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Modulation of competence for genetic transformation in Streptococcus pneumoniae.

The spontaneous development of competence by cultures of Streptococcus pneumoniae in casein hydrolysate medium was strongly dependent on the initial pH of the culture medium. Cells growing in cultures beginning with a wide range of initial pH values (6.8 to 8.0) all developed competence, as measured by [3H]DNA uptake, [3H]DNA degradation and genetic transformation; but the initial pH of the medium affected both the timing of the occurrence of competence and the number of times the culture became competent. In cultures grown in media of lower initial pH, competence occurred only once, at high population densities, while in more alkaline media a succession of competence cycles occurred, beginning at lower cell densities. The critical population density required for the initiation of competence varied tenfold over the pH range studied. Successive competence cycles in an alkaline medium were not equivalent: while the percentage of competent cells in the first competence cycle was high (approximately 80%), that in the second competence cycle was lower (approximately 12%). Correspondingly, competence-specific proteins were less prominent in the labelled-protein pattern of the second competence cycle than in that of the first. These features of the physiology of competence control make it possible to adjust the expression of competence to suit various experimental requirements.

DNA, Bacterial↗

Sequencing, targeted mutagenesis and expression of a recA gene required for the extreme radioresistance of Deinococcus radiodurans.

Deinococcus radiodurans and other members of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA. A DNA damage-sensitive and natural transformation-deficient strain generated by chemical mutagenesis (strain rec30) was found to be defective in a gene that has extended homology with recA of Escherichia coli. Upon transformation with a chromosomal DNA fragment that contained this deinococcal recA gene from wild-type (wt) D. radiodurans both DNA damage resistance and full transformation competence were restored in the rec30 mutant. Targeted insertional mutagenesis of the deinococcal recA gene was used to construct a mutant isogenic with the wt. The insertional mutant was phenotypically indistinguishable from strain rec30, indicating that the recA defect alone was responsible for observed phenotypic alterations. For example, in the case of ionizing radiation, the D37 of the wt was about 1.75 Mrad, while the D37 of rec30 and the insertional mutant were both 25 krad, a 70-fold decrease. Evidence is presented that expression of the deinococcal recA gene in E. coli is lethal, suggesting that the mode of interaction of the deinococcal RecA protein with nucleic acids or other cellular proteins differs at least in part from RecA of E. coli.

Amino Acid Sequence↗

Possible mechanism for donor DNA binding and transport in Haemophilus.

Morphological differences were observed in competent and noncompetent Haemophilus parainfluenzae and Haemophilus influenzae when thin sections of these cells were examined by electron microscopy. The membranous extensions present on the surface of competent H. parainfluenzae cells disappeared on treatment with transforming DNA, while vacuole-like structures appeared in the periplasm. Noncompetent cells had 1/5th as many extensions on their surface as competent cells, and no vacuoles were observed after treatment with homologous DNA. Competent cells treated with radiolabeled DNA were disrupted and the clarified lysate was centrifuged on CsCl density gradients. Material having a density of 1.34 g/ml was found to contain the majority of the DNase-resistant radioactive DNA recovered from the bacteria and was shown by electron microscopy to be composed of membrane vesicles. The polypeptide composition of this dense membrane fraction was similar to that of H. parainfluenzae outer membrane.

Biological Transport↗

Inhibition of resistance plasmid transfer in Escherichia coli by ionophores, chlortetracycline, bacitracin, and ionophore/antimicrobial combinations.

Medicinal feed additives bacitracin, chlortetracycline (CTC), laidlomycin, lasalocid, and salinomycin inhibited the transfer of multiresistance-conferring plasmid pBR325 (Tet(r) Amp(r) Cp(r), 6.0 kb) into selected gram-negative strains with the use of an in vitro model. High concentrations of ampicillin-sensitive competence-pretreated Escherichia coli HB 101 cells were exposed to 10% (v/v) of 1:10 dimethyl sulfoxide/agent : water containing test mixtures for 0.5 hr prior to plasmid addition and transforming conditions. Transformation was inhibited for all antimicrobials and showed a positive association wich higher concentration. Additional testing of ionophore compounds separately and in combination with bacitracin, chlortetracycline, lincomycin, roxarsone, tylosin, and virginiamycin at representative feed concentrations demonstrated 80.6% to >99.9% inhibition (P < 0.001) of resistance transfer. Bacitracin alone inhibited transformation within the range of 50-500 ppm. No increase in resistance transfer was observed when poultry-derived and reference gram-negative isolates having low or no transformation efficiency were additionally tested. The results suggest that these compounds, at relevant concentrations used in animal feed, may interfere with cell envelope-associated DNA uptake channels or other transformation competence mechanisms. Through these mechanisms, ionophores and cell membrane-interactive feed agents such as CTC and bacitracin may act to inhibit resistance transfer mechanisms within poultry and livestock.

Bacitracin↗

[Construction of eukaryotic expression plasmid pcDNA3-gtfB expressing glucosyltransferase B of Streptococcus mutans].

OBJECTIVE: Glucosyltransferase (GTF) of Streptococcus mutans is considered as a cariogenic virulence factor due to its ability to synthesize glucan, which facilitate sucrose-depended adherence and cell-to-cell accumulation of bacteria. In this study, gtfB, the target gene fragment which encodes multiple catalytic sites and antigen epitopes of GTF, was recombined into eukaryotic expression vector pcDNA3. The feasibility of the recombination plasmid pcDNA3-gtfB used as gene vaccine will be investigated in further study. METHODS: The target gene fragment gtfB (904-4578 bp) was obtained by standard PCR amplification while genome DNA of streptococcus mutans GS-5 was used as template. Then the PCR products were extracted and purified from low-melting temperature agarose. The gtfB and plasmid pcDNA3 were cut by Kpn I, Xho I, and the digested products were extracted and purified again for recombination. The purified gtfB and plasmid pcDNA3 were recombined by T4 DNA ligase, ligation products were transformed into competent cell, Escherichia coli JM109. Transformed colonies were screened by Ampr LB plate, then recombined plasmids were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing. RESULTS: Identified by agarose gel electrophoresis, the target gene-gtfB obtained PCR amplification had the same molecular size (36 kb) as predicted. It was indicated that recombined plasmids contained inserted gtfB gene fragment by restricted endonuclease cut analysis, the sequencing data also indicated that inserted gtfB gene had correct DNA sequence and orientation according to DNA sequence of Streptococcus mutans GS-5 (gene bank M17361). CONCLUSION: Inserted gene-gtfB of recombined plasmid pcDNA3-gtfB encoded multiple catalytic sites and epitopes. It was proved that these epitopes had high immune antigenicity and that antiserum could significantly inhibit the synthesis of water-insoluble glucans and water-soluble glucan. In vitro adherence experiment also indicated that it could inhibit streptococcus mutans adherence to saliva-coated hydroxyapatite. Vector pcDNA3 was high expressing eukaryotic vector, and could stimulate antigen-representing cell. It was suggested that recombined plasmid pcDNA3-gtfB had high immune antigenicity and immune responsiveness, and this supported its use as gene vaccine candidates in the development of anti-caries vaccines.

Dental Caries↗

Release of DNA into the medium by competent Streptococcus pneumoniae: kinetics, mechanism and stability of the liberated DNA.

The release of chromosomal DNA into culture media has been reported for several naturally transformable bacterial species, but a direct link between competence development and the liberation of DNA is generally lacking. Based on the analysis of strains with mutations in competence-regulatory genes and the use of conditions favouring or preventing competence, we provide evidence that DNA release is triggered by the induction of competence in Streptococcus pneumoniae. Kinetic analyses revealed that whereas competence was maximal 20 min after addition of competence-stimulating peptide, and then decreased, the amount of liberated DNA continued to increase and reached a maximum in stationary phase, when cells are no longer competent for DNA uptake. These data are not consistent with the proposal that release of DNA by a fraction of the population is coordinated with uptake by the remainder. Moreover, we observed that an unidentified DNase was specifically induced or released in competent cultures, and that together with the major pneumococcal endonuclease, EndA, it could degrade released DNA. Nearby complete abolition of release in a mutant lacking both the major autolysin, LytA, and the autolytic lysozyme, LytC, indicated that DNA liberation occurs by LytA-LytC-dependent cell lysis. These observations suggest that competence-dependent DNA release is one facet of a more general phenomenon of sensitization to autolysis that reaches its maximum in stationary phase.

DNA, Bacterial↗

Altered expression of adenovirus 12 DNA-binding protein but not DNA polymerase during abortive infection of hamster cells.

Replication of human adenovirus type 12 DNA is blocked in abortively infected baby hamster kidney cells. The activity and accumulation of adenovirus 12 DNA polymerase is equivalent in infected hamster and human cell extracts. However, the accumulation of adenovirus type 12 DNA-binding protein is approximately 120-fold lower in extracts from infected hamster cells when compared to infected permissive human cells. This difference in accumulation is not due to replication of viral DNA during productive infection, since this difference is observed in the presence of hydroxyurea. The DNA-binding protein from infected hamster cells retains the ability to bind denatured DNA-cellulose. An adenovirus 5 early region 1 transformed hamster cell line competent to complement the adenovirus 12 DNA replication defect also stimulates accumulation of the DNA-binding protein even when the cells are treated with hydroxyurea. Thus, the reduced expression of the viral DNA-binding protein may play a role in the mechanism of abortive infection of hamster cells by adenovirus 12.

Adenovirus Early Proteins↗

Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.

Binding, Competitive↗

Generation and release of DNA-binding vesicles by Haemophilus influenzae during induction and loss of competence.

Genetic transformation of bacterial cells required the induction of a state of competence to bind and absorb free DNA molecules. Induction of competence in Haemophilus influenzae was accompanied by the generation on the cell surface of membrane extensions ("blebs") 80 to 100 nm in diameter. When competent cells were returned to normal growth conditions, they shed these structures as free vesicles with a concomitant loss of cellular DNA-binding activity. Purified vesicle preparations retained the ability to bind double-stranded DNA in a nuclease-resistant, salt-stable form. Binding was specific for DNA molecules containing the 11-base pair Haemophilus uptake sequence, required Na+ and divalent cations (Mg2+, Ca2+, or Mn2+), and was inhibited by the presence of EDTA or high concentrations of salt (greater than 0.5 M NaCl). Binding was not stimulated by nucleotide triphosphates and was insensitive to the uncoupling agents dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. Vesicles contained the major Haemophilus outer membrane proteins and were enriched in several minor proteins.

Cations, Divalent↗

Fate of free DNA and transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva.

Competitive PCR was used to monitor the survival of a 520-bp DNA target sequence from a recombinant plasmid, pVACMC1, after admixture of the plasmid with freshly sampled human saliva. The fraction of the target remaining amplifiable ranged from 40 to 65% after 10 min of exposure to saliva samples from five subjects and from 6 to 25% after 60 min of exposure. pVACMC1 plasmid DNA that had been exposed to degradation by fresh saliva was capable of transforming naturally competent Streptococcus gordonii DL1 to erythromycin resistance, although transforming activity decreased rapidly, with a half-life of approximately 50 s. S. gordonii DL1 transformants were obtained in the presence of filter-sterilized saliva and a 1-microg/ml final concentration of pVACMC1 DNA. Addition of filter-sterilized saliva instead of heat-inactivated horse serum to S. gordonii DL1 cells induced competence, although with slightly lower efficiency. These findings indicate that DNA released from bacteria or food sources within the mouth has the potential to transform naturally competent oral bacteria. However, further investigations are needed to establish whether transformation of oral bacteria can occur at significant frequencies in vivo.

Base Sequence↗

Structural organization, nucleotide sequence, and regulation of the Haemophilus influenzae rec-1+ gene.

The Haemophilus influenzae rec-1+ protein plays a central role in DNA metabolism, participating in general homologous recombination, recombinational (postreplication) DNA repair, and prophage induction. Although many H. influenzae rec-1 mutants have been phenotypically characterized, little is known about the rec-1+ gene at the molecular level. In this study, we present the genetic organization of the rec-1+ locus, the DNA sequence of rec-1+, and studies of the transcriptional regulation of rec-1+ during cellular assault by DNA-damaging agents and during the induction of competence for genetic transformation. Although little is known about promoter structure in H. influenzae, we identified a potential rec-1+ promoter that is identical in 11 of 12 positions to the bacterial sigma 70-dependent promoter consensus sequence. Results from a primer extension analysis revealed that the start site of rec-1+ transcription is centered 6 nucleotides downstream of this promoter. We identified potential DNA binding sites in the rec-1+ gene for LexA, integration host factor, and cyclic AMP receptor protein. We obtained evidence that at least one of the proposed cyclic AMP receptor protein binding sites is active in modulating rec-1+ transcription. This finding makes rec-1+ control circuitry novel among recA+ homologs. Two H. influenzae DNA uptake sequences that may function as a transcription termination signal were identified in inverted orientations at the end of the rec-1+ coding sequence. In addition, we report the first use of the Escherichia coli lacZ operon fusion technique in H. influenzae to study the transcriptional control of rec-1+. Our results indicate that rec-1+ is transcriptionally induced about threefold during DNA-damaging events. Furthermore, we show that rec-1+ can substitute for recA+ in E. coli to modulate SOS induction of dinB1 expression. Surprisingly, although 5% of the H. influenzae genome is in the form of single-stranded DNA during competence for genetic transformation, an event that could be a potent SOS-inducing signal, we failed to detect significant changes in rec-1+ transcription during the induction of genetic competence.

Amino Acid Sequence↗

Relationship between competence for transformation of Bacillus subtilis with native and single-stranded deoxyribonucleic acid.

The response of populations of Bacillus subtilis to both native deoxyribonucleic acid (DNA) and denatured DNA was investigated at maximal competence and at various times during the development of compentency. The results indicate that competence for transformation with native and denatured DNA increases and decreases simultaneously. Competition occurs between native and single-stranded DNA during transformation, and the same cells in a population can be doubly transformed by DNA molecules of both configurations.

Bacillus subtilis↗

Bioassay for specific DNA sequences using a non-radioactive probe.

A novel method for detecting specific DNA sequences is described. The method uses a non-radioactive DNA probe, called a probe-vector, that can transform competent Escherichia coli cells at high efficiency only when it has hybridized to a specific DNA target, thus forming a circular, double-stranded, plasmid-like molecule. The probe-vector carries a plasmid origin of replication and a gene that confers antibiotic resistance on transformed E. coli. The output of the assay--colored bacterial colonies on an agar plate--is quantitative and proportional over a wide range of target concentrations. The utility of the probe-vector method for detecting hepatitis B virus (HBV) DNA in human serum is demonstrated. The assay can detect as little as 0.1 pg HBV DNA. The presence of an internal standard monitors DNA recovery and E. coli transformation efficiency for each sample. The assay has the potential to simultaneously measure the DNA of two or more pathogens within the same clinical sample.

Biological Assay↗

Effective targeted cytotoxicity of neuroblastoma cells.

BACKGROUND/PURPOSE: Despite aggressive treatment with surgery, chemotherapy, and radiotherapy, the prognosis for many children with neuroblastoma remains poor. Targeted toxins represent novel cancer therapeutics designed to selectively target and kill cancer cells. The authors have developed a novel fusion toxin, DT5F11, consisting of truncated diphtheria toxin (DT(A)) linked to a single chain antibody (sc5F11) targeting the GD(2) antigen found on most neuroblastoma cells. This report describes the construction, expression, and in vitro function of DT5F11. METHODS: Utilizing restriction enzyme digestion, polymerase chain reaction amplification, and gel electrophoresis, the prkDTL5F11 plasmid was created by the fusion of distinct coding sequences for a single-chain GD(2) targeting antibody (sc5F11) and truncated diphtheria toxin (DT(A)). DH5alpha Escherichi coli-competent cells were transformed with prkDTL5F11; DNA was amplified, isolated, and sequenced. The fusion protein was expressed and assayed by Western blot. Targeted cytotoxicity was analyzed on GD(2)-positive (SK-N-AS, IMR-32, SK-N-MC, LAN-1) and GD(2)-negative (HeLa) cells. RESULTS: Fluorescent dye-labeled cycle sequencing identified the constructed fusion toxin gene. Western blot analysis using a mouse antihuman DT(A) antibody showed a 69-kD band identifying the fusion toxin, DT5F11. Targeted cell killing with DT5F11 was seen only in GD(2) positive cells. CONCLUSIONS: This study demonstrates creation of a novel fusion toxin with effective GD(2)-targeted cellular toxicity. Further investigation of this fusion toxin as a therapeutic agent in the management of neuroblastoma is warranted.

Antibodies, Monoclonal↗

Isolation and properties of Bacillus subtilis strains lysogenized by a clear plaque mutant of bacteriophage phi 105.

A clear plaque mutant of the temperate Bacillus phage phi105 lysogenized a small fraction of infected cells forming an integrated prophage at or near the normal phi105 insertion site. These lysogens exhibited a spontaneous induction rate approximately 1,000-fold lower than wild type and were noninducible (ind(-)) by mitomycin C. Prophage was induced, however, when competent cultures were incubated with transforming DNA. The ind(-) phenotype could not be attributed solely to the clear plaque mutation and appears to involve a cell-specific factor. Lysogenization by the clear plaque mutant, in contrast to wild-type phage, did not cause a marked reduction in transformation efficiency.

Bacillus subtilis↗

Studies on transformations of Hemophilus influenzae. I. Competence.

A procedure has been developed for obtaining Hemophilus influenzae of such competence that 1 to 10 per cent transform to any of several genetic factors by utilizing a period of aerobic growth followed by a non-aerobic period. Differences in levels of competence were not due to differences in genetic background. Competence was due to at least one factor intrinsic to the cell or site on the cell and was not transferable to non-competent cells. Competence was affected by salt concentration, pH, and temperature. Washing competent cells reduces their ability to transform, but not their capacity to bind DNA reversibly. The irreversible step could be restored with little or no accompanying growth. These facts suggest that reversible and irreversible binding represent separate biochemical steps. DNA initiates a reaction in cells leading to a loss of competence. In the absence of DNA the cells remain competent for at least an hour. Competence correlates quantitatively with predictability of multiple transformations. The observed and calculated values of multiple transformations are in closer agreement, the higher the frequency of transformation for single markers. The correction needed to bring the two figures into agreement is a measure of the fraction of non-competent cells.

DNA, Bacterial↗

Plasmid transformation of Azotobacter vinelandii OP.

Azotobacter vinelandii OP which had been naturally induced to competence by growth in iron- and molybdenum-limited medium was transformed with the broad-host-range cloning vector pKT210. However, the transformation frequency at nearly saturating levels of DNA was 1000-fold lower for pKT210 than for a single chromosomal DNA marker (nif+). Plasmid- and chromosomal-DNA-mediated transformation events were competitive, magnesium-dependent, 42 degrees C-sensitive processes specific to double-stranded DNA, suggesting a common mechanism of DNA binding and uptake. The low frequency of plasmid transformation was not related to restriction of transforming DNA or to the growth period allowed for phenotypic expression. Covalently-closed-circular and open-circular forms of pKT210 transformed cells equally well whereas EcoRI- or HindIII-linearized pKT210 transformed cells with two to three times greater efficiency. Genetic transformation was enhanced 10- to 50-fold when pKT210 contained an insert fragment of A. vinelandii nif DNA, indicating that A. vinelandii possessed a homology-facilitated transformation system. However, all transformants failed to maintain the plasmid-encoded antibiotic resistance determinants, and extrachromosomal plasmid DNA was not recovered from these cells. Flush-ended pKT210 was not active in transformation; however, competent cells were transformed to Nif+ by HincII-digested plasmid DNA containing the cloned A. vinelandii nif-10 marker.

Azotobacter↗

Deoxyribonucleic acid-binding properties and membrane protein composition of a competence-deficient mutant of Haemophilus influenzae.

A mutant of Haemophilus influenzae was isolated which was completely unable to take up double-stranded homologous deoxyribonucleic acid (DNA) at normal physiological conditions but which took up DNA equally as well as the wild type at low pH (pH 4.4). The properties of the mutant provide evidence for the existence of two different mechanisms for DNA entry in the H. influenzae transformation system. With the aid of the mutant the optimal conditions for entry of DNA by these two mechanisms were determined, and the dependence of entry and the specific transforming activity of the entered DNA on competence was examined. The mechanism of entry of DNA at neutral pH, which is not functioning in the mutant, effected entry of homologous DNA only, whereas the mechanism involved in entry of DNA at low pH also effected entry of heterologous DNA. This suggests that the mutant is lacking a protein which recognizes the specific base sequence(s) required for entry. Comparison of the protein composition of the membranes of mutant cells subjected to a growth regimen provoking competence in wild-type cells with that of competent wild-type cells revealed that the mutant is impaired in the synthesis of a protein with a molecular weight of 22,500.

Bacterial Proteins↗