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Results for “DNA Transformation Competence”

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PubMed1997

Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae--a review.

In Neisseria gonorrhoea (Ngo), the processes of type-4 pilus biogenesis and DNA transformation are functionally linked and play a pivotal role in the life style of this strictly human pathogen. The assembly of pili from its main subunit pilin (PilE) is a prerequisite for gonococcal infection since it allows the first contact to epithelial cells in conjunction with the pilus tip-associated PilC protein. While the components of the pilus and its assembly machinery are either directly or indirectly involved in the transport of DNA across the outer membrane, other factors unrelated to pilus biogenesis appear to facilitate further DNA transfer across the murein layer (ComL, Tpc) and the inner membrane (ComA) before the transforming DNA is rescued in the recipient bacterial chromosome in a RecA-dependent manner. Interestingly, PilE is essential for the first step of transformation, i.e., DNA uptake, and is itself also subject to transformation-mediated phase and antigenic variation. This short-term adaptive mechanism allows Ngo to cope with changing micro-environments in the host as well as to escape the immune response during the course of infection. Given the fact that Ngo has no ecological niche other than man, horizontal genetic exchange is essential for a successful co-evolution with the host. Horizontal exchange gives rise to heterogeneous populations harboring clones which better withstand selective forces within the host. Such extended horizontal exchange is reflected by a high genome plasticity, the existence of mosaic genes and a low linkage disequilibrium of genetic loci within the neisserial population. This led to the concept that rather than regarding individual Neisseria species as independent traits, they comprise a collective of species interconnected via horizontal exchange and relying on a common gene pool.

Antigenic Variation↗

PubMed1996

Metabolic activation of benzo[a]pyrene in two fetal mouse hepatocyte lines: induction of DNA adducts and micronuclei.

We have studied the metabolic competence of two non-transformed epithelial-like cell lines derived from fetal mouse liver, C 6 and C 2.8, to activate the promutagen benzo[a]pyrene by measuring both the induction of DNA adducts through the nuclease P1-enhanced 32P-postlabeling assay and the formation of micronuclei. The pattern and level of DNA adducts detected in C 6 and C 2.8 cells treated with benzo[a]pyrene were compared with those obtained in human peripheral blood lymphocytes treated with the same compound and with [3H]anti-benzo[a]pyrene diolepoxide. In both the cell lines and in human lymphocytes we observed a consistent induction of distinct DNA adducts. In C 6 and C 2.8 cells, the most evident adduct showed a position similar to that of the main adduct induced by [3H]-anti-benzo[a]pyrene diolepoxide in human lymphocytes. In addition, benzo[a]pyrene caused a significant increase of micronucleated C 6 and C 2.8 cells, whereas the frequency of micronuclei did not increase in CHO cells treated, for comparison, in the same way.

Animals↗

PubMed2002

Construction of a marker rescue system in Bacillus subtilis for detection of horizontal gene transfer in food.

A marker rescue system based on the repair of the kanamycin resistance gene nptII was constructed for use in Gram-positive bacteria and established in Bacillus subtilis 168. Marker rescue was detected in vitro using different types of donor DNA containing intact nptII. The efficiency of marker rescue using chromosomal DNA of E. coli Sure as well as plasmids pMR2 or pSR8-30 ranged from 3.8 x 10(-8) to 1.5 x 10(-9) transformants per nptII gene. Low efficiencies of ca. 10(-12) were obtained with PCR fragments of 792 bp obtained from chromosomal DNA of E. coli Sure or DNA from a transgenic potato. B. subtilis developed competence during growth in milk and chocolate milk, and marker rescue transformation was detected with frequencies of ca. 10(-6) and 10(-8), respectively, using chromosomal DNA of E. coli Sure as donor DNA. Although the copy number of nptII genes of the plant DNA exceeded that of chromosomal E. coli DNA in the marker rescue experiments, a transfer of DNA from the transgenic plant to B. subtilis was detectable neither in vitro nor in situ.

Bacillus subtilis↗

PubMed1973

Transposition of the arsenate resistance locus of Bacillus subtilis strains 23 and 168.

Wild-type Bacillus subtilis strains 23 and 168 are resistant to high concentrations of sodium arsenate. The genetic configurations of the arsenate resistance loci of these two related strains of B. subtilis have been characterized. The transformable 168 strain has a single resistance locus which maps between phe and aroD in the terminal third of the genome. In contrast, strain 23 is shown to have its single arsenate resistance locus between purB and thr in the first third of the bacterial chromosome. Moreover, in strain 23 the chromosomal segment equivalent to the phe-linked asa region of 168 strains is missing. DNA isolated from 23 strains is able to transform 168 arsenate-sensitive strains to resistance and the heterologous 23 DNA is found to preferentially establish a new purB linked asa locus in such transformed cells. Thus, the majority of phenotypically arsenate-resistant cells recovered after exposure of competent 168 sensitive mutants to 23 DNA are "heterozygous" and still retain their phe-linked mutated asa locus. The tolerance of several of these heterologously transformed hybrid strains to arsenate suggests that the 168 and 23 asa gene products are similar, and a transposition model for the evolution of arsenate resistance in B. subtilis is proposed.

Arsenic↗

PubMed1987

LiCl treatment releases a nickase implicated in genetic transformation of Streptococcus pneumoniae.

When cells competent for genetic transformation of Streptococcus pneumoniae which could bind and enable entry of extracellular DNA molecules were treated with LiCl, they released a nickase that introduced nicks into a double-stranded DNA in the presence of EDTA. The nickase was specific for competent cells and coupled with DNA-binding activity. Furthermore, when noncompetent cells were treated with LiCl, they released the putative receptors for the competence activator.

Chlorides↗

PubMed1981

A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication.

Deletion mutants within the Py DNA region between the replication origin and the beginning of late protein coding sequences have been constructed and analysed for viability, early gene expression and viral DNA replication. Assay of replicative competence was facilitated by the use of Py transformed mouse cells (COP lines) which express functional large T-protein but contain no free viral DNA. Viable mutants defined three new nonessential regions of the genome. Certain deletions spanning the PvuII site at nt 5130 (67.4 mu) were unable to express early genes and had a cis-acting defect in DNA replication. Other mutants had intermediate phenotypes. Relevance of these results to eucaryotic "enhancer" elements is discussed.

Animals↗

PubMed1991

Novel plasmid vectors for gene cloning in Pseudomonas.

Novel host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. We found that a new Pseudomonas strain, Pseudomonas flavida IF-4, isolated from soil, carried two small cryptic plasmids, named pNI10 and pNI20. They were multi-copy, but not self-transmissible, and the genome size was 3.7 kb for pNI10 and 2.9 kb for pNI20. Several types of cloning vectors containing a kanamycin or streptomycin resistance (Kmr or Smr) gene were constructed from pNI10 and pNI20. These plasmid vectors were efficiently transformed into several strains of Pseudomonas at a frequency up to 4 x 10(5) transformants per 1 microgram plasmid DNA by the usual competent cell method. The vectors derived from pNI10 replicated not only in Pseudomonas but also in some other Gram-negative enteric bacteria such as Escherichia coli, Enterobacter aerogenes, and Proteus mirabilis.

Blotting, Southern↗

PubMed1982

Correlation of the Genetic Map and the Endonuclease Site Map of Bacillus subtilis Bacteriophage SP02.

By marker rescue of bacteriophage SP02 sus mutants with purified bacteriophage SP02 DNA fragments, 11 of the 17 known bacteriophage SP02 sus loci were assigned to discrete DNA fragments. The left-most genetic locus, susA, was found to reside near one bacteriophage SP02 terminus (EcoRI-C1 fragment), whereas the right-most genetic locus, susP, was found to reside near the other bacteriophage SP02 terminus (EcoRI-C2 fragment). The physical locations of the intervening genetic loci were found to be consistent with the previously determined genetic order. Evidence was also obtained which suggested that at least one end of a transforming DNA fragment is degraded during DNA uptake by the competent bacterium.

Journal Article↗

PubMed1983

Genetic transformation of Bacillus brevis 47, a protein-secreting bacterium, by plasmid DNA.

A method has been developed for introducing plasmid DNA into Bacillus brevis 47, a protein-secreting bacterium. Treatment of B. brevis 47 cells with 50 mM Tris-hydrochloride buffer of alkaline pH was effective for inducing DNA uptake competence. In the presence of polyethylene glycol, the Tris-treated cells incorporated plasmid DNA with a frequency of 10(-4) (transformants per viable cell) when 1 microgram of plasmid DNA was added to 10(9) Tris-treated cells. The pH of Tris-hydrochloride buffer as well as the concentration and molecular weight of the polyethylene glycol affected the transformation frequency. The growth phase of B. brevis 47 cells strongly influenced the frequency. Two plasmids, pHW1 and pUB110, have been introduced into B. brevis 47 by this method. The mechanism of induction of competence for DNA uptake in connection with removal of the outer two protein layers of the cell wall by treatment of B. brevis 47 cells with Tris-hydrochloride buffer is discussed.

Bacillus↗

PubMed1989

Epstein-Barr virus nuclear protein 2 is a key determinant of lymphocyte transformation.

Epstein-Barr virus (EBV) efficiently transforms B lymphocytes to perpetual proliferation. The EBV laboratory strain P3HR-1 is transformation-incompetent and lacks a DNA segment that includes the EBV nuclear antigen 2 (EBNA-2) gene and a portion of the EBNA leader protein (EBNA-LP) gene. These two genes are expressed in transformed B lymphocytes. Recombinant transformation-competent EBVs were produced by transfecting P3HR-1-infected cells with a cosmid containing the DNA deleted in P3HR-1. Deletion of 105 nucleotides from the middle of the EBNA-2 gene had no discernible affect on transformation. Two larger EBNA-2 deletions abolished transformation but did not affect EBNA-2 nuclear localization. Two naturally occurring EBV variants (EBV types 1 and 2) differ extensively in their growth-transformation phenotype and in their EBNA-LP, EBNA-2, and EBNA-3A, -3B, and -3C genes. Recombinant P3HR-1 carrying EBV-1 EBNA-2 has many of the EBV-1 in vitro growth-transforming effects; recombinant P3HR-1, isogenic except for EBV-2 EBNA-2, has many of the EBV-2 growth-transforming effects including slow emergence of transformants, growth in tight clumps with few surrounding viable cells, and early sensitivity to dilution with fresh medium. Thus, EBNA-2 is an essential molecule in lymphocyte growth transformation by EBV and a major determinant of the differences between EBV-1 and EBV-2 in lymphocyte growth transformation.

Antigens, Viral↗

PubMed2004

[The study of optimal conditions of electroporation in Pseudomonas aeruginosa].

A P. aeruginosa strain PA68 isolated from the sputum of a patient suffering from bronchiectasis was used as the recipient strain. Optimum conditions including growth stage of the strain, electroshock voltage, concentration and preservation of competent cell were defined for the electroporation of PA68 with plasmid pSMC28. It was showed that the highest transformation efficiency was up to 1.68 x 10(3) CFU/microgram DNA under the optimum conditions in which the competent cells were collected at logarithmic growth phase (OD(540) = 0.7-0.8) and concentrated to about 10(11) cells/ml, the mixture of the competent cells and plasmid pSMC28 was eletroporated at 2.6 kV. With this optimal condition, Mu transponson complexes have been successfully transformed into P. aeruginosa strain PA68 and the obtained efficiency was 2.47 x 10(4) CFU/microgram DNA. This is the first time to electroporate Mu transposon complexes into Pseudomonas spp. The artificial Mu transposons could integrate into bacterial genomes at a single site randomly. Then the phenotype change was the result of the gene inactivation caused by Mu transposon insertion. That will be very helpful for the study of genomic function of Pseudomonas spp.

DNA Transposable Elements↗

PubMed1994

Identification of Moraxella bovis and related species from calves with IBK and goats by qualitative genetic transformation assay.

Eight Moraxella bovis strains isolated from bovine pink eye, 16 Moraxella bovis related strains isolated from healthy goats nasal flora, one nonhemolytic Moraxella sp. isolated from bovine conjunctivitis and different collection strains of Moraxella and Branhamella genus were studied through the combined use of qualitative genetic transformation assay and the ability to grow on a moraxella bovis defined medium (medium MB). Crude DNA extracted from the strains studied were used to transform two mutant auxotroph competent strains of Moraxella bovis CIP 103741 and CIP 103743. Non-specific positive transformation was obtained with mutant assay strain CIP 103743 when treated with DNA from caprine or bovine Moraxella strains, collection strains of Moraxella bovis, Moraxella lacunata, Moraxella nonliquefaciens and strains of Branhamella genus, whereas specific transformation was observed with mutant assay CIP 103741 when treated only with DNA from all the Moraxella bovis (collection and clinical isolates) and 14 of the 16 caprine Moraxella bovis related strains. The specificity and the simplicity of the test make it suitable for use in clinical laboratories.

Animals↗

PubMed2000

[Construction of a transformation-competent artificial chromosome (TAC) library of a wheat-Haynaldia villosa translocation line].

Transformation-competent artificial chromosome (TAC) vector is able to clone and transfer large DNA fragments in plants and is a powerful tool for plant gene isolation and transformation. To clone important genes from wheat, a TAC genomic library was constructed from nuclear DNA of a 6VS/6AL wheat-Haynaldia villosa translocation line that harbor the gene Pm21 for resistance to powdery mildew. The library consists of 2.1 x 10(6) clones with an average DNA insert size of 35 kb, and represents in total 4.9 genome equivalents. The library was stored as clone pools in 96-well plates, and each pool contained about 1000 clones. TAC clones containing gene(s) of interest can be screened by a pooled-PCR/colony-hybridization strategy.

Chromosomes, Artificial↗

PubMed1985

Streptococcus faecalis R plasmid pJH1 contains a pAM alpha 1 delta 1-like replicon.

Streptococcus faecalis R plasmid pJH1 did not transform competent strains of Streptococcus sanguis. A hybrid plasmid, pDL310, consisting of virtually all of the S. faecalis hemolysin-bacteriocin plasmid pJH2 and a segment of pJH1 DNA that included the tetracycline resistance determinant, yielded tetracycline-resistant transformants at a frequency of less than 10(-8) transformants per CFU, when it was added to a competent culture of S. sanguis Wicky. Four of the transformants contained a 4.7-kilobase plasmid (pDL316) that transformed strain Wicky at a frequency of 8.6 X 10(-8). Restriction endonuclease digests, agarose gel electrophoresis, and Southern blot hybridizations indicated that pDL316 consisted entirely of pJH1-derived DNA. Additional restriction studies, Southern blot hybridizations, and heteroduplex analyses indicated that pDL316 was very closely related to 4.6-kilobase tetracycline resistance plasmid pAM alpha 1 delta 1, a derivative of 9.0-kilobase S. faecalis plasmid pAM alpha 1.

Bacteriocins↗

PubMed1976

Single-stranded regions in transforming deoxyribonucleic acid after uptake by competent Haemophilus influenzae.

About 15% of donor deoxyribonucleic acid (DNA) is single stranded immediately after uptake into competent Haemophilus influenzae wild-type cells, as judged by its sensitivity to S1 endonuclease. This amount decreases to 4 to 5% by 30 min after uptake. Mutants which are defective in the covalent association of recipient and donor DNA form little or no S1 endonuclease-sensitive donor. At 17 C donor DNA taken up by the wild type contains single-stranded regions although there is no observable association, either covalent or noncovalent. The single-stranded regions are at the ends of donor DNA molecules, as judged by the unchanged sedimentation velocity after S1 endonuclease digestion. The amount of single-stranded donor remains constant at 17 C for more than 60 min after uptake, suggesting that the decrease observed at 37 C is the result of association of single-stranded ends with single-stranded regions of recipient cell DNA. Three sequential steps necessary for the integration of donor DNA into recipient DNA are proposed: the synthesis of single-stranded regions in recipient DNA, the interaction of donor DNA with recipient DNA resulting in the production of single-stranded ends on donor DNA, and the stable pairing of homologous single-stranded regions.

DNA, Bacterial↗

PubMed1991

Release of transforming plasmid and chromosomal DNA from two cultured soil bacteria.

The release of chromosomal and plasmid DNA from Acinetobacter calcoaceticus and Bacillus subtilis cultivated in minimal medium and broth over a period of 50 h was monitored and related to growth phase, autolysis, DNase production and natural competence. The released DNAs were biologically active in natural transformation. In addition, the circular integrity of a released B. subtilis shuttle vector (pHV14) was demonstrated by artificial transformation of Escherichia coli. In cultures of both strains high molecular weight DNA accumulated, particularly during the stationary and death phase (up to 30 micrograms ml-1). Generally, despite the presence in culture fluids of DNase activity (and of an intracellular enzyme, catalase, indicating some cell lysis) there was high transforming activity of chromosomal and plasmid DNA even 40 h after the cultures reached the stationary phase. In cultures of B. subtilis in minimal medium a presumably active release of intact plasmids and chromosomal DNA occurred during the competence phase. The release of biologically functional DNA during essentially all growth phases of a gram-positive and a gram-negative member of soil bacteria might facilitate horizontal gene transfer by transformation in natural habitats.

Acinetobacter calcoaceticus↗

PubMed1998

A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation.

The introduction of binary plasmids into Agrobacterium hosts for Agrobacterium-mediated transformation of plants is most readily achieved by electroporation. However, occasionally, no transformed colonies are recovered and the transformation program is delayed. Poor transformation rates are commonly associated with particular combinations of Agrobacterium strains and plasmid-selection markers. In order to avoid this problem, it is important for the bacteria to have a highly competent status for reception of plasmid DNA. It is also important to optimize the level of antibiotic for the selection of transformed colonies. In this article, we demonstrate that transformation competence is strongly related to the phase of growth at which a bacterial culture is prepared for electroporation, and we describe a simple procedure that allows the level of transformation-competent cells to be maximized. We have observed that there is significant variation between transformed Agrobacterium strains in the levels of antibiotic tolerance; we define the antibiotic levels that are appropriate for selection of three Argobacterium tumefaciens (EHA101, LBA4404, C58) and two Agrobacterium rhizogenes (LBA9402, Ar2626) strains, transformed with three alternative resistance markers (spectinomycin(res), kanamycin(res), and gentamycin(res)).

Agrobacterium tumefaciens↗

PubMed2003

Type IV pilus gene homologs pilABCD are required for natural transformation in Actinobacillus actinomycetemcomitans.

Some clinical isolates of the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans are naturally competent for DNA uptake. In this study, we examined the sequence and the function of a type IV pilus-like pilABCD gene cluster and its downstream region in a naturally transformable A. actinomycetemcomitans strain D7S. Specific knockout mutants of pilABCD of strain D7S were constructed by replacing individual genes with an antibiotic resistance cassette. The transformation frequency of chromosome markers in the wildtype strain D7S was approximately 10(-3) per CFU. In contrast, the delta pilA, delta pilB, delta pilC, delta pilBC or delta pilD mutants were non-transformable (transformation frequency <10(-8)). Disruption of an ORF downstream of pilD had no apparent effect on the transformability of this bacterium. The pilA or pilBC deletion did not seem to affect fimbria expression or cell surface structure in either rough or smooth strains as determined by scanning and transmission electron microscopy examinations. RT-PCR analysis showed that pilA was expressed in strain D7S under a competence-inducing growth condition. The expression of pilA was barely detectable in strain D7S cultured under a non-competence-inducing condition or in the non-transformable strain JP-2. The results indicate that pilABCD are required for competence but are apparently not involved in fimbria expression of A. actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans↗