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Results for “transposon insertion sequencing”

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PubMed1999

Streptococcus mutans ffh, a gene encoding a homologue of the 54 kDa subunit of the signal recognition particle, is involved in resistance to acid stress.

The ability of Streptococcus mutans, a bacterial pathogen associated with dental caries, to tolerate rapid drops in plaque pH (acidurance), is considered an important virulence factor. To study this trait, Tn917 mutants of S. mutans strain JH1005 which display acid sensitivity have been isolated and partially characterized. In this paper, the characterization of one of these mutants, AS17, is reported. Preliminary sequence analysis revealed that the transposon insertion in AS17 occurred in the intergenic region of a two-gene locus which has been named sat for secretion and acid tolerance. This locus displays a high degree of homology to the ylxM-ffh operon of Bacillus subtilis. The sat+ locus was cloned by complementation of a conditional Escherichia coli ffh mutant with an S. mutans genomic library. Sequencing of the complementing clone identified the intact ylxM and ffh genes as well as a partial ORF with homology to the proUlopuAC gene of B. subtilis which encodes the binding protein of the ProU/OpuA osmoregulated glycine betaine transport system. RNA dot blot experiments indicated steady-state levels of ffh mRNA in the mutant that were approximately eightfold lower compared to parental levels. This suggests a partial polar effect of the sat-1::Tn917 mutation on ffh expression. Upon acid shock (pH 5), wild-type ffh mRNA levels were found to increase approximately four- to eightfold compared to unstressed (pH 7.5) levels. Mutant levels remained unaltered under the same conditions. Experiments designed to investigate the origins of the acid-sensitivity of the mutant revealed a lack of an acid-adaptive/tolerance response. Assays of proton-extruding ATPase (H+/ATPase) specific activity measured with purified membranes derived from acid-shocked AS17 showed twofold lower levels compared to the parent strain. Also, AS17 was found to be unable to ferment sorbitol although it was able to grow in glucose and a variety of other sugar substrates. These findings suggest that Ffh may be involved in the maintenance of a functional membrane protein composition during adaptation of S. mutans to changing environmental conditions.

Bacterial Proteins↗

PubMed2006

Genetic analysis of a multiresistant strain of Pseudomonas aeruginosa producing PER-1 beta-lactamase.

A multiresistant strain of Pseudomonas aeruginosa, PA2345, belonging to serotype O:1, was isolated at the Teaching Hospital of Besançon, France. Resistance to beta-lactams, including third-generation cephalosporins, depended upon a chromosomally-located composite transposon carrying the bla(PER-1) gene encoding extended-spectrum beta-lactamase PER-1. PA2345 was unrelated genotypically to two previous PER-1-producing isolates of P. aeruginosa. Sequence analysis of the transposon in PA2345 revealed the presence of two insertion sequences (ISPa23 and ISPa24) with very different predicted transposases (TnpA1, TnpA2), which were both bordered by closely related 16-bp inverted repeats. High resistance of PA2345 to aminoglycosides was caused, in part, by a chromosomal class-I integron containing gene cassettes aadB, encoding an ANT(2'') enzyme, and aadA11, encoding a new ANT(3'') enzyme with 281 amino-acids that conferred elevated resistance to streptomycin and spectinomycin. Stable overproduction of efflux system MexXY contributed to resistance to amikacin, while mutations in the quinolone resistance-determining regions of gyrA and parC accounted for the high resistance of PA2345 to fluoroquinolones. The study indicates that multidrug resistance in P. aeruginosa might arise from sequential acquisition of a variety of mechanisms provided by both horizontal gene transfers and mutations in chromosomal genes.

Drug Resistance, Multiple, Bacterial↗

PubMed1987

Detection and characterization of IS256, an insertion sequence in Staphylococcus aureus.

Resistance to the aminoglycosides gentamicin (Gmr), tobramycin (Tmr) and kanamycin (Kmr) in strains of Staphylococcus aureus isolated in Australia is mediated by the transposon Tn4001. The 1.35 kb inverted repeat of this transposon exhibits many of the characteristics of an insertion sequence and has consequently been designated IS256. Tandem duplication of IS256 contiguous with Tn4001 results in an increase in the level of GmrTmrKmr, thereby implying that the element possesses strong promoter sequences. Both contiguous and independent insertions of IS256 into the staphylococcal chromosome have been observed, the latter suggesting that the element may play a role in molecular rearrangements of the genome.

Chromosome Mapping↗

PubMed1990

The pheR gene of Escherichia coli encodes tRNA(Phe), not a repressor protein.

Nucleotide sequence analysis and transposon 5 (Tn5) insertional mutagenesis indicate that the Escherichia coli gene pheR encodes tRNA(Phe) and not a repressor protein as previously reported. The coding region of pheR is identical to that of three other cloned tRNA(Phe) genes, pheU, pheV, and pheW. Multicopy plasmids carrying pheR, like those carrying pheU, pheV, or pheW, complement a temperature-sensitive lesion in the gene for the alpha-subunit of phenylalanyl-tRNA synthetase (pheS). The nucleotide sequences of the 5'-flanking DNA of pheR, pheU, and pheW are almost identical but are quite different from the same region of pheV. By comparison with pheV, which has two tandem promoters, pheR was found to have a single promoter. The expression of pheA (encoding chorismate mutase/prephenate dehydratase) in strains carrying the pheR374 allele was decreased to similar extents by multicopy plasmids containing either pheR or pheV. It is proposed that this decrease in pheA expression and the increase in expression of pheA previously reported for chromosomal pheR mutants are both mediated through the attenuation control mechanism that regulates pheA.

Base Sequence↗

PubMed1991

Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones.

The 264 bp mini-transposon Tn5supF was constructed to sequence DNAs cloned in phage lambda without extensive shotgun subcloning or primer walking. Unique sequences near each transposon end serve as primer binding sites, and a supF gene is used to select transposition to lambda. We describe here PCR methods that facilitate Tn5supF-based sequencing. In a first pass, insertions are mapped relative to the ends of the cloned fragment using pairs of primers specific for vector DNA next to the cloning site and for a Tn5supF end. Most insertions not mapped in this step are near the center of the cloned fragment or in the vector arms, and are then mapped relative to the two innermost insertions by 'crossover' PCR. This involves amplification from primers on different DNA molecules, and generates hybrid DNA products whose lengths correspond to the distances between the two insertions. We routinely amplified more than 6 kb in direct PCR and 3 kb in crossover PCR; at the limit we amplified up to approximately 10 kb in direct PCR and approximately 6 kb in crossover PCR, but not reproducibly. Crossover PCR products were also obtained with insertions separated by only 200 bp, indicating that no rare sites are needed to switch templates. PCR products were purified by adsorption and then elution from glass slurry, and sequenced directly. Ladders of more than 400 bp were obtained from primer sites on each DNA strand; 2 kb was read from crossover PCR products, and showed that they were amplified with fidelity. In conclusion, direct and crossover PCR methods expedite transposon insertion mapping, and yield templates for accurate sequencing of both DNA strands.

Autoradiography↗

PubMed2006

Mariner mutagenesis of Brucella melitensis reveals genes with previously uncharacterized roles in virulence and survival.

BACKGROUND: Random gene inactivation used to identify cellular functions associated with virulence and survival of Brucella spp has relied heavily upon the use of the transposon Tn5 that integrates at G/C base pairs. Transposons of the mariner family do not require species-specific host factors for efficient transposition, integrate nonspecifically at T/A base pairs, and, at a minimum, provide an alternative approach for gene discovery. In this study, plasmid vector pSC189, containing both the hyperactive transposase C9 and transposon terminal inverted repeats flanking a kanamycin resistance gene, were used to deliver Himar1 transposable element into the B. melitensis genome. Conjugation was performed efficiently and rapidly in less than one generation in order to minimize the formation of siblings while assuring the highest level of genome coverage. RESULTS: Although previously identified groups or classes of genes required for virulence and survival were represented in the screen, additional novel identifications were revealed and may be attributable to the difference in insertion sequence biases of the two transposons. Mutants identified using a fluorescence-based macrophage screen were further evaluated using gentamicin-based protection assay in macrophages, survival in the mouse splenic clearance model and growth in vitro to identify mutants with reduced growth rates. CONCLUSION: The identification of novel genes within previously described groups was expected, and nearly two-thirds of the 95 genes had not been previously reported as contributing to survival and virulence using random Tn5-based mutagenesis. The results of this work provide added insight with regard to the regulatory elements, nutritional demands and mechanisms required for efficient intracellular growth and survival of the organism.

Animals↗

PubMed1999

High-resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234.

Most of the bacterial genes involved in nodulation of legumes (nod, nol and noe ) as well as nitrogen fixation (nif and fix ) are carried on pNGR234a, the 536 kb symbiotic plasmid (pSym) of the broad-host-range Rhizobium sp. NGR234. Putative transcription regulators comprise 24 of the predicted 416 open reading frames (ORFs) contained on this replicon. Computational analyses identified 19 nod boxes and 16 conserved NifA-sigma54 regulatory sequences, which are thought to co-ordinate the expression of nodulation and nitrogen fixation genes respectively. To analyse transcription of all putative ORFs, the nucleotide sequence of pNGR234a was divided into 441 segments designed to represent all coding and intergenic regions. Each of these segments was amplified by polymerase chain reactions, transferred to filters and probed with radioactively labelled RNA. RNA was extracted from bacterial cultures grown under various experimental conditions, as well as from bacteroids of determinate and indeterminate nodules. Generally, genes involved in the synthesis of Nod factors (e.g. the three hsn loci) were induced rapidly after the addition of flavonoids, whereas others thought to act within the plant (e.g. those encoding the type III secretion system) responded more slowly. Many insertion (IS) and transposon (Tn)-like sequences were expressed strongly under all conditions tested, while a number of loci other than those known to encode nod, noe, nol, nif and fix genes were also transcribed in nodules. Many more diverse transcripts were found in bacteroids of determinate as opposed to indeterminate nodules.

Base Sequence↗

PubMed2002

Association of different mobile elements to generate novel integrative elements.

Among the more important problems in modern hospitals is the prevalence of bacterial pathogens expressing resistance to multiple antimicrobial agents. The frequency of multiresistance suggests mechanisms by which bacterial species can concentrate and efficiently exchange a variety of resistance determinants. Mechanisms by which this occurs include insertion of transposons within transposons, coalescence through the activity of insertion sequences and the employment of integrons. In some instances, more than one of these mechanisms is involved in creating large multiresistance genetic elements. The association of the elements with transferable elements or transposons may promote rapid dissemination among clinical strains, and create further opportunities for inclusion of additional resistance determinants.

Bacteria↗

PubMed2004

Proteus mirabilis genes that contribute to pathogenesis of urinary tract infection: identification of 25 signature-tagged mutants attenuated at least 100-fold.

Proteus mirabilis, a common cause of urinary tract infections (UTI) in individuals with functional or structural abnormalities or with long-term catheterization, forms bladder and kidney stones as a consequence of urease-mediated urea hydrolysis. Known virulence factors, besides urease, are hemolysin, fimbriae, metalloproteases, and flagella. In this study we utilized the CBA mouse model of ascending UTI to evaluate the colonization of mutants of P. mirabilis HI4320 that were generated by signature-tagged mutagenesis. By performing primary screening of 2088 P. mirabilis transposon mutants, we identified 502 mutants that ranged from slightly attenuated to unrecoverable. Secondary screening of these mutants revealed that 114 transposon mutants were reproducibly attenuated. Cochallenge of 84 of these single mutants with the parent strain in the mouse model resulted in identification of 37 consistently out-competed P. mirabilis transposon mutants, 25 of which were out-competed >100-fold for colonization of the bladder and/or kidneys by the parent strain. We determined the sequence flanking the site of transposon insertion in 29 attenuated mutants and identified genes affecting motility, iron acquisition, transcriptional regulation, phosphate transport, urease activity, cell surface structure, and key metabolic pathways as requirements for P. mirabilis infection of the urinary tract. Two mutations localized to a approximately 42-kb plasmid present in the parent strain, suggesting that the plasmid is important for colonization. Isolation of disrupted genes encoding proteins with homologies to known bacterial virulence factors, especially the urease accessory protein UreF and the disulfide formation protein DsbA, showed that the CBA mouse model and mutant pools are a reliable source of attenuated mutants with mutations in virulence genes.

Animals↗

PubMed1996

The integrons In0, In2, and In5 are defective transposon derivatives.

The class 1 integrons In0, In2, and In5, found in different locations in pVS1, Tn21, and pSCH884, have closely related structures. All three integrons contain an insertion sequence, IS1326, that is a new member of the IS21 family. IS1326 has caused deletions of adjacent 3'-conserved segment and transposition module sequences, and all three integrons retain a complete copy of only one of four genes required for transposition of related transposons and are thus defective transposon derivatives. In2 contains an additional insertion sequence, IS1353, located within IS1326. IS1353 is a member of the IS3 family and appears to have been acquired after the integron was inserted into an ancestral mercury resistance transposon to create the ancestor of Tn21 and several other transposons that are close relatives of Tn21.

Amino Acid Sequence↗

PubMed2004

Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNA.

The bacterial transposon Tn10 inserts preferentially into specific target sequences. This insertion specificity appears to be linked to the ability of target sites to adopt symmetrically positioned DNA bends after binding the transposition machinery. Target DNA bending is thought to permit the transposase protein to make additional contacts with the target DNA, thereby stabilizing the target complex so that the joining of transposon and target DNA sequences can occur efficiently. In the current work, we have asked whether the introduction of a discontinuity in a target DNA strand, a modification that is expected to make it easier for a DNA molecule to bend, can enhance or rescue target capture under otherwise suboptimal reaction conditions. We show that either a nick or a missing phosphate specifically at the site of reaction chemistry increases the ability of various target DNAs to form the target capture complex. The result suggests that the bends in the target DNA are highly localized and include the scissile phosphates. This raises the possibility that strand transfer is mechanistically linked to target capture. We have also identified specific residues in the target DNA and in transposase that appear to play an important role in target DNA bending.

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↗

PubMed1980

[Tn9 integration sites and their effect on transposon properties].

The insertion of transposable deoxyribonucleic acid sequence that specifies chloramphenicol acetyltransferase (Tn9) occurs in the preferential sites of the Escherichia coli K-12 chromosome. It is found that some strains of E. coli K-12 have different preferential sites for the Tn9 insertion, located in various regions of the chromosome. The IS1 element inserted into the galT gene of the N116 strain (insertion mutation galT 116:IS1) serves as a strongly preferential site for the Tn9 insertion in this strain. However, being transferred into the chromosome of the KS836 strain this element loses such preference. It was demonstrated that the transposons (Tn9) inserted into different chromosomal sites are distinguished by such properties as stability, different capability to transposition into genomes of bacteriophages and plasmids, and by the choice of the preferential sites of integration in the course of subsequent insertions.

Acetyltransferases↗

PubMed1998

An isoflavonoid-inducible efflux pump in Agrobacterium tumefaciens is involved in competitive colonization of roots.

Agrobacterium tumefaciens 1D1609, which was originally isolated from alfalfa (Medicago sativa L.), contains genes that increase competitive root colonization on that plant by reducing the accumulation of alfalfa isoflavonoids in the bacterial cells. Mutant strain I-1 was isolated by its isoflavonoid-inducible neomycin resistance following mutagenesis with the transposable promoter probe Tn5-B30. Nucleotide sequence analysis showed the transposon had inserted in the first open reading frame, ifeA, of a three-gene locus (ifeA, ifeB, and ifeR), which shows high homology to bacterial efflux pump operons. Assays on alfalfa showed that mutant strain I-1 colonized roots normally in single-strain tests but was impaired significantly (P < or = 0.01) in competition against wild-type strain 1D1609. Site-directed mutagenesis experiments, which produced strains I-4 (ifeA::gusA) and I-6 (ifeA::omega-Tc), confirmed the importance of ifeA for competitive root colonization. Exposure to the isoflavonoid coumestrol increased beta-glucuronidase activity in strain I-4 21-fold during the period when coumestrol accumulation in wild-type cells declined. In the same test, coumestrol accumulation in mutant strain I-6 did not decline. Expression of the ifeA-gusA reporter was also induced by the alfalfa root isoflavonoids formononetin and medicarpin but not by two triterpenoids present in alfalfa. These results show that an efflux pump can confer measurable ecological benefits on A. tumefaciens in an environment where the inducing molecules are known to be present.

Agrobacterium tumefaciens↗

PubMed1996

Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus.

Shiga-toxigenic Escherichia coli strains belonging to serotype O157 are important human pathogens, but the genetic basis of expression of the O157 antigen and the role played by the lipopolysaccharide O side chain in the adherence of this organism to epithelial cells are not understood. We performed TnphoA mutagenesis on E. coli O157:H7 strain 86-24 to identify a mutant (strain F12) deficient in O-antigen expression. Nucleotide sequence analysis demonstrated that the transposon inserted within an open reading frame with significant homology to rfbE of Vibrio cholerae O1 (U. H. Stroeher, L. E. Karageorgos, R. Morona, and P. A. Manning, Proc. Natl. Acad. Sci. USA 89:2566-2570, 1992), which is postulated to encode perosamine synthetase. This open reading frame was designated rfbE(EcO157:H7). The guanine-plus-cytosine fraction (0.35) suggests that rfbE(EcO157:H7) may have originated in a species other than E. coli. rfbE(EcO157:H7) is conserved in nontoxigenic E. coli O157 strains expressing a variety of other flagellar antigens but is not found in E. coli O55:H7 strains, which are more closely related to E. coli O157:H7. Strain F12 was significantly more adherent to HeLa cells in a quantitative adherence assay than was its E. coli O157:H7 parent, but they did not differ in other phenotypes. Restoration of the expression of the O side chain by complementation of the TnphoA mutation in strain F12 by a plasmid expressing intact rfbE(EcO157:H7) reduced the adherence of the hyperadherent strain F12. We conclude that rfbE(EcO157:H7) is necessary for the expression of the O157 antigen, that acquisition of E. coli rfb genes occurred independently in E. coli O157:H7 and unrelated O157 strains, and that the O side chain of E. coli O157:H7 lipopolysaccharide interferes with the adherence of E. coli O157:H7 to epithelial cells.

Amino Acid Sequence↗

PubMed1998

Identification of a hotspot for transformation of Neisseria meningitidis by shuttle mutagenesis using signature-tagged transposons.

Shuttle mutagenesis using signature-tagged transposons was employed to generate a library of individually tagged mutants of the Neisseria meningitidis strain B1940, which belongs to serogroup B. The use of tagged transposons allowed us to monitor for enrichment for single mutants during the process of shuttle mutagenesis, by amplification of the tags and subsequent sequence determination. Enrichment of a single clone occurred during the transformation of the meningococci with transposon-containing plasmid DNA. Sequence determination around the site of transposon insertion revealed that the transposon had mutagenized a previously unknown locus, which was designated hrtA (high rate of transformation). hrtA-mediated transformation was independent of TnMax5 and tag sequences, and it most probably involved recombination events. The hrtA locus is restricted to meningococci and gonococci and is present in few apathogenic neisserial species. Chromosomal mapping of hrtA and six further hrt sites revealed a random distribution of highly transforming DNA fragments on the meningococcal chromosome. In conclusion, our data demonstrate that shuttle mutagenesis of naturally competent bacteria using signature-tagged transposons allows the isolation of chromosomal DNA fragments, which exhibit a high transformation efficiency, and which, therefore, are likely to be involved in horizontal gene transfer.

Base Sequence↗

PubMed1996

The devR gene product is characteristic of receivers of two-component regulatory systems and is essential for heterocyst development in the filamentous cyanobacterium Nostoc sp. strain ATCC 29133.

Strain UCD 311 is a transposon-induced mutant of Nostoc sp. strain ATC C 29133 that is unable to fix nitrogen in air but does so under anoxic conditions and is able to establish a functional symbiotic association with the hornwort Anthoceros punctatus. These properties of strain UCD 311 are consistent with previous observations that protection against oxygen inactivation of nitrogenase is physiologically provided within A. punctatus tissue. Upon deprivation of combined nitrogen, strain UCD 311 clearly differentiates heterocysts and contains typical heterocyst-specific glycolipids; it also makes apparently normal akinetes upon phosphate starvation. Sequence analysis adjacent to the point of the transposon insertion revealed an open reading frame designated devR. Southern analysis established that similar sequences are present in other heterocyst-forming cyanobacteria. devR putatively encodes a protein of 135 amino acids with high similarity to the receiver domains of response regulator proteins characteristics of two-component regulatory systems. On the basis of its size and the absence of other functional domains, DevR is most similar to CheY and Spo0F. Reconstruction of the mutation with an interposon vector confirmed that the transposition event was responsible for the mutant phenotype. The presence of wild-type devR on a plasmid in strain UCD 311 restored the ability to fix nitrogen in air. While devR was not essential for differentiation of akinetes, its presence in trans in Nostoc sp. strain ATCC 29133 stimulated their formation to above normal levels in aging medium. On the basis of RNA analysis, devR is constitutively expressed with respect to the nitrogen source for growth. The devR gene product is essential to the development of mature heterocysts and may be involved in a sensory pathway that is not directly responsive to cellular nitrogen status.

Amino Acid Sequence↗

PubMed2006

Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants.

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.

Animals↗