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Biomedical subjects

M Espinosa

Publications and source records attributed to M Espinosa.

At least 127 records · Page 7Linked to original sources
PubMed1983

Transformation in Bacillus subtilis: different affinities for DNA-binding to competent cells and to membrane vesicles.

Comparison between dose-response curves to homologous and to T*4 (non-glucosylated) DNA in cells and in membrane vesicles, isolated from competent Bacillus subtilis, indicated the presence of two kinds of DNA receptors in the membrane vesicles system. This was confirmed by competition experiments. In addition, concentration dependence for binding of low-molecular weight homologous DNA also revealed the existence of more than one DNA receptor site in competent cells of B. subtilis. However, no differences in the uptake of sheared or intact DNA was observed, which indicates that only one kind of receptors is involved in the entry of donor DNA. Competition experiments in binding, uptake, and transformation, with the possible combinations of sheared or intact DNAs, suggested that the former has more affinity for the binding sites than the latter. This finding is also supported by the results obtained in membrane vesicles, either in competition or in chasing experiments.

Bacillus subtilis↗
PubMed1983

Influence of temperature-induced competence in the genetic transformation of Bacillus subtilis.

Competence of Bacillus subtilis can be induced or repressed by decreasing or raising the temperature of incubation from 42 degrees C to 37 degrees C or vice-versa. Concomitantly with changes in transformability of the cultures, changes in the cell ability to bind 3H-transforming DNA and to degrade the donor DNA was discovered. These alterations reflect changes in the processing of donor DNA rather than at the level of integration of the transforming DNA, since phage SPP1 DNA transfection was affected to the same extent as transformation. Induction of competence development by temperature shift from 42 degrees C to 37 degrees C requires continuous synthesis of protein(s) involved in the binding of donor DNA to the competent cells, whereas the nucleolytic activity related to DNA entry into the cells is already synthesized at the moment of the induction of competence.

Bacillus subtilis↗
PubMed1982

Uptake and fate of bacteriophage phi W-14 DNA in competent Bacillus subtilis.

Phage phi W-14 DNA (in which one-half of the thymine residues are replaced by alpha-putrescinyl thymine) was taken up by competent Bacillus subtilis cells at a rate threefold higher than the rate of homologous DNA uptake. In contrast to other types of heterologous DNA, the amount of phi W-14 DNA taken up in 15 min exceeded the amount of homologous DNA taken up by a factor of two to three, as measured in terms of acid-precipitable material. The amount of phi W-14 DNA taken up was even greater than this analysis indicated if allowance was made for the fact that phi W-14 DNA was degraded more rapidly after uptake than homologous DNA. Competition experiments showed that the affinity of phi W-14 DNA for homologous DNA receptors was lower than the affinity of homologous DNA and was similar to the affinities of other types of heterologous DNA. The more rapid and more extensive uptake of phi W-14 DNA appeared to occur via receptors other than the receptors for homologous DNA, and these receptors (like those for homologous DNA) were an intrinsic property of competent cells. Uptake of phi W-14 DNA was affected by temperature, azide, EDTA, and chloramphenicol, as was uptake of homologous DNA. This was consistent with entry of both DNAs by means of active transport. After uptake, undegraded phi W-14 [3H]DNA was found in the cells in a single-stranded form, whereas a portion of the label was associated with recipient DNA, presumably as a result of incorporation of monomers resulting from degradation. Acetylation of the amino groups of the putrescine side chains in phi W-14 DNA decreased the affinity of this DNA for its receptors without affecting its ability to compete with homologous DNA.

Azides↗
PubMed1982

Facilitation of plasmid transfer in Streptococcus pneumoniae by chromosomal homology.

The frequency of plasmid establishment in the transformation of Streptococcus pneumoniae by plasmid DNA was increased more than 10-fold when the plasmid carried DNA homologous to the host chromosome. Perfect homology was not necessary for such facilitation; small additions or deletions were tolerated, but extensive deletions in the homologous segment of either plasmid or chromosome reduced or eliminated facilitation. The facilitated plasmid transfer showed a linear dependence on monomeric plasmid concentration rather than the quadratic dependence found in the absence of homology, which indicated that entering plasmid fragments interacted with the chromosome rather than with each other to establish a plasmid replicon. Restriction enzyme cleavage of the plasmid in the nonhomologous segment destroyed its activity, but cleavage in the homologous segment or even enzymatic removal of part of that segment did not prevent plasmid transfer, and plasmids of the original size were established. In facilitated transfer, chromosomal markers (additions and deletions as well as single-site mutations) entered the plasmid with a frequency ranging from 10 to 90% depending on the marker location. Several possible mechanisms for the establishment of plasmids in the presence of chromosomal homology and for the transfer of chromosomal information are considered. They depend on synapsis of the newly entered single-strand plasmid fragment with the host chromosome and subsequent copying of, donation from, or integration into the homologous chromosomal segment. After plasmid establishment, equilibration of donor and chromosomal markers between the chromosome and the plasmid pool, presumably by homologous recombination events, was observed.

Alleles↗
PubMed1981

Detection of different types of receptors for deoxyribonucleic acid in competent Bacillus subtilis.

Competent cells of Bacillus subtilis are able to bind but not to take up DNA in magnesium-free medium. Wall-membrane complexes, but not cell walls, bind homologous DNA. Binding of DNA to membrane vesicles suggests that DNA-receptor sites are located at the membrane level. On the basis of competition experiments with homologous and heterologous DNAs, in cells and in membrane vesicles, different types of DNA receptors are detected.

Bacillus subtilis↗
PubMed1981

Cloning of chromosomal genes in Streptococcus pneumoniae.

A system for molecular cloning in Streptococcus pneumoniae was developed. The multicopy plasmids pMV158 (5.4 kilobases) and pLS1 (4.3 kilobases), which confer tetracycline resistance, were used as vectors to clone chromosomal genes of S. pneumoniae in host cells of this species. A 3.3-kilobase restriction fragment containing the malM gene, which codes for amylomaltase, was cloned in a deletion mutant lacking chromosomal homology with the fragment. The recombinant plasmid pLS70, could transform over 50% of a recipient population to maltose utilization. Amylomaltase constituted up to 10% of the protein of cells containing pLS70. A derivative with a deletion, pLS69, appeared to gain a selective advantage by producing less enzyme. A 10-kilobase restriction fragment containing the sul-d gene for sulfonamide resistance was cloned in the presence of the homologous chromosomal gene. De novo establishment of a recombinant plasmid was just as frequent as transformation in an endogenous plasmid. Despite the processing of DNA during uptake in the transformation of S. pneumoniae, recombinant plasmids can be introduced. Models for the reconstruction of recombinant DNA in cells of S. pneumoniae and Bacillus subtilis are considered and compared.

Chromosomes, Bacterial↗
PubMed1981

Phi W-14 DNA inhibits transfection of Bacillus subtilis by SPP1 DNA.

The DNA of bacteriophage phi W-14 is unusual in that half of the thymine residues are replaced with the hypermodified pyrimidine alpha-putrescinylthymine (Kropinski et al., Biochemistry 12:151-157, 1973). Bacteriophage phi W-14 DNA and Bacillus subtilis DNA exhibited comparable competing abilities for the uptake of transfecting bacteriophage SPP1 DNA by competent cells of B. subtilis. B. subtilis DNA decreased transfection and uptake to the same extent, indicating that it merely competed with SPP1 DNA for uptake. Phi W-14 DNA, however, decreased transfection up to 30 times more effectively than it inhibited uptake. Phi W-14 DNA did not alter the kinetics of transfection. The degree of inhibition of transfection was dependent upon the time of addition of Phi W-14 DNA relative to the time of addition of SPP1 DNA. If failed to inhibit when added 30 min after SPP1 DNA. It had a fourfold-greater effect when added 10 min before, rather than simultaneously with, SPP1, but this enhancement was abolished by high concentrations of SPP1 DNA. The nature of the transfection process was not altered in those cells escaping inhibition by Phi W-14 DNA: two molecules of transfecting SPP1 DNA were required to form a transfectant with or without Phi W-14 DNA. Free putrescine did not affect transfection by SPP1 DNA. It was concluded that the putrescine groups covalently attached to phi W-14 DNA allowed this DNA to interfere with the transfection process at the intracellular level.

Bacillus subtilis↗
PubMed1981

The effect of magnesium of transfection and transformation in Bacillus subtilis.

The influence of Mg2+ on phage SPP1 DNA-mediated transfection as compared with chromosomal transformation was studied. A differential influence of this cation in both processes was detectable by analyzing the competence development and the kinetics of appearance of transformants and transfectants. Binding and uptake of DNA and release of acid-soluble products by competent cells in high- and low-Mg2+ media was measured for 3H-labelled SPP1 and 3H-labelled Bacillus subtilis DNAs. Phage SPP1 DNA was shown to be subjected to endonucleolytic cleavage after exposure to competent cells.

Bacillus subtilis↗
PubMed1980

DNA binding and deoxyribonuclease activity in Bacillus subtilis during temperature-induced competence development.

Rapid development of competence can be induced in cultures of Bacillus subtilis by incubation at 37 degrees C after previous growth at 42 degrees C. This temperature-induced competence was accompanied by an increase in DNA binding capacity and breakdown of donor DNA. Inhibition of protein synthesis prevented the rapid increase of competence. This is probably due to the inhibition of de novo synthesis of a constituent that enables the bacteria to bind DNA.

Bacillus subtilis↗
PubMed1980

Interactions of homologous and heterologous deoxyribonucleic acids and competent Bacillus subtilis cells.

Glucosylated and nonglucosylated bacteriophage T4 deoxyribonucleic acids (DNAs) are able to bind to competent cells of Bacillus subtilis, although the former does so in a rather unstable fashion, probably because of the glucosylation. Several heterologous DNAs compete with homologous DNA for the same receptors in binding and in transformation. A different pattern in competition for DNA binding was observed for homologous and T4 glucosylated DNAs in intact cells as compared with protoplasts or membrane vesicles. The results are consistent with the existence of two types of receptor sites on the membrane of competent B. subtilis cells.

Bacillus subtilis↗
PubMed1980

Influence of bacteriophage PBS1 and phi W-14 deoxyribonucleic acids on homologous deoxyribonucleic acid uptake and transformation in competent Bacillus subtilis.

Both bacteriophage PBS1 deoxyribonucleic acid (DNA) (in which all the thymine residues are replaced by uracil) and phage phiW-14 DNA [in which half the thymine residues are replaced by 5-(aminobutylaminomethyl)uracil or 5-putrescinylthymine] exhibit comparable competing abilities for uptake of homologous DNA in a Bacillus subtilis competent system. But, whereas PBS1 DNA leads to a decrease in transformation frequencies compatible with its competing ability for DNA uptake, phiW-14 DNA decreases transformation frequencies by a factor up to eightfold higher. The effect of phiW-14 DNA on transformation frequencies is visible even at a concentration level that does not decrease transforming DNA uptake. No such effect was observed with heterologous DNA containing presumably ionically bound putrescine. Low concentrations of phiW-14 DNA decreased the number of double (nonlinked) transformants more than single transformants. The influence on transformation was abolished when phiW-14 DNA was added 20 min after addition of transforming DNA, i.e., when the recombination process was terminated. The putrescine-containing DNA also decreased retention of trichloroacetic acid-precipitable radioactivity of homologous DNA taken up. We conclude that phiW-14 DNA inhibits some intracellular process(es) at the level of recombination. In addition, there is evidence that phiW-14 DNA, but not heterologous DNA with ionically bound putrescine, binds also to site(s) on the cell surface other than receptors for homologous DNA.

Bacillus subtilis↗
PubMed1980

Influence of some antineoplastic agents on genetic exchange in Bacillus subtilis.

The influence of three categories of anti-cancer agents on competence development and on the binding of homologous DNA to cells have been studied in the Bacillus subtilis transformation system. Treatment of cells developing competence with testolactone resulted in a slight enhancement of transforamtion at high doses (50 and 100 micrograms/ml) of the drug, but this process was inhibited at 25 micrograms/ml. DNA binding was unaffected by this drug. DNA-interacting agents (daunomycin, prospidine, peptichemio and mithramycin) usually inhibited DNA-mediated transformation in a higher extent than DNA binding. Antimetabolites of DNA synthesis were also tested: dacarbazine did not greatly inhibit the binding and expression of donor DNA. 5-Fluorouracil appeared to slightly enhance transformation at low levels 1 microgram/ml), although it was inhibited at higher doses.

Antineoplastic Agents↗
PubMed1978

Further studies on the competence development in exponentially growing cultures of Bacillus subtilis.

Bacillus subtilis, growing in Bott and Wilson's medium, develops two peaks of competence in batch cultures. The first maximum developed during the exponential growth phase and it has been studied comparatively with the competence level reached in continuous culture at Dt = 2.5 h. In both cases, 100 microgram/ml of arginine inhibited competence. Continuous cultures treated with arginine recovered competence specifically after the addition of Mn2+. In addition, a reduction in the synthesis of aconitase and fumarase was observed in the arginine-inhibited continuous cultures.

Aconitate Hydratase↗