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PubMed1994

Bacterial gene transfer by natural genetic transformation in the environment.

Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation.

DNA, Bacterial↗

PubMed1988

Genetically transformed maize plants from protoplasts.

Genetically transformed maize plants were obtained from protoplasts treated with recombinant DNA. Protoplasts that were digested from embryogenic cell suspension cultures of maize inbred A188 were combined with plasmid DNA containing a gene coding for neomycin phosphotransferase (NPT II) next to the 35S promoter region of cauliflower mosaic virus. A high voltage electrical pulse was applied to the protoplasts, which were then grown on filters placed over feeder layers of maize suspension cells (Black Mexican Sweet) and selected for growth in the presence of kanamycin. Selected cell lines showed NPT II activity. Plants were regenerated from transformed cell lines and grown to maturity. Southern analysis of DNA extracted from callus and plants indicated the presence of the NPT II gene.

Cell Membrane Permeability↗

PubMed1999

[Factors affecting Agrobacterium-mediated genetic transformation of Poncirus trifoliata (L.) Raf].

OBJECTIVE: Establishing an effective system for the Agrobacterium-mediated genetic transformation of Poncirus trifoliata, so as to lay a foundation for improving breeds and introducing foreign genes. METHOD: The explants used for the genetic transformation were the epicotyls from Poncirus trifoliata. The Agrobacterium tumefaciens strain was EHA101, containing vector plasmid PGA482GG. The GUS gene and NPT II gene were introduced into the transformation plasmid. RESULTS: 20-day epicotyls were suitable for transformation. 2-day precultivation enhanced shoot regeneration. Shoot regeneration frequency was high when the cocultivation lasted 2-3 days. With the presence of acetosyringone during cocultivation, the efficiency of transformation could be greatly enhanced, and the frequency of shoot regeneration rose as high as 27.5%. After the explants were cocultivated with Agrobacterium, 2-day delayed selection could stimulate shoot regeneration. CONCLUSION: An effective genetic transformation system has been established for Poncirus trifoliata on the basis of factors affecting Agrobacterium-mediated genetic transformation.

Acetophenones↗

PubMed2006

Natural genetic transformation: A novel tool for efficient genetic engineering of the dairy bacterium Streptococcus thermophilus.

Streptococcus thermophilus is widely used for the manufacture of yoghurt and Swiss or Italian-type cheeses. These products have a market value of approximately 40 billion dollars per year, making S. thermophilus a species that has major economic importance. Even though the fermentation properties of this bacterium have been gradually improved by classical methods, there is great potential for further improvement through genetic engineering. Due to the recent publication of three complete genome sequences, it is now possible to use a rational approach for designing S. thermophilus starter strains with improved properties. Progress in this field, however, is hampered by a lack of genetic tools. Therefore, we developed a system, based on natural transformation, which makes genetic manipulations in S. thermophilus easy, rapid, and highly efficient. The efficiency of this novel tool should make it possible to construct food-grade mutants of S. thermophilus, opening up exciting new possibilities that should benefit consumers as well as the dairy industry.

DNA, Bacterial↗

PubMed2005

[Preparation and Regeneration of Protoplasts from Monascus purpureus and Genetic Transformation System.].

Generation of fungal protoplast is an essential tool for genetic transformation system. To establish protoplast-mediated genetic transformation system of Monascus purpureus, conditions for the protoplast isolation and regeneration of the mycelia of various enzymes and osmotic stabilizers were examined. To investigate suitable cell age for the protoplast preparation of mycelia of M. purpureus, the mycelia were cultured in different ways at 30oC. Mycelia obtained through cellophane - mediated culture for 30~40h were adequate to protoplast preparation. When lysing enzyme, cellulase and snailase were added to the mycelia in combination or alone, combination of lysing enzyme, cellulase and snailase accordingly at the concentration of 0.3%, 0.1% and 1% was most benefit for protoplast yield. When we applied various osmotic stabilizers at different concentrations to protoplast preparation, 1 mol/L MgSO4 was most effective for the protoplast release. The suitable incubation time with enzyme for the maximum release of protoplasts was 2.5-hr. When we investigate various osmotic stabilizers for the regeneration of the protoplasts of mycelia of strain M34 and N18, the complete medium containing 0.6 mol/L sucrose induced highest hyphal growth with regeneration frequency of 8.5% and 36.4%, respectively. PEG and CaCl2- mediated protoplast co-transformation of strain M34 with pBC-Hygro and pNL1, hygromycin B as selective marker, was fulfilled and 100 stable transformants per microgram DNA were obtained.

Hygromycin B↗

PubMed1978

[The role of a protonmotive force in genetic transformation of Bacillus subtilis].

The hypothesis on the role of protonmotive force in the transport of DNA through the membrane of Bac. subtilis cell during initial stages of genetic transformation was tested. A genetic transformation of arsenate-treated cells was observed. Treatment of cells by the protonophorous uncoupler of oxidative phosphorylation-carbonylcyanide dichlorophenyl--hydrazone-led to the inhibition of initial stages of genetic transformation having no significant effect on the level of intracellular ATP concentration and on the viability of cells. The dissipation of protonmotive force by means of K+ and H+ fluxes catalyzed by valinomycin and nigericin also caused the inhibition of initial stages of genetic transformation. The inhibitory effect of cationic penetrant tetraphenyl phosphonium was observed, the effect being potentiated by low concentrations of anionic penetrant phenyldicarbaundecaborate. The value of the membrane potential in the energized valinomycin-treated cells calculated from the distribution of K+ was within the range of 70--100 mV (inside minus). These results support the conception that a protonmotive force drives DNA transport through the membrane of Bac. subtilis cells.

Adenosine Triphosphate↗

PubMed2005

Stable genetic transformation of the ectomycorrhizal fungus Pisolithus tinctorius.

In the present work the genetic transformation and the expression of gene markers in transgenic Pisolithus tinctorius are reported. The ectomycorrhizae are facultative symbionts of plant roots, which are capable of affording mineral nutrients to its co-host in exchange of fixed carbon. Given the importance of this association (more than 80% of gymnosperms are associated with these fungi), its study from both basic and applied viewpoints is relevant. We have transformed this fungus with reporter genes and analyzed their expression in its saprophytic state. Genetic transformation was performed by microprojectile bombardment and Agrobacterium-mediated transformation. This last method proved to be the more efficient. Southern analysis of biolistic-transformed fungi revealed the random integration of the transgene into the genome. The accumulation of the transcript of the reporter gene was demonstrated by RT-PCR. The visualization of GFP-associated fluorescence in saprophytic mycelia confirmed the expression of the reporter gene. This is the first report on the stable transformation and expression of GFP in the ectomycorrhizal fungus P. tinctorius.

Agrobacterium tumefaciens↗

PubMed1988

Highly efficient genetic transformation of Bacillus subtilis attached to sand grains.

Genetic transformation at the solid/liquid interface was studied using Bacillus subtilis 1G20 (trpC2) with a flow-through system of columns filled with chemically pure sea sand. Studies were done at 23 degrees C. In one type of experiment, competent cultures were incubated with sand-adsorbed DNA, and in another, competent cultures were exposed to sand and then incubated with dissolved DNA for transformation. Of the applied cells, around 10% were retained in columns filled with DNA-loaded sand and around 1% in columns with pure sand. Reversible attachment of some of the cells to surfaces of sand grains could be demonstrated. The overall transformation frequencies obtained were 25- to 50-fold higher than in a standard liquid culture procedure. In this standard procedure, transformation was sensitive to DNAase I concentrations above 50 ng ml-1, whereas in sand columns it was resistant to DNAase I concentrations up to 1 microgram ml-1. Quantification of transformants eluting from columns indicated that sand-attached cells detach at some point after DNA binding or uptake.

Adsorption↗

PubMed2004

Identification of ComW as a new component in the regulation of genetic transformation in Streptococcus pneumoniae.

Regulation of competence for genetic transformation in Streptococcus pneumoniae depends on a quorum-sensing system, genes involved in DNA uptake and recombination and a link between these two gene sets. The alternative sigma factor ComX provides this link. ComE, the response regulator of the quorum-sensing system, is required for expression of ComX and other early genes. However, an unknown ComE-dependent regulator is also required for competence when comX is expressed under control of the raffinose-responsive promoter of the aga operon. The gene comW (SP0018) is required for a high level of competence and is regulated by the quorum-sensing system, but its function is unknown. To explore its role further, comW was cloned into the multicopy plasmid pMSP3535, under the control of a nisin-inducible promoter (P(N)), and transformed into pneumococcal strains containing a raffinose-inducible comX gene (P(R)::comX). Further introduction of a comE deletion blocked the endogenous CSP signal transduction pathway. In the resulting strain, competence was independent of CSP but depended on treatment with both nisin and raffinose, showing that coexpression of comW and comX complemented the comE deficiency. ComX protein accumulation and expression of a late competence gene in the above strain support the conclusion that ComW is a new positive factor involved in competence regulation.

Bacterial Proteins↗

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↗

PubMed1984

Genetic transformation in Streptococcus sanguis. Simultaneous variation of surface-spreading, competence, hemagglutination and polar fimbriation in selected strains.

Strains of Streptococcus sanguis expressing spreading zones around the colonies and competence in genetic transformation, were subcultivated in the laboratory, and in three strains a variation to non-spreading colony morphology on human blood agar plates was observed. These variants were isolated and compared with the original strains by genetic transformation, hemagglutination and electron microscopy. In genetic transformation with streptomycin resistance as genetic marker, the non-spreading variants showed a decrease to 1/50 or less of the transformation frequencies compared to the wild types. Hemagglutination of guinea-pig erythrocytes is a common characteristic of S.sanguis strains, but the non-spreading variants did not hemagglutinate. In electron microscopy studies the wild-type strains of S.sanguis, but not the non-spreading variants, presented long, polar fimbriae. One of the non-spreading variants had short, peritrichous fimbriae. The co-variation in surface-spreading, competence in genetic transformation, hemagglutination and polar fimbriation is discussed.

Fimbriae, Bacterial↗

PubMed2002

An inhibitor of DNA binding and uptake events dictates the proficiency of genetic transformation in Neisseria gonorrhoeae: mechanism of action and links to Type IV pilus expression.

Although natural genetic transformation is a widely disseminated form of genetic exchange in prokaryotic species, the proficiencies with which DNA recognition, uptake and processing occur in nature vary greatly. However, the molecular factors and interactions underlying intra- and interspecies diversity in levels of competence for natural genetic transformation are poorly understood. In Neisseria gonorrhoeae, the Gram-negative aetiologic agent of gonorrhoea, DNA binding and uptake involve components required for Type IV pilus (Tfp) biogenesis as well as those which are structurally related to Tfp biogenesis components but dispensable for organelle expression. We demonstrate here that the gonococcal PilV protein, structurally related to Tfp pilin subunits, is an intrinsic inhibitor of natural genetic transformation which acts ultimately by reducing the levels of sequence-specific DNA uptake into the cell. Specifically, we show that DNA uptake is enhanced in strains bearing pilV mutations and reduced in strains overexpressing PilV. Furthermore, we show that PilV exerts its effect by acting as an antagonist of ComP, a positive effector of sequence-specific DNA binding. As it prevents the accumulation of ComP at a site where it can be purified by shear extraction of intact cells, the data are most consistent with PilV either obstructing ComP trafficking or altering ComP stability. In addition, we report that ComP and PilV play overlapping and partially redundant roles in Tfp biogenesis and document other genetic interactions between comP and pilV together with the pilE and pilT genes required for the expression of retractile Tfp. Together, the results reveal a novel mechanism by which the levels of competence are governed in prokaryotic species and suggest unique ways by which competence might be modulated.

Amino Acid Sequence↗

PubMed1983

Genetic transformation of obligately chemolithotrophic thiobacilli.

Genetic transformation of Thiobacaillus thioparus auxotrophs to prototrophy was obtained at frequencies of up to 10(-2) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent strain of the same bacterium. The rate at which transformation occurred depended on recipient growth rate and could be drastically reduced by depriving otherwise competent cells of either nitrogen or exogenous energy substrate. Interspecies marker transfer was also shown among several obligately chemolithotrophic members of the genus.

Genetic Markers↗

PubMed2003

Natural genetic transformation in monoculture Acinetobacter sp. strain BD413 biofilms.

Horizontal gene transfer by natural genetic transformation in Acinetobacter sp. strain BD413 was investigated by using gfp carried by the autonomously replicating plasmid pGAR1 in a model monoculture biofilm. Biofilm age, DNA concentration, and biofilm mode of growth were evaluated to determine their effects on natural genetic transformation. The highest transfer frequencies were obtained in young and actively growing biofilms when high DNA concentrations were used and when the biofilm developed during continuous exposure to fresh medium without the presence of a significant amount of cells in the suspended fraction. Biofilms were highly amenable to natural transformation. They did not need to advance to an optimal growth phase which ensured the presence of optimally competent biofilm cells. An exposure time of only 15 min was adequate for transformation, and the addition of minute amounts of DNA (2.4 fg of pGAR1 per h) was enough to obtain detectable transfer frequencies. The transformability of biofilms lacking competent cells due to growth in the presence of cells in the bulk phase could be reestablished by starving the noncompetent biofilm prior to DNA exposure. Overall, the evidence suggests that biofilms offer no barrier against effective natural genetic transformation of Acinetobacter sp. strain BD413.

Acinetobacter↗

PubMed2005

In vitro plant regeneration and genetic transformation of Dichanthium annulatum.

Optimization of in vitro plant regeneration and genetic transformation of apomictic species such as Dichanthium annulatum would enable transfer of desirable genes. Seven genotypes of this grass species were screened through mature seed explant for embryogenic callus induction, callus growth and quality (color and texture), and shoot induction. Genotype IG-1999, which produced highly embryogenic, rapidly growing good-quality callus capable of regenerating at a high frequency, was selected for transformation experiments. Using a binary vector (pCAMBIA1305), frequency of GUS expression was compared between two methods of transformation. Bombardment of embryogenic calli with gold particles coated with pCAMBIA1305 at a distance of 11 cm, pressure of 4 bars, and vacuum of 27 Hg passing through 100 muM mesh produced maximum GUS expression (23%). Agrobacterium infection was maximum at an optical density of 2.0 when cocultured under vacuum for 15 min and cocultivated for 3 days at 28 degrees C in constant dark on MS medium of pH 5.8 with 3 mg/l 2,4-D, and 400 muM acetosyringone. Among two binary vectors used for Agrobacterium-mediated transformation, pCAMBIA1301 showed higher frequency of GUS expression while pCAMBIA1305 recorded more of the GUS spots per callus. Supplementation of acetosyringone in the cocultivation medium was found indispensable for Agrobacterium-mediated transformation. Injuring the calli through gold particle bombardment before their cocultivation with Agrobacterium improved the transformation efficiency. Several transgenic plants were developed using the PIG method, while stable GUS-expressing calli were multiplied during selection on MS medium containing 250 mg/l cefotaxime and 50 mg/l hygromycin, incubated in constant dark. A highly significant difference was observed between two methods of transformation for both frequency of GUS expression and GUS spots per callus. PIG-mediated transformation resulted in higher GUS expression compared to the Agrobacterium method. These results demonstrate that Dichanthium annulatum is amenable to Agrobacterium-mediated genetic transformation using a binary vector.

Acetophenones↗

PubMed2006

An unstable competence-induced protein, CoiA, promotes processing of donor DNA after uptake during genetic transformation in Streptococcus pneumoniae.

Natural genetic transformation in Streptococcus pneumoniae entails transcriptional activation of at least two sets of genes. One set of genes, activated by the competence-specific response regulator ComE, is involved in initiating competence, whereas a second set is activated by the competence-specific alternative sigma factor ComX and functions in DNA uptake and recombination. Here we report an initial characterization of CoiA, a ComX-dependent gene product that is induced during competence and is required for transformation. CoiA is widely conserved among gram-positive bacteria, and in streptococci, the entire coiA locus composed of four genes is conserved. By use of immunoblot assay, we show that, similar to its message, CoiA protein is transient, appearing at 10 min and largely disappearing by 30 min post-competence induction. Using complementation analysis, we establish that coiA is the only gene of this induced locus needed for transformability. We find no indication of CoiA having a role in regulating competence. Finally, using 32P- and 3H-labeled donor DNA, we demonstrate that a coiA mutant can internalize normal amounts of donor DNA compared to the wild-type strain but is unable to process it into viable transformants, suggesting a role for CoiA after DNA uptake, either in DNA processing or recombination.

Bacterial Proteins↗

PubMed1985

[Spontaneous genetic transformation in Pseudomonas pseudomallei].

Spontaneous genetic transformation has been registered in Pseudomonas pseudomallei cells. Transforming frequencies registered reach 10(-4)-10(-5). Spontaneous transformation has been simulated for Pseudomonas pseudomallei in soil. Intrageneric spontaneous transformation has been demonstrated to occur between Pseudomonas pseudomallei and Pseudomonas mallei.

DNA, Bacterial↗

PubMedDate not supplied

Method and parameters for genetic transformation of Streptococcus sanguis Challis.

A simple procedure for genetic transformation of Streptococcus sanguis Challis was developed and standardized. During the exponential phase of growth, cells became competent while growing as diplococci in broth containing 10% foetal calf serum. High levels of competence were maintained by the cultures for 60 min. Competent cells could be stored frozen without loss of competence for at least three years. Using total chromosomal DNA as donor, the dose-response curve for transformation of a point mutation (streptomycin resistance) showed one-hit kinetics, as the DNA concentration varied from 0.000001 to 10 micrograms/ml. At 10 micrograms/ml, more than 2.2% of the colony-forming units were transformed to streptomycin resistance, while transforming activity remained detectable with 1 pg of DNA/ml. Optimal time of exposure of competent cells to transforming DNA was 30 min. The transformation reaction was inhibited at 0 and 4 degrees C, whereas it occurred efficiently both at 25 and 37 degrees C.

DNA, Bacterial↗