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PubMed2005

[Enrichment of giant panda microsatellite markers using dynal magnet beads].

The 400 -600 bp DNA fractions of giant panda containing STR sequences were captured by hybridization with the oligonucleotide probes attached to streptavadin coated magnetic beads (Dynal). The enriched DNA were ligated into pGEM-T and then transformed into E. coil JM109 competent cells. In total 260 positive clones were identified from 2 880 transformants in the libraries which were screened by gamma-32 P radiolabelled probes. Finally, we got 54 sequences and successfully designed 37 pairs of STR primers for giant panda. The results showed that this method is very efficient to isolate microsatellite markers.

Animals↗
PubMed2003

ComX is a unique link between multiple quorum sensing outputs and competence in Streptococcus pneumoniae.

Natural competence for genetic transformation in Streptococcus pneumoniae is achieved directly by specific proteins that are involved in DNA uptake and chromosomal recombination, and is regulated indirectly by a quorum-sensing system encoded by two loci, comAB and comCDE. The alternative sigma factor, ComX, is thought to be the unique link between quorum sensing and competence-specific genes. To test this hypothesis, we replaced the quorum-sensing inducible promoter (PQ) of the comX gene with either a constitutive promoter (PC) or a raffinose-inducible promoter (PR), so that comX transcription would be independent of quorum sensing. Surprisingly, both competence and expression of late genes, such as ssbB, cglA or celB, were found to depend on CSP in these mutants. An unknown, CSP-dependent regulator was needed when comX was expressed from these ectopic promoters, and it appears to act post-transcriptionally. However, when a multicopy nisin-inducible ComX-overexpressing plasmid was introduced, pneumococcal cells developed competence in the presence of nisin even despite deletion of comE. At 1% of the normal protein peak level, ComX protein stimulated competence without the participation of the pheromone response circuit. Thus, ComX is a unique link to competence-specific genes, but depends on multiple outputs of quorum sensing for maximal expression.

Bacterial Proteins↗
PubMed1982

Intramolecular recombination during plasmid transformation of Bacillus subtilis competent cells.

We have constructed plasmids carrying direct internal repeats 260-2000 bp long. Monomers of such plasmids transformed Bacillus subtilis competent cells. The efficiency of transformation varied with the square of the length of repeats. The transformed clones harbored either the entire transforming plasmid and the plasmid arising by recombination between the repeats, or only the latter plasmid. Internally-repeated plasmids linearized by in vitro cleavage with restriction endonuclease could transform, yielding clones which exclusively harbored a plasmid resulting from recombination between the repeats. When the transforming plasmid carried repeats which differed slightly, conversion of one repeat into the other could occur. The following model of plasmid transformation accounts for these data: (1) plasmid DNA is cleaved and rendered linear in contact with competent cells; (2) a linear, at least partially double-stranded plasmid molecule is introduced or formed by repair within the cell; (3) a circular viable plasmid is produced by recombination between repeats carried on this molecule; (4) alternatively, a viable plasmid is produced by repairing the cut within one of the repeats by DNA synthesis which uses the other repeat as a template.

Bacillus subtilis↗
PubMed1973

Kinetic analysis of the products of donor deoxyribonucleate in transformed cells of Bacillus subtilis.

This paper describes the major transmutations of donor deoxyribonucleic acid (DNA) after uptake by competent Bacillus subtilis cells. Kinetic experiments confirm that after exposure to competent cells, donor DNA is converted to double-stranded fragments (DSF) which can be isolated as early as 30 s from the beginning of the reaction. At this time, DSF represent the only identifiable product of donor origin. After 1 to 2 min, DSF are converted to deoxyribonuclease-resistant forms, identified as single-stranded DNA fragments (SSF). SSF are intermediates in the transformation process leading to the formation of donor-recipient complex. This component makes its appearance between 2 to 4 min from the beginning of the transformation process. All the donor-recipient complexes found at the end of the reaction can be accounted for quantitatively by the DSF and the SSF found in the initial stages of transformation. A quantitative discussion of the transformation process is included.

Bacillus subtilis↗
PubMed2005

Chitin induces natural competence in Vibrio cholerae.

The mosaic-structured Vibrio cholerae genome points to the importance of horizontal gene transfer (HGT) in the evolution of this human pathogen. We showed that V. cholerae can acquire new genetic material by natural transformation during growth on chitin, a biopolymer that is abundant in aquatic habitats (e.g., from crustacean exoskeletons), where it lives as an autochthonous microbe. Transformation competence was found to require a type IV pilus assembly complex, a putative DNA binding protein, and three convergent regulatory cascades, which are activated by chitin, increasing cell density, and nutrient limitation, a decline in growth rate, or stress.

Animals↗
PubMed2002

Instability of repeated DNAs during transformation in Escherichia coli.

Escherichia coli has provided an important model system for understanding the molecular basis for genetic instabilities associated with repeated DNA. Changes in triplet repeat length during growth following transformation in E. coli have been used as a measure of repeat instability. However, very little is known about the molecular and biological changes that may occur on transformation. Since only a small proportion of viable cells become competent, uncertainty exists regarding the nature of these transformed cells. To establish whether the process of transformation can be inherently mutagenic for certain DNA sequences, we used a genetic assay in E. coli to compare the frequency of genetic instabilities associated with transformation with those occurring in plasmid maintained in E. coli. Our results indicate that, for certain DNA sequences, bacterial transformation can be highly mutagenic. The deletion frequency of a 106 bp perfect inverted repeat is increased by as much as a factor of 2 x 10(5) following transformation. The high frequency of instability was not observed when cells stably harboring plasmid were rendered competent. Thus, the process of transformation was required to observe the instability. Instabilities of (CAG).(CTG) repeats are also dramatically elevated upon transformation. The magnitude of the instability is dependent on the nature and length of the repeat. Differences in the methylation status of plasmid used for transformation and the methylation and restriction/modification systems present in the bacterial strain used must also be considered in repeat instability measurements. Moreover, different E. coli genetic backgrounds show different levels of instability during transformation.

DNA Replication↗
PubMed1975

Fate of transforming bacteriophage HP1 deoxyribonucleic acid in Haemophilus influenzae lysogens.

The biological fate of temperate phage HP1 deoxyribonucleic acid (DNA) was followed after uptake by defectively lysogenic competent Haemophilus influenzae cultures. The similar inactivation kinetics of three single phage genetic markers and of their triple combination indicated a complete rather than partial destruction of about half of the adsorbed DNA molecules. Intracellular DNA breakdown products were tentatively identified by hydroxyapatite column chromatography as short single strands and extensively damaged short double strands. Integrated donor DNA (after single-strand insertion?) was still highly efficient for triple-marker co-transformation. This suggests that whole or nearly whole donor DNA molecules were integrated. Some donor DNA was never integrated but remained largely unaltered. This DNA fraction did not contain significant amounts of recipient prophage marker activity. It is concluded that it had not participated in some kind of reciprocal recombination event involving the recipient chromosome. Since very similar phage DNA marker inactivation rates were observed after adsorption by competent nonlysogenic recipients (transfection), the relationship between biological inactivation of adsorbed donor phage DNA and its integration in lysogenic recipients is not clear.

Bacteriophages↗
PubMed1990

Genetic transformation of intact cells of Bacillus subtilis by electroporation.

Plasmid DNAs were introduced by electroporation into Bacillus subtilis PB1424 as an alternative to competent-cell or protoplast transformation. The maximum electroporation efficiency was 10(4) transformants/microgram DNA. Parameters including growth phase of cells, ionic strength of the suspending medium, concentration and size of plasmid DNAs, amplitude and duration of the pulse, were evaluated in order to determine conditions that improved transformation efficiency.

Bacillus subtilis↗
PubMed2004

Exploring the mechanism of competence development in Escherichia coli using quantum dots as fluorescent probes.

The mechanism of divalent Ca2+ cation induction of Escherichia coli competence is still not fully understood, though it is a common method for introducing recombinant DNA into bacterial cells in gene engineering. Quantum dots (QDs), as a new fluorescent probe of being applied in biology research, have aroused great interest. In this paper, we explored the mechanism of E. coli competence development using QDs for the first time. Results showed that water-soluble QDs of diameter 3-4 nm could go into competent cells, but could not enter noncompetent cells. This result was further confirmed using atomic force microscopy and DNA transforming experiments, suggesting that nonphysiological, high concentrations of Ca2+ enhanced the penetrability of cell membranes so that QDs, which cannot enter cells normally due to their greater diameter (3-4 nm), can do so easily into competent cells. Therefore, we believe that, at least for E. coli, the mechanism of Ca2+-induced competence development is mediated physicochemically rather than physiologically.

Biochemistry↗
PubMed2004

DNA transport into Bacillus subtilis requires proton motive force to generate large molecular forces.

Bacteria can acquire genetic diversity, including antibiotic resistance and virulence traits, by horizontal gene transfer. In particular, many bacteria are naturally competent for uptake of naked DNA from the environment in a process called transformation. Here, we used optical tweezers to demonstrate that the DNA transport machinery in Bacillus subtilis is a force-generating motor. Single DNA molecules were processively transported in a linear fashion without observable pausing events. Uncouplers inhibited DNA uptake immediately, suggesting that the transmembrane proton motive force is needed for DNA translocation. We found an uptake rate of 80 +/- 10 bp s(-1) that was force-independent at external forces <40 pN, indicating that a powerful molecular machine supports DNA transport.

Bacillus subtilis↗
PubMed2000

Efficient transformation of primary human amniocytes by E1 functions of Ad5: generation of new cell lines for adenoviral vector production.

Primary human cells are relatively refractory to transformation by adenoviral E1 functions. For almost two decades, human embryonic kidney (HEK)-derived 293 cells have been the only E1-complementing cell line suitable for production of E1-deleted adenoviral vectors. More recently, new vector production cell lines have been derived from human embryonic retina (HER) cells, a cell type that is difficult to obtain. We were surprised to find that readily available primary human amniocytes are efficiently transformed by adenoviral E1 functions. We selected cell lines that allow high-titer production of recombinant adenoviral vectors. The generation of replication-competent adenovirus (RCA) during production, caused by homologous recombination between vector and cellular DNA, was excluded by designing the transforming plasmid to lack sequence overlap with current adenoviral vectors. In addition, we generated an infectious plasmid that can be used for convenient generation of first-generation adenoviral vectors in Escherichia coli and that matches the E1 complementation in the new production cell lines.

Adenoviridae↗
PubMed1985

Fate of exogenous recombinant plasmids introduced into mouse and human cells.

We have constructed a number of plasmids selectable in both E. coli and mouse or human cells. Human DNA sequences were inserted and the recombinant plasmids were used to transfect either mouse or human cells by the Ca-phosphate precipitation technique. We have observed that: (i) competent cells uptake large amounts of plasmid DNA; (ii) input plasmids persist in transformed mammalian cells as free unreplicating circular molecules for up to 20 generations; such persistence does not depend on the presence of selective markers; (iii) plasmids incorporated into mouse L-cells undergo widespread rearrangements (in the absence of replication) entailing mostly deletions of both human and bacterial sequences which yield smaller products; the latter appear to be more stable in a subsequent transformation cycle. Surprisingly such rearrangements are almost totally absent in transformed human KB-cells. This property of human KB-cells may prove useful for the development of a vector apt at cloning and expressing human DNA sequences. Unlike what has been observed in yeast, no "autonomously replicating sequence" can be detected in mammalian cells by randomly cloning human DNA sequences into a selectable plasmid and screening for an increased transformation efficiency.

Animals↗
PubMed1993

Physiological characterization of natural transformation in Acinetobacter calcoaceticus.

Acinetobacter calcoaceticus BD413 develops competence for natural transformation immediately after the start of the exponential growth-phase and remains competent up to e few hours into the stationary phase, after which competence gradually declines. The transformation frequencies obtained strongly depend on the kind of transforming DNA and the incubation time with DNA. Up to 25% of the cells in a culture can be transformed. DNA uptake in Acinetobacter does not display sequence specificity, is Mg(2+)-, Mn(2+)- or Ca(2+)-dependent and is uncoupler sensitive. The transforming DNA enters the cells in single-stranded form. These properties constitute a unique combination, not previously observed in other bacteria, and make A. caloaceticus ideally suited for detailed studies of the bioenergetics of DNA translocation.

Acinetobacter calcoaceticus↗
PubMed2001

The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403.

Lactococcus lactis is a nonpathogenic AT-rich gram-positive bacterium closely related to the genus Streptococcus and is the most commonly used cheese starter. It is also the best-characterized lactic acid bacterium. We sequenced the genome of the laboratory strain IL1403, using a novel two-step strategy that comprises diagnostic sequencing of the entire genome and a shotgun polishing step. The genome contains 2,365,589 base pairs and encodes 2310 proteins, including 293 protein-coding genes belonging to six prophages and 43 insertion sequence (IS) elements. Nonrandom distribution of IS elements indicates that the chromosome of the sequenced strain may be a product of recent recombination between two closely related genomes. A complete set of late competence genes is present, indicating the ability of L. lactis to undergo DNA transformation. Genomic sequence revealed new possibilities for fermentation pathways and for aerobic respiration. It also indicated a horizontal transfer of genetic information from Lactococcus to gram-negative enteric bacteria of Salmonella-Escherichia group.

Amino Acids↗
PubMed2006

Antibiotic stress induces genetic transformability in the human pathogen Streptococcus pneumoniae.

Natural transformation is a widespread mechanism for genetic exchange in bacteria. Aminoglycoside and fluoroquinolone antibiotics, as well as mitomycin C, a DNA-damaging agent, induced transformation in Streptococcus pneumoniae. This induction required an intact competence regulatory cascade. Furthermore, mitomycin C induction of recA was strictly dependent on the development of competence. In response to antibiotic stress, S. pneumoniae, which lacks an SOS-like system, exhibited genetic transformation. The design of antibiotherapy should take into consideration this potential of a major human pathogen to increase its rate of genetic exchange in response to antibiotics.

Aminoglycosides↗
PubMed1969

Killing of Haemophilus influenzae cells by integrated ultraviolet-induced lesions from transforming deoxyribonucleic acid.

Highly competent cultures of Haemophilus influenzae are inactivated by exposure to transforming deoxyribonucleic acid (DNA) irradiated with ultraviolet light (UV). As a function of UV dose to the DNA, the killing goes to a maximum and then decreases. The killing of H. influenzae by unirradiated H. parainfluenzae DNA, reported by other workers, is enhanced by low doses of UV, but drops off at high doses. Since there are no such lethal effects in a strain of H. influenzae that takes up DNA normally but does not integrate it, it is concluded that the killing is associated with integrated UV lesions. All the killing of wild-type cells due to irradiated DNA is eliminated by photoreactivation of the DNA. The killing of an excisionless strain of H. influenzae, however, is not eliminated by maximal photoreactivation of the irradiated transforming DNA. The nonphotoreactivable fraction of killing in the excisionless strain increases with increasing dose. The kinetics of the killing-dose curves may be explained only partially in terms of UV-induced loss of integration. It is postulated that the number of pyrimidine dimers relative to other DNA components integrated decreases at higher UV doses.

DNA, Bacterial↗
PubMed1984

DNA-mediated cotransfer of excision repair capacity and drug resistance into chinese hamster ovary mutant cell line UV-135.

We have investigated DNA-mediated transfer of aminopterin resistance conferred by plasmid and UV resistance conferred by genomic DNA to the Chinese hamster ovary (CHO) cell line UV-135, a UV-sensitive mutant defective in nucleotide excision repair. Plasmid pSV2gpt-CaPO4 coprecipitates induced aminopterin resistance with equal efficiency in the 6-thioguanine-resistant, aminopterin-sensitive, repair-proficient parental line AA8-4(tg-1) and in UV-135(tg-2). Genetic and molecular evidence for genomic DNA-mediated transformation of UV-135(tg-2) cells with a putative excision repair gene were obtained by demonstrating that: (i) UV resistance transformation is dependent upon and specific for genomic DNA from excision repair-competent CHO cells: (ii) UV and drug coresistant colonies are bona fide transferants as verified by hybridization and Southern blotting analysis of pSV2gpt sequences in their genomic DNAs: (iii) confirmed transferants exhibit partial to near normal UV resistances for colony formation: and (iv) UVr transferants have near normal levels of excision repair capacity. The overall frequency of drug and UV resistance cotransformation was 8 X 10(8) per cell plated. This frequency was ca. 200- to 500-fold greater than that expected from coincident but independent UVr reversion and plasmid gene transfer events. DNA transfer techniques with this CHO system will be useful for further analysis of the essential structural DNA sequences, gene cloning, and expression of functional excision repair genes.

Aminopterin↗