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PubMed2004

Release of transforming plasmid DNA from actively growing genetically engineered Escherichia coli.

We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria.

Colony Count, Microbial↗

PubMed1998

Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor.

Competence is a physiological state, distinct from sporulation and vegetative growth, that enables cells to bind and internalize transforming DNA. The transcriptional regulator ComK drives the development of competence in Bacillus subtilis. ComK is directly required for its own transcription as well as for the transcription of the genes that encode DNA transport proteins. When ComK is sequestered by binding to a complex of the proteins MecA and ClpC, the positive feedback loop leading to ComK synthesis is interrupted. The small protein ComS, produced as a result of signaling by a quorum-sensing two-component regulatory pathway, triggers the release of ComK from the complex, enabling comK transcription to occur. We show here, based on in vivo and in vitro experiments, that ComK accumulation is also regulated by proteolysis and that binding to MecA targets ComK for degradation by the ClpP protease in association with ClpC. The release of ComK from binding by MecA and ClpC, which occurs when ComS is synthesized, protects ComK from proteolysis. Following this release, the rates of MecA and ComS degradation by ClpCP are increased in our in vitro system. In this novel system, MecA serves to recruit ComK to the ClpCP protease and connects ComK degradation to the quorum-sensing signal-transduction pathway, thereby regulating a key developmental process. This is the first regulated degradation system in which a specific targeting molecule serves such a function.

Bacillus subtilis↗

PubMed2006

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis.

Neisseria meningitidis is the causative agent of epidemic meningococcal meningitis and septicaemia. Type IV pili are surface organelles that mediate a variety of functions, including adhesion, twitching motility, and competence for DNA binding and uptake in transformation. The secretin PilQ is required for type IV pilus expression at the cell surface, and forms a dodecameric cage-like macromolecular complex in the meningococcal outer membrane. PilQ-null mutants are devoid of surface pili, and prevailing evidence suggests that the PilQ complex facilitates extrusion and retraction of type IV pili across the outer membrane. Defining the orientation of the meningococcal PilQ complex in the membrane is a prerequisite for understanding the structure-function relationships of this important protein in pilus biology. In order to begin to define the topology of the PilQ complex in the outer membrane, polyhistidine insertions in N- and C-terminal regions of PilQ were constructed, and their subcellular locations examined. Notably, the insertion epitopes at residues 205 and 678 were located within the periplasm, whereas residue 656 was exposed at the outer surface of the outer membrane. Using electron microscopy with Ni-NTA gold labelling, it was demonstrated that the insertion at residue 205 within the N-terminus mapped to a site on the arm-like features of the 3D structure of the PilQ multimer. Interestingly, mutation of the same region gave rise to an increase in vancomycin permeability through the PilQ complex. The results yield novel information on the PilQ N-terminal location and function in the periplasm, and reveal a complex organization of the membrane-spanning secretin in vivo.

Bacterial Outer Membrane Proteins↗

PubMed1972

Competence of pneumococcal isolates and bacterial transformations in man.

A survey of pneumococci isolated from 19 healthy carriers and from 23 patients with pneumococcal disease showed that, for both groups, 25 to 30% of the isolates were competent for transformation by soluble deoxyribonucleic acid (DNA) in vitro. Untransformable type 3 and type 8 pneumococci, whose capsules had been hydrolyzed by the Bacillus palustris enzymes prior to exposure to DNA, remained untransformable. Thus, at least for these isolates, it was not the presence of capsule that prevented transformation. Type 9 pneumococci in a healthy human carrier were transformed by DNA released from living unencapsulated pneumococci sprayed onto the pharynx. The donor bacteria were resistant to 1,000 mug of streptomycin/ml. Two types of streptomycin-resistant bacteria were recovered from the carrier's pharynx: a type 9 pneumococcus and an alpha-hemolytic streptococcus. No streptomycin-resistant, gram-positive cocci were isolated from this individual prior to inoculation of the pharynx with the resistant organisms. It seems possible that transformations can occur in the natural environment of some gram-positive cocci.

Journal Article↗

PubMed2005

[Construction of the eukaryote expression vector of human soluble tumor necrosis factor receptor].

OBJECTIVE: Human soluble tumor necrosis factor receptor (sTNFR) can interfere with the biological functions of interleukin-1, which may be appropriate to the treatment of periodontitis. The eukaryote expression vector of the human sTNFR gene must be constructed prior to conducting transgene therapy of periodontitis. METHODS: Both sTNFR gene and plasmid pcDNA 3.1 (+) DNA were digested with Kpn I and Xho I. After purification, the two fragments were ligated by TakaRa DNA Ligation Kit (Ver 2.0). This recombinant DNA was then transformed into E. Coli Competent Cells JM109 and positive clones were selected on the LB agarose plate containing ampicillin (80 microg/ul). RESULTS: Six single clones were indentified by double digestion with kpn I and xho I and two fragments with the size of 5.4 kb and 1.0 kb were produced as expected. CONCLUSION: The sTNFR gene was successfully inserted into the eukaryote expression vector plasmid pcDNA 3.1 (+) by recombination technology in vitro.

Escherichia coli↗

PubMed1997

Ability of high hydrostatic pressure treated plasmids and cells of Escherichia coli to genetically transform.

The exposure of plasmid pUC18 and pBR322 DNA to high hydrostatic pressure increased the ability of plasmids to transform competent Escherichia coli cells. For pUC18 plasmid, a pressure of 400 MPa, and for pBR322, a pressure of 200 MPa was found to provide the highest transformation efficiency. The DNA duplexes of the two plasmids were found to be the most stable for melting conditions at these pressures. At pressures higher than these, both the stability of the duplex DNA and the transformation efficiency were affected. The stabilizing effect of high hydrostatic pressure on the hydrogen bond may be responsible for the observed increase in transformation efficiency of the pressure-exposed plasmid DNA. The possibility of pressure-induced changes in the structure and conformation of DNA was studied using various techniques. In agarose gel electrophoresis, pressure-treated plasmids (pUC18 at 400 MPa and pBR322 at 200 MPa) consistently showed visibly distinct higher mobility compared to untreated plasmids. Pressure-treated pUC18 as well as pBR322 DNA showed significant reduction in ethidium bromide binding as is evident from the reduced intensity of fluorescence of the dye bound pressure-treated DNA. Spectroscopic studies using circular dichroism and Fourier transform infrared (FTIR) spectroscopy also showed significant differences in the absorption profiles of pressure-treated plasmids as compared to an untreated control. These studies revealed that the pressure-induced changes in the conformation of these DNAs may be responsible for the observed increase in the transformation ability of the plasmids. On the other hand, the exposure of competent cells of E. coli to a high hydrostatic pressure of 50 MPa not only reduced their colony-forming ability but also drastically reduced their ability to take up plasmid DNA.

Journal Article↗

PubMed1963

INCORPORATION OF DEOXYRIBONUCLEIC ACID IN THE BACILLUS SUBTILIS TRANSFORMATION SYSTEM.

Young, F. E. (Western Reserve University Cleveland, Ohio) and John Spizizen. Incorporation of deoxyribonucleic acid in the Bacillus subtilis transformation system. J. Bacteriol. 86:392-400. 1963.-The optimal conditions for the incorporation of deoxyribonucleic acid (DNA) were studied. In competent cells, the irreversible binding of DNA was influenced by temperature, hydrogen ion concentration, and aeration. Divalent cations, such as barium, strontium, calcium, or magnesium, were required. Under suboptimal environmental conditions and with metabolic inhibitors, the process of transformation was decreased to a greater extent than was incorporation of DNA. Under conditions of phosphate depletion, the incorporation of P(32) increased. However, the frequency of transformation decreased. This inducible process was not related to competence.

Bacillus subtilis↗

PubMed1974

The chemical and biological effects of cis-dichlorodiammineplatinum (II), an antitumor agent, on DNA.

cis-Dichlorodiammineplatinum (II) binds irreversibly to the bases in DNA; the amount of platinum complex bound can be determined from changes in the ultraviolet absorption spectrum. As the ratio of platinum to phosphate is increased, an increasing inactivation of bacterial transforming DNA is observed. At a ratio that corresponds to spectrometric saturation, transforming activity is inactivated >10(5)-fold. The trans isomer of the platinum complex, which is not effective against tumors, induces a similar inactivation of transforming DNA but with half the efficiency, indicating a different mode of binding. The sensitivity to inactivation by cis isomer varies slightly with the genetic marker assayed but is not dependent on the excision repair system. Uptake of DNA by competent cells is unaffected by bound platinum complex; however, integration of platinum-bound transforming DNA into the host genome decreases as the mole fraction of platinum increases. This loss of integration parallels the decreased transforming activity of the DNA. Although the drug induces interstrand crosslinks in DNA in vitro, these crosslinks are relatively rare events and cannot account for the observed inactivation.

Cisplatin↗

PubMed1992

Modification in penicillin-binding proteins during in vivo development of genetic competence of Haemophilus influenzae is associated with a rapid change in the physiological state of cells.

By using whole-cell labeling assay with 125I-penicillin V, we observed a reduction in the binding of the radiolabeled beta-lactam to four or five penicillin-binding proteins (PBPs) in Haemophilus influenzae cells cultivated under specific conditions. PBPs 3A, 3B, 4, and 6 were altered after the growth of bacteria in diffusion chambers implanted in the peritoneal cavity of rats. PBP 2 was also modified when cells were cultivated in human cerebrospinal fluids. Because this observation may have important consequences on the efficacy of beta-lactams during antibiotic therapy, we characterized the physiological state of bacteria cultivated in animals in the hope of explaining how such important changes in cell properties develop in vivo. Since the development of natural genetic competence occurs at the stationary phase of growth in H. influenzae, we used a DNA transformation assay to evaluate the physiological state of bacteria grown in diffusion chambers implanted in rats. Chromosomal DNA isolated from an antibiotic-resistant donor strain was mixed with bacteria in diffusion chambers. At different times during a 5-h incubation period, recipient bacteria were collected from the chambers, CFU were determined by plate counting, and antibiotic-resistant transformants were isolated on selective plates. Genetic competence rapidly developed in cells grown in rats, and the frequency of transformation by test DNA was elevated. Electron microscopy revealed an irregular cell shape and blebs at the surface of bacteria cultivated in animals and in cerebrospinal fluids. In an attempt to induce a similar physiological state in vitro, we supplemented broth cultures with cyclic AMP or synchronized cultures by a nutritional upshift. No changes in PBPs were observed with supplemental cyclic AMP or during a single cell cycle. Finally, a reduction in the affinity of PBPs for 125I-penicillin V identical to that observed in bacteria grown in rats was observed in cells isolated from the stationary phase of growth in vitro. These results clearly indicate that H. influenzae cells grown in animals undergo a rapid change to a physiological state similar to that found in late-stationary-phase cultures in vitro. This observation indicates that the rational design of future and improved antibiotic therapy of H. influenzae infections should consider cell properties of slow-growing or latent bacteria.

Animals↗

PubMed1989

Large T-antigen mutants define multiple steps in the initiation of simian virus 40 DNA replication.

The biochemical activities of a series of transformation-competent, replication-defective large T-antigen point mutants were examined. The assays employed reflect partial reactions required for the in vitro replication of simian virus 40 (SV40) DNA. Mutants which failed to bind specifically to SV40 origin sequences bound efficiently to single-stranded DNA and exhibited nearly wild-type levels of helicase activity. A mutation at proline 522, however, markedly reduced ATPase, helicase, and origin-specific unwinding activities. This mutant bound specifically to the SV40 origin of replication, but under certain conditions it was defective in binding to both single-stranded DNA and the partial duplex helicase substrate. This suggests that additional determinants outside the amino-terminal-specific DNA-binding domain may be involved in nonspecific binding of T antigen to single-stranded DNA and demonstrates that origin-specific DNA binding can be separated from binding to single-stranded DNA. A mutant containing a lesion at residue 224 retained nearly wild-type levels of helicase activity and recognized SV40 origin sequences, yet it failed to function in an origin-specific unwinding assay. This provides evidence that origin recognition and helicase activities are not sufficient for unwinding to occur. The distribution of mutant phenotypes reflects the complex nature of the initiation reaction and the multiplicity of functions provided by large T antigen.

Antigens, Polyomavirus Transforming↗

PubMed1990

Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene.

The protein encoded by the wild-type p53 proto-oncogene has been shown to suppress transformation, whereas certain mutations that alter p53 become transformation competent. Fusion proteins between p53 and the GAL4 DNA binding domain were made to anchor p53 to a DNA target sequence and to allow measurement of transcriptional activation of a reporter plasmid. The wild-type p53 stimulated transcription in this assay, but two transforming mutations in p53 were unable to act as transcriptional activators. Therefore, p53 can activate transcription, and transformation-activating mutations result in a loss of function of the p53 protein. The inability of the p53 mutant proteins to activate transcription may enable them to be transformation competent.

Base Sequence↗

PubMed1995

Reducing the complexity of the transforming Epstein-Barr virus genome to 64 kilobase pairs.

Transformation-competent, replication-defective Epstein-Barr virus (EBV) recombinants which are deleted for 18 kbp of DNA encoding the largest EBNA intron and for 58 kbp of DNA between the EBNA1 and LMP1 genes were constructed. These recombinants were made by transfecting three overlapping cosmid-cloned EBV DNA fragments into cells infected with a lytic replication-competent but transformation-defective EBV (P3HR-1 strain) and were identified by clonal transformation of primary B lymphocytes into lymphoblastoid cell lines. One-third of the lymphoblastoid cell lines were infected with recombinants which had both deletions and carried the EBNA2 and EBNA3 genes from the transfected EBV DNA and therefore are composed mostly or entirely from the transfected EBV DNA fragments. The deleted DNA is absent from cells infected with most of these recombinants, as demonstrated by Southern blot and sensitive PCR analyses for eight different sites within the deleted regions. Cell growth and EBNA, LMP, and BZLF1 gene expression in lymphoblastoid cell lines infected with these recombinants are similar to those in cells infected with wild-type EBV recombinants. Together with previous data, these experiments reduce the complexity of the EBV DNA necessary for transformation of primary B lymphocytes to 64 kbp. The approach should be useful for molecular genetic analyses of transforming EBV genes or for the insertion of heterologous fragments into transforming EBV genomes.

Antigens, Viral↗

PubMed1976

Physical size of the donor locus and transmission of Haemophilus influenzae ampicillin resistance genes by deoxyribonucleic acid-mediated transformation.

The properties of donor deoxyribonucleic acid (DNA) from three clinical isolates and its ability to mediate the transformation of competent Rd strains to ampicillin resistance were examined. A quantitative technique for determining the resistance of individual Haemophilus influenzae cells to ampicillin was developed. When this technique was used, sensitive cells failed to tolerate levels of ampicillin greater than 0.1 to 0.2 mug/ml, whereas three resistant type b beta-lactamase-producing strains could form from the colonies in 1- to 3-mug/ml levels of the antibiotic. DNA extracted from the resistant strains elicited transformation of the auxotrophic genes in a multiply auxotrophic Rd strain. For two of the donors, transformation to ampicillin resistance occurred after the uptake of a single DNA molecule approximately 104-fold less frequently than transformation of auxotrophic loci and was not observed to occur at all with the third. The frequency of transformation to ampicillin resistance was two- to fivefold higher in strain BC200 (Okinaka and Barnhart, 1974), which was cured of a defective prophage. All three clinical ampicillin-resistant strains were poor recipients, but the presence of the ampicillin resistant genes in strain BC200 did not reduce its competence. Sucrose gradients of DNA from ampicillin-resistant transformants of BC200 and from the original ampicillin-resistant strains showed that: (i) all the DNA preparations had high molecular weights; (ii) donor activity for ampicillin resistance sedimented heterogeneously and in parallel with genome DNA up to the highest molecular weights observed (100 x 106 to 200 x 106); and (iii) genetic transformation of ampicillin resistance from strain BC200-amp90383 required the physical integrity of a linearly integrated segment of DNA having a molecular weight of about 25 x 106 to 30 x 106.

Ampicillin↗

PubMed1972

Competence Mutant of Haemophilus influenzae with Abnormal Ratios of Marker Efficiencies in Transformation.

In studies of competence-deficient mutants of Haemophilus influenzae which absorb deoxyribonucleic acid (DNA) but fail to produce transformants, it was observed that in some mutants the residual transforming activity for different markers varied widely, i.e., produced a ratio effect. One of these mutants, com(-56), was studied intensively to determine the cause of the residual efficiency of transformation and the reason for the ratio effect. The residual frequency of transformation was higher for markers considered single-site mutations (like naladixic acid resistance), whereas the least efficient markers tested were those conferring resistance to high levels of streptomycin or novobiocin which are more complex than single-site mutations. Measurement of frequencies of cotransformation indicated that overall genetic linkage was reduced. Transfection was fairly efficient with phage S2 DNA, but not prophage DNA. Donor marker activity could be detected in transformed cell lysates, but not linked to recipient markers in recombinant molecules. Sucrose gradient analysis of such lysates revealed that donor material was associated with recipient DNA in at least normal quantities, but lacked detectable genetic activity. Material from donor DNA labeled with heavy isotopes was incorporated into recipient chromosomal fragments having a density indistinguishable from normal density, unlike the hybrid density recombinant material found in normal cells. No excessive solubilization or nicking of unincorporated donor was detected. It is postulated that this strain contains a hyperactive nuclease, which reduces the effective size of the input DNA during the integration process.

Journal Article↗

PubMed1986

Effect of glycerol on plasmid transfer in genetically competent Haemophilus influenzae.

The small plasmid pAT4 transformed at characteristically low frequencies those competent Haemophilus influenzae Rd strains that had no DNA homology with this plasmid. Transformation was increased up to 100 times, however, when the recipient cells were exposed to 30% glycerol before plating for transformants. Expression of plasmid resistance markers was then immediate. Ultraviolet irradiation experiments indicated that this large increase was due to release by the glycerol of double-stranded plasmid molecules, presumably from transformasomes. Several other plasmids exhibited the same phenomenon. Dimethylsulfoxide also stimulated plasmid transformation but lysolecithin and high concentrations of NaCl or glucose were ineffective. Glycerol did not increase the efficiency of transformation by either chromosomal DNA or linearized plasmid DNA.

Dose-Response Relationship, Radiation↗

PubMed1978

Bacillus subtilis 168 genetic transformation mediated by outgrowing spores: necessity for cell contact.

Transforming activity released in sequential genetic order during the first synchronous cycle of DNA replication during outgrowth of spores of Bacillus subtilis 168 was investigated. A transformation assay was used consisting of outgrowing spores as DNA donors and multiply marked competent cells as recipients. DNA synthesis inhibitors known to stop DNA release were used during and subsequent to DNA transfer to recipient cells. The released DNA sedimented with the outgrowing cells after low-speed centrifugation, and it was discovered that markers released both early and late were resistant to up to 500 microgram of deoxyribonuclease per ml under conditions in which the transforming capacity of purified DNA was eliminated by 5 microgram of the nuclease per ml. Inaccessibility to deoxyribonuclease was increased and maintained during the transformation event while detergents and proteolytic attack did not expose the released chromosome to nuclease action. The results indicate that tight physical contact between outgrowing spores and competent cells is required for transformation in this system.

Bacillus subtilis↗

PubMed1983

Isolation and characterization of three new classes of transformation-deficient mutants of Streptococcus pneumoniae that are defective in DNA transport and genetic recombination.

Transformation-deficient mutants of Streptococcus pneumoniae were isolated after nitrosoguanidine mutagenesis. Seventeen mutants developed normal peaks of competence, as tested by their ability to degrade one strand of donor DNA, but they yielded transformants for chromosomal point markers at efficiencies from less than 0.001 to 0.04 that of the wild type. Some of the mutants were defective in DNA uptake and are described as entry defective (Ent-). Others took up DNA in normal quantities, but they failed to give stable transformants and are described as recombination defective (Rec-). In two of the Rec- mutants, normal levels of transformation by plasmid DNA occurred; in the others, it was reduced as much as chromosomal transformation. Conjugative transfers of a chromosomal omega (cat tet) element and of the plasmid pIP501 occurred at normal levels both to and from Rec- mutants. Transfer of chloramphenicol resistance by transformation with omega (cat tet) donor DNA, however, was blocked in Rec- mutants to about the same extent as was transformation for point markers.

Conjugation, Genetic↗

PubMed1990

Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization.

The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24 h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40-50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kb Hind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells of Escherichia coli, and colonies were recovered that carried pGEM with Hind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transform E. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning into E. coli.

Chloramphenicol O-Acetyltransferase↗