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PubMed1993

Plasmid DNA in a groundwater aquifer microcosm--adsorption, DNAase resistance and natural genetic transformation of Bacillus subtilis.

Prokaryotes can exchange chromosomal and plasmid genes via extracellular DNA in a process termed genetic transformation. This process has been observed in the test tube for several bacterial species living in the environment but it is not clear whether transformation occurs in natural bacterial habitats. A major constituent of terrestrial environments are solid particles such as quartz, silt and clay, which have considerable surface areas and which make up the solid-liquid interfaces of the habitat. In previous experiments the adsorption of DNA to chemically purified quartz and clay minerals was shown and the partial protection of adsorbed DNA against DNAase I. In a microcosm consisting of natural groundwater aquifer material (GWA) sampled directly from the environment and groundwater (GW) both linear duplex and supercoiled plasmid DNA molecules bound rapidly and quantitatively to the minerals. The divalent cations required to form the association were those present in the GWA/GW microcosm. The association was stable to extended elution over one week at 23 degrees C. Upon adsorption, the DNA became highly resistant against enzymatic degradation. About 1000 times higher DNAase I concentrations were needed to degrade bound DNA to the same extent as DNA dissolved in GW. Furthermore, chromosomal and plasmid DNA bound on GWA transformed competent cells of Bacillus subtilis. However, in contrast to DNA in solution, on GWA the chromosomal DNA was more active in transformation than the plasmid DNA. The studies also revealed that in the transformation of B. subtilis Mg2+ can be replaced by Na+, K+ or NH4+. The observations suggest that in soil and sediment environments, mineral material with inorganic precipitates and organic matter can harbour extracellular DNA leaving it available for genetic transformation.

Bacillus subtilis↗

PubMed1997

Streptococcal competence for genetic transformation: regulation by peptide pheromones.

Although the capacity for genetic transformation is perhaps the most famous attribute of pneumococcus, use of this genetic phenomenon as a tool for study of the biology of the organism and of its pathogenicity has been largely restricted to a few favored unencapsulated strains, both by the delicacy of the conditions required for development of competence, and by experience that encapsulated strains transformed poorly. We discuss here the recent discovery of a small stable inexpensive peptide pheromone that acts as a quorum-sensing signal and that induces competence under a wide variety of conditions and in encapsulated strains. Its use circumvents some if not all limitations to the expression of transformability in pneumococcus and therefore expands opportunities for application of tools molecular genetics to many strains of pneumococcus without prior genetic manipulation.

Peptides↗

PubMed2006

Agrobacterium-mediated genetic transformation of plants: biology and biotechnology.

Agrobacterium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Extensive research aimed at understanding and improving the molecular machinery of Agrobacterium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Agrobacterium strains, plasmids and technologies currently used for the successful transformation of numerous plant species. Unlike the role of bacterial proteins, the role of host factors in the transformation process has remained obscure for nearly a century of Agrobacterium research, and only recently have we begun to understand how Agrobacterium hijacks host factors and cellular processes during the transformation process. The identification of such factors and studies of these processes hold great promise for the future of plant biotechnology and plant genetic engineering, as they might help in the development of conceptually new techniques and approaches needed today to expand the host range of Agrobacterium and to control the transformation process and its outcome during the production of transgenic plants.

Biotechnology↗

PubMed1987

Competence for genetic transformation in Streptococcus pneumoniae: molecular cloning of com, a competence control locus.

To identify and map genes involved in competence for genetic transformation in Streptococcus pneumoniae, we have cloned DNA surrounding an ermB insertion mutation that causes a competence factor deficiency. We recovered the insert and approximately 500 base pairs of neighboring pneumococcal DNA in pMB9. Larger pieces of DNA from this region were unstable in pMB9 and pBR325. However, larger pieces were stable in pKK232-8, an Escherichia coli vector containing strong transcription terminators. Overlapping pieces of wild-type DNA from this competence control region were cloned and mapped in this vector. Insertion mutations were constructed in vitro throughout the cloned region. When crossed into the pneumococcus chromosome, they showed that the com locus was 4.2 to 5.2 kilobases long.

Bacterial Proteins↗

PubMed1979

Genetic transformation in Streptococcus sanguis. Spontaneous and induced competence of selected strains.

Nine strains of Streptococcus sanguis were examined for competence in genetic transformation with streptomycin resistance (str-r) as marker. Eight strains belonged to serogroup H and one to the newly-described serogroup W. Seven of the strains, one of which was the reference strain NCTC 7868 (strain Challis), were competent with str-r DNA from strain Challis. Strains NCTC 9124 (strain Wicky) and 480 were incompetent. The efficiency of transformation was examined in four different media. Use of Todd Hewitt broth gave frequencies of transformants as high as the more complex media. Addition of serum to the transformation media was not essential for the development of competence. The presence of a competence factor (CF) in the culture filtrate of strain Challis was confirmed. The factor transferred strain Wicky to competence with a great variation in the number of transformants and had no influence on strain 480. On the other hand, this spontaneously incompetent strain became competent after addition of culture filtrate from the competent strain 13b, in contrast to Wicky which now remained incompetent. Thus, it is suggested that several factors are involved in the induction of competence of S. sanguis.

Bacterial Proteins↗

PubMed1992

A reorganized Candida albicans DNA sequence promoting homologous non-integrative genetic transformation.

In order to develop plasmids adequate for non-integrative genetic transformation of Candida albicans, a DNA fragment of 15.3 kb was cloned from this organism on the basis of its capacity to convert the integrative Saccharomyces cerevisiae vector YIp5 into a non-integrative one. Southern hybridization analysis, carried out with a labelled DNA probe of 3.6 kb derived from the cloned fragment, showed that it consisted of C. albicans DNA, the hybridization pattern indicating that the corresponding sequences were homologous to several chromosomal regions. The size of the C. albicans DNA promoting autonomous replication in S. cerevisiae was substantially reduced by subcloning. A 5.1 kb subfragment, defined by BamHI and SalI restriction sites, retained autonomous replication sequences (ARS) functional in the heterologous S. cerevisiae system and in C. albicans, when inserted in plasmid constructions that carried a S. cerevisiae trichodermin-resistance gene (tcm1) as selection marker. C. albicans transformants were both of the integrative and the non-integrative type and the plasmids recovered from the latter very often carried a reorganized ARS, indicating that recombination of the inserted ARS DNA had occurred in the homologous host. Successive reorganizations of the ARS insert in C. albicans eventually led to a more stable and much smaller fragment of 687 bp that was subsequently recovered unchanged from transformants. Sequence analysis of the 687 bp fragment revealed four 11-base blocks, rich in A+T, that carried the essential consensus sequence considered relevant for yeast ARS elements in addition to other features also described as characteristic of yeast replication origins.

Base Sequence↗

PubMed1998

Genetic transformation in Haemophilus parainfluenzae clinical isolates.

Haemophilus parainfluenzae isolates recovered from patients with respiratory diseases were studied for their ability to undergo genetic transformation by isogenic DNA. Two chromosomal markers, streptomycin resistance and nalidixic acid resistance, were tested for transformation efficiencies in H. parainfluenzae recipients from three biotypes. Most efficient in transformation was biotype II, followed by biotype I, while biotype III was nontransformable. Lack of transformation was not owing to poor donor activity of DNA, but to inability of the cells to develop competence. Strains that formed clumps in liquid media were nontransformable. Since the transformable biotype II is one of the prevalent biotypes world wide, one can speculate that DNA transformation probably plays a major role in the spread of drug resistance in H. parainfluenzae.

Cell Adhesion↗

PubMed1962

Release of genetic transforming agent from pneumococcal cultures during growth and disintegration.

Genetic transformations of pneumococcus mediated by streptomycin-induced lysates were studied to gain some insight into the nature of freshly released transforming principle, and the influence of the physiologic state of the donor population on the transformation process. It was found that streptomycin could make the DNA of sensitive cells available for transformation of other cells. Living cultures of pneumococcus growing exponentially in ordinary media were also found to discharge significant quantities of genetically active DNA. Such cultures, not treated with any drug, showed no evidence of concomitant cell disintegration or death. Both single markers and small linkage groups could be transferred in transformations mediated by drug-induced lysates and by filtrates of living cultures. The quantity of DNA liberated is small (less than 0.1 microg per ml), but these transformations are at least as efficient as transformations mediated by purified DNA, when compared on the basis of total DNA available. Up to 1 per cent of the cells in an average recipient culture can be transformed by a small quantity of culture fluid. Both in drug-treated and in untreated cultures the amount of transforming activity increased and then decreased during growth of the culture. Although the source of transforming DNA in growing cultures could not be established, the decline in the transforming activity of aging drug-treated or untreated cultures was attributed to the presence of deoxyribonuclease. The release of this nuclease by pneumococcal cultures midway in exponential growth is sufficient to result in a mild degradation of the low concentration of freshly released transforming agent present. Maximal release of active transforming agent by a living culture coincided in time with the development of maximal receptivity to exogenous DNA by that culture. As a result, recombinants could be recovered from appropriately genetically marked strains growing in each other's presence. In view of these results, it seems possible that DNA-mediated transformations might provide, or might have provided, a mechanism of genetic recombination in nature for some bacterial species in which sexual mechanisms may not be available.

DNA, Bacterial↗

PubMed2001

Genetic transformation of major wine grape cultivars of Vitis vinifera L.

We have developed an Agrobacterium-mediated transformation system for a number of important grapevine cultivars used in wine production. Transgenic plants were obtained for the seven cultivars: Cabernet Sauvignon, Shiraz, Chardonnay, Riesling, Sauvignon Blanc, Chenin Blanc and Muscat Gordo Blanco. Embryogenic callus was initiated from anther filaments and genotypic differences were observed for initiation and subsequent proliferation with Chardonnay responding most favourably to culture conditions. The transformation system allowed the recovery of germinating transgenic embryos 10-12 weeks after Agrobacterium inoculation and plants within 18 weeks. Examination of the expression patterns of the green fluorescent protein gene under the control of the CAMV35S promoter in leaf tissue of transgenic plants showed that for up to 35% of plants the pattern was not uniform. The successful transformation of a genetically diverse group of wine grape cultivars indicates that the transformation system may have general application to an even wider range of Vitis vinifera cultivars.

Blotting, Southern↗

PubMed2000

An efficient method for the production of transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium tumefaciens-mediated genetic transformation.

Cotyledon explants from mature peanut seeds (Arachis hypogaea L.) were optimized to obtain adventitious shoot buds with high frequencies (>90%). Efficient transformation of these cotyledons by using Agrobacterium tumefaciens strain C58 carrying neomycin phosphotransferase II (nptII) and ß-glucuronidase (GUS; uidA), or coat protein gene of the Indian peanut clump virus (IPCVcp) and nptII on binary vectors (pBI121; pROKII:IPCVcp) led to the production of a large percentage (55%) of transgenic plants. Transformed individuals were obtained through selection on medium containing 125 mg l(-1) kanamycin. A large number of independently transformed plants (over 75) were successfully transplanted to the glasshouse. Integration of the transgenes and stable genetic transformants in the progeny were assessed by PCR amplification of 700-bp fragment of nptII and 585-bp of IPCVcp genes, and Southern blot hybridizations in the T1 generation of transgenic plants. Analysis of 35 transgenic plants of T1 generation from the progeny of a single transformation event suggested the segregation of a single copy insert in a 3:1 Mendelian ratio. On an average, 120-150 days were required between the initiation of explant transformation and transfer of rooted plants to the greenhouse. The cotyledon regeneration system proved to be an excellent vehicle for the production of a large number of independently transformed peanut plants. Shoot formation was rapid and prolific, and a large proportion of these shoots developed into fertile plants. The method reported here provides new opportunities for the crop improvement of peanut via genetic transformation.

Journal Article↗

PubMed2004

Agrobacterium-mediated genetic transformation of Perilla frutescens.

A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla). The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA). The effects of preculture and extent of cocultivation were examined by assaying beta-glucuronidase (GUS) activity in explants infected with A. tumefaciens strain EHA105 harboring the plasmid pIG121-Hm. The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation. Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin. The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene. The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.

Agrobacterium tumefaciens↗

PubMed2007

Maize (Zea mays) genetic transformation by co-cultivating germinating seeds with Agrobacterium tumefaciens.

A novel plant genetic transformation method for maize (Zea mays) is reported. Using a scalpel, germinating seeds were wounded in the meristem and co-cultivated with an Agrobacterium tumefaciens strain harbouring a Ti plasmid. Seedlings produced from the treatment were screened by hygromycin selection. Fertile transgenic T(0) and T(1) plants were obtained. PCR amplification, PCR-Southern and Southern-blot analysis showed that the foreign gene had been introduced into the inbreds of maize. About 29% of T(0) seedlings examined were found to be transgenic, although the overall transformation was only 0.6% when total treated seeds were taken into account. The method circumvented tedious and prolonged tissue-culture steps, is simple and can be readily integrated into conventional plant breeding programs.

Agrobacterium tumefaciens↗

PubMed1998

Is natural genetic transformation a mechanism of horizontal gene transfer in Yersinia?

We investigated the capacity of different Yersinia strains with emphasis to Yersinia enterocolitica to take up and incorporate DNA by natural genetic transformation. Our studies were initiated by the observation of partial homology between the virulence plasmid of a pathogenic Y. enterocolitica strain (O: 3, biovar 4) and plasmids indiginous to different a pathogenic Y. enterocolitica biovar 1A strains revealed by hybridization studies. Furthermore, an observation of natural genetic transformation in a strain of Y. enterocolitica has been published by CALLAHAN and KOROMA (1979). To detect an uptake and incorporation of DNA, we incubated potential recipient strains with naked DNA under varying experimental conditions. The parameters tested were--the recipient strain,--the markers used to detect a DNA transfer,--the condition of the transforming DNA,--the nutrient availability,--the temperature,--the growth phase, and--the influence of stress. In our experiments, we could not identify conditions under which Y. enterocolitica could be naturally transformed. We thus conclude that natural transformation is unlikely to be an important mechanism for horizontal gene transfer in Yersinia.

Electroporation↗

PubMed1992

Detection of the neor gene expression in animal cells after genetic transformation.

A reliable assay is reported for the detection of the marker gene aminoglycoside phosphotransferase activity in cells that express this enzyme transiently or as a result of stable genetic transformation. This method combines the simplicity of the dot assays with the reliability of the more elaborate and time consuming electrophoretic or chromatographic methods. Inhibition of phosphatases and protein kinases during the reaction reduces labeled ATP consumption by these enzymes. As a result, this assay allows the detection of approx 10 times lower levels of the enzyme than currently used methods. To detect the expression of reporter genes in transformed cells aminoglycoside phosphotransferase can be used as well as the widely used chloramphenicol acetyltransferase enzyme.

Animals↗

PubMed1978

Genetic fate of DNA in a strain of Bacillus subtilis which is impaired in genetic transformation.

A mutation in Bacillus subtilis call recC4 which results in an impairment of genetic transformation was transferred to a new strain using the closely linked marker mit-2 (mitomycin C-resistance) for selection. This derived strain was in turn impaired in transformation but showed normal levels of sensitivity to ultraviolet irradiation and methyl methane sulfonate. The genetic and molecular fate of transforming DNA in the recC4 strain was studied. Normal amounts of DNA were taken up by the cells and this DNA or parts of it became associated with recipient DNA. Linkage between genes on donor and recipient molecules was, however, not established and transformants were not generated. The recC4 mutation therefore affects a step in the recombination pathway during transformation. Either the association between donor and recipient DNA molecules is abnormal or the cells are deficient in the further processing of the associated complex.

Bacillus subtilis↗

PubMed1991

High Frequency of Natural Genetic Transformation of Pseudomonas stutzeri in Soil Extract Supplemented with a Carbon/Energy and Phosphorus Source.

Agar medium (SME) prepared from aqueous soil extract was used to examine genetic transformation of Pseudomonas stutzeri JM302 (his-1) by homologous his DNA in a plate transformation assay. Growth studies indicated that SME was strongly limited in carbon and nitrogen sources. Transformation was observed on SME supplemented with pyruvate, phosphate, and ammonium. A 25-fold increase of the transformation frequency was obtained with nitrogen limitation when SME was supplemented with only pyruvate plus phosphate. Similar results were obtained with artificial soil extract medium prepared on the basis of the chemical analysis of the soil extract. On a standard minimal medium, transformation frequencies also increased (10- to 60-fold) when ammonium, phosphate, or pyruvate was growth limiting. Limitation of two or three nutrients did not stimulate transformation. The size of the inoculum (2 x 10 to 2 x 10 cells) was irrelevant to the enhanced transformation under nitrogen limitation on SME or standard minimal medium. We further show that P. stutzeri can use a variety of carbon and energy sources for competence development. It is concluded that genetic transformation of P. stutzeri is possible in the chemical environment of soil upon supply of nutrients and may be strongly stimulated by a growth-limiting concentration of single nutrients including sources of C, N, or P.

Journal Article↗

PubMed1989

Genetic transformation in encapsulated clinical isolates of Haemophilus influenzae type b.

Haemophilus influenzae type b strains isolated from children with meningitis, septicaemia and pharyngitis were studied for their ability to undergo genetic transformation by two chromosomal markers, streptomycin resistance and nalidixic acid resistance. Fifty-eight percent of the strains were non-transformable while the remaining 42% showed considerable strain variation with regard to their transformation frequencies, which ranged from 8 x 10(-4) to 1 x 10(-6). The effect of type b capsule on competence development and transformation activity was studied by comparing encapsulated strains with their non-encapsulated variants. Type b capsule did not inhibit either competence development or transforming efficiency. The lack of transformability in the majority of strains was not due to the presence of a capsule.

Cell Membrane↗

PubMed2002

In vitro regeneration and genetic transformation of the berberine-producing plant, Thalictrum flavum ssp. glaucum.

Protocols have been developed for the in vitro regeneration and Agrobacterium-mediated genetic transformation of meadow rue, Thalictrum flavum ssp. glaucum. Ten-day-old seedlings were bisected along the embryonic axis and the cotyledons were co-cultured with various Agrobacterium tumefaciens strains for 3 days. The cotyledons were cultured on a shoot induction medium (B5 salts and vitamins, 30 g l-1 sucrose, 2 mg l-1 kinetin, and 3 g l-1 Gelrite) containing 25 mg l-1 hygromycin B as the selection agent and 250 mg l-1 timentin to facilitate the elimination of Agrobacterium. Only the oncogenic A. tumefaciens strains A281 and C58 produced transgenic T. flavum callus tissues. A281 was the most effective strain producing hygromycin-resistant callus on 85% of the explants. Transgenic callus was subcultured on the shoot induction medium every 2 weeks. After 12 weeks, hygromycin-resistant shoots that formed on explants exposed to strain A281 were transferred to a root induction medium (B5 salts and vitamins, 25 mg l-1 hygromycin B, 250 mg l-1 timentin, and 3 g l-1 Gelrite). Detection of the beta-glucuronidase (GUS) gene using a polymerase chain reaction assay, the high levels of GUS mRNA and enzyme activity, and the cytohistochemical localization of GUS activity confirmed the genetic transformation of callus cultures and regenerated plants. The transformation process did not alter the normal content of berberine in transgenic roots or cell cultures; thus, the reported protocol is valuable to study the molecular and metabolic regulation of protoberberine alkaloid biosynthesis.

Journal Article↗