other transconjugant, designated NUM-T29, demonstrated defective haemolytic activity, but had black pigmentation and protease activity. Cell surface protein analysis by SDS-PAGE indicated that 40 and 18 kD proteins were lost or reduced and that 45 and 36 kD proteins were made to appear in both NUM-T14 and NUM-T29 transconjugants.

Blotting, Southern↗

PubMed1997

High-frequency transposition of IS1373, the insertion sequence delimiting the amplifiable element AUD2 of Streptomyces lividans.

IS1373 is the putative insertion sequence delimiting the amplifiable unit AUD2 of Streptomyces lividans. Two IS1373-derived thiostrepton-resistant transposons, Tn5492 and Tn5494, transposed into multiple sites of the S. lividans chromosome at frequencies as high as 0.4 and 1%, respectively. Hence, IS1373 is a functional insertion sequence and its unique open reading frame, insA, encodes the transposase.

Anti-Bacterial Agents↗

PubMed1999

Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases.

DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase-like proteins. Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044. Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans. To our knowledge, this is the first case when a site-specific recombination system outside a transposon has been shown to be involved in transposition.

Amino Acid Sequence↗

PubMed1979

Sites of insertion of TnA and TnM in RP1 and its derivatives.

The sites of insertion of TnM into the plasmid RP1, into derivatives of RP1 lacking TnA, and into derivatives in which the location of TnA within RP1 has been altered, were determined. Similarly, the sites of insertion of TnA into derivatives of RP1 from which TnA had been deleted, both with and without copies of TnM, have been examined. These studies show the presence in the plasmids of 'hot-spots' for TnA and TnM insertion. It is clear from the observations, however, that a particular DNA sequence in the recipient replicon is not sufficient to definite a 'hot-spot' since particular sequences sometimes do, and sometimes do not, contain many sites of insertion for a given transposon.

Base Sequence↗

PubMed1986

DNA sequence analysis of ARS elements from chromosome III of Saccharomyces cerevisiae: identification of a new conserved sequence.

Four fragments of Saccharomyces cerevisiae chromosome III DNA which carry ARS elements have been sequenced. Each fragment contains multiple copies of sequences that have at least 10 out of 11 bases of homology to a previously reported 11 bp core consensus sequence. A survey of these new ARS sequences and previously reported sequences revealed the presence of an additional 11 bp conserved element located on the 3' side of the T-rich strand of the core consensus. Subcloning analysis as well as deletion and transposon insertion mutagenesis of ARS fragments support a role for 3' conserved sequence in promoting ARS activity.

Base Sequence↗

PubMed2001

Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis.

A GFP excision assay was developed to monitor the excision of Ac introduced into rice by Agrobacterium-mediated transformation. The presence of a strong double enhancer element of the CaMV 35S promoter adjacent to the Ac promoter induced very early excision, directly after transformation into the plant cell, exemplified by the absence of Ac in the T-DNA loci. Excision fingerprint analysis and characterization of transposition events from related regenerants revealed an inverse correlation between the number of excision events and transposed Ac copies, with single early excisions after transformation generating Ac amplification. New transpositions were generated at a frequency of 15-50% in different lines, yielding genotypes bearing multiple insertions, many of which were inherited in the progeny. The sequence of DNA flanking Ac in three representative lines provided a database of insertion tagged sites suitable for the identification of mutants of sequenced genes that can be examined for phenotypes in a reverse genetics strategy to elucidate gene function. Remarkably, two-thirds of Ac tagged sites showing homology to sequences in public databases were in predicted genes. A clear preference of transposon insertions in genes that are either predicted by protein coding capacity or by similarity to ESTs suggests that the efficiency of recovering knockout mutants of genes could be about three times higher than random. Linked Ac transposition, suitable for targeted tagging, was documented by segregation analysis of a crippled Ac element and by recovery of a set of six insertions in a contiguous sequence of 70 kb from chromosome 6 of rice.

Base Sequence↗

PubMed2002

Determination of Trichuris muris from murid hosts and T. arvicolae (Nematoda) from arvicolid rodents by amplification and sequentiation of the ITS1-5.8S-ITS2 segment of the ribosomal DNA.

Trichuris muris has been isolated from murid hosts ( Apodemus sylvaticus and Mus musculus) and Trichuris arvicolae from arvicolid rodents in Barcelona, Spain. Genomic DNA was isolated and the ITS1-5.8S-ITS2 segment from the ribosomal DNA (rDNA) was amplified and sequenced using polymerase chain reaction techniques. The ITS2 of both populations isolated from Apodemus and Mus was 382 nucleotides in length and had a GC content of about 60.73%, while the ITS2 of T. arvicolae was 442 nucleotides in length and had a GC content of about 59.8%. Furthermore, the ITS1 of Trichuris from murids was 448 nucleotides in length and had a GC content of about 56.47%, while T. arvicolae was 446 nucleotides in length and had 57.62% of GC content. A total of 161 and 173 nucleotides were observed along the 5.8S gene of T. murisand T. arvicolae, respectively; This difference in nucleotides was due to the insertion of a DNA segment (transposon) in the 5.8S sequence of the latter species. Slight intraindividual and intraspecific variations were detected in the rDNA of both species. The presence of microsatellites was observed in all of the individuals assayed. Sequence analysis of the internal transcribed spacers and the 5.8S gene demonstrated no sequence differences between T. muris isolated from both of its murid hosts. Nevertheless, clear differences were detected between the ITS2, ITS1 and 5.8S gene of T. muris and T. arvicolae. This corroborates the existence of two separate Trichuris species in murid and arvicolid hosts. Furthermore, a phylogenetic analysis was carried out and endonucleases restriction maps were elaborated for both species.

Animals↗

PubMed1996

Evidence that the hanA gene coding for HU protein is essential for heterocyst differentiation in, and cyanophage A-4(L) sensitivity of, Anabaena sp. strain PCC 7120.

The highly pleiotropic, transposon-generated mutant AB22 of Anabaena sp. strain PCC 7120 exhibits slow growth, altered pigmentation, cellular fragility, resistance to phage A-4(L), and the inability to differentiate heterocysts. Reconstruction of the transposon mutation in the wild-type strain reproduced the phenotype of the original mutant. Sequencing of the flanking DNA showed that the transposon had inserted at the beginning of a gene, which we call hanA, that encodes Anabaena HU protein (R. Nagaraja and R. Haselkorn, Biochimie 76:1082-1089, 1994). Mapping of the transposon insertion by pulsed-field gel electrophoresis showed that hanA is located at ca. 4.76 Mb on the physical map of the chromosome and is transcribed clockwise. Repeated subculturing of AB22 resulted in improved growth and loss of filament fragmentation, presumably because of one or more compensatory mutations; however, the mutant retained its A-4(L)r Het- phenotype. The mutation in strain AB22 could be complemented by a fragment of wild-type DNA bearing hanA as its only open reading frame.

Amino Acid Sequence↗