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Biomedical subjects

R Curtiss

Publications and source records attributed to R Curtiss.

At least 163 records · Page 9Linked to original sources
PubMed1974

R6K plasmid replication: influence of chromosomal genotype in minicell-producing strains of Escherichia coli K-12.

Alkaline sucrose velocity sedimentation and cesium chloride-ethidium bromide equilibrium centrifugation have been used to determine the number of copies per chromosomal equivalent of the relaxedly replicating R6K plasmid (a conjugative plasmid conferring ampicillin and streptomycin resistance) in two minicell-producing strains of Escherichia coli K-12. In one strain, the average number of covalently closed circular R6K molecules per chromosomal equivalent is 13 in log-phase and 35 in stationary-phase cells. In the other strain, there is an average of six covalently closed circular R6K molecules per chromosomal equivalent in both log- and stationary-phase cells. Selection from this strain of spontaneously occurring mutants resistant to high concentrations of ampicillin has been accomplished and such mutants show a two- to threefold increase in the number of R6K copies per chromosomal equivalent. Relative to the parental strain, mutants display the following properties: (i) elevated streptomycin resistance, (ii) a 10-fold increase in R6K conjugal transfer, (iii) a 10-fold increase in the amount of R6K plasmid deoxyribonucleic acid segregated into minicells, and (iv) a two- to threefold increase in R6K-specified beta-lactamase. The mutation(s) responsible for the increase in the number of R6K molecules per chromosomal equivalent is located on the bacterial chromosome. No R6K-linked mutations conferring the above phenotypes have been obtained. The mutations are presumed to be in chromosomal genes which play a role in the regulation of R6K replication in this strain.

Cell-Free System↗
PubMed1973

Cryptic plasmids in a minicell-producing strain of Salmonella typhimurium.

A minicell-producing strain of Salmonella typhimurium contains two cryptic plasmids. One has a molecular weight of 2.6 x 10(6) to 2.8 x 10(6), is present in multiple copies per cell, and segregates into minicells. The other has a molecular weight of 130 x 10(6), is present in few copies per cell, and probably does not segregate into minicells.

Bromides↗
PubMed1973

Molecular nature of R-factor deoxyribonucleic acid isolated from Salmonella typhimurium minicells.

In earlier reports it was shown that a variety of extrachromosomal elements harbored in the Escherichia coli minicell producer segregate into the minicells. We show in this report that the fi(+) R factor R-100-1 (derepressed derivative of R-100) similarly segregates into minicells produced by a Salmonella typhimurium strain. Four distinct classes of covalently closed circular deoxyribonucleic acid molecules are found in minicells derived from the R(+)Salmonella minicell producer. The sum of the average molecular weights or contour lengths of the circular molecules in two of the classes is equal to the average molecular weight or contour length of those in a third class. The data suggest that R-100-1 dissociates into resistance determinants (i.e., genes that specify the molecules that confer resistance) and the resistance transfer factor (i.e., genes responsible for the transferability of the R factor to a recipient). In contrast, only one molecular species is found in minicells derived from the R(+) (R-100-1) Escherichia coli minicell producer. The fourth size class consists of small covalently closed circles (minicircles), which were originally found in the R(-)Salmonella minicell producer and are shown in this report to be enhanced in number in R(+)Salmonella minicells.

Bromides↗
PubMed1973

Derepression of anthranilate synthase in purified minicells of Escherichia coli containing the Col-trp plasmid.

Purified minicells of Escherichia coli K-12 containing the plasmid Col-trp(+) or Col-trpA2 could be derepressed for the synthesis of anthranilate synthase, the first enzyme encoded in the trp operon. Non-plasmid-containing, deoxyribonucleic acid-deficient minicells could not be derepressed. Derepressed enzyme synthesis was initiated by l-tryptophan starvation. The kinetics of derepression were studied with minicells containing the Col-trpA2 plasmid. The derepression curves were biphasic with a rapid initial rate of enzyme synthesis followed by a slower rate of synthesis. The presence of l-tryptophan (20 to 50 mug/ml) or chloramphenicol (200 mug/ml) abolished enzyme synthesis. The presence of rifamycin SV (280 mug/ml) partially inhibited enzyme synthesis after at least 3.5 min of exposure. The ratio of minicell-to-cell synthetic capacity was 1:2.4 when compared on the basis of derepressed enzyme activity per unit cell volume. This work demonstrates that plasmid-containing minicells are capable of considerable functional protein and messenger ribonucleic acid synthesis and that the regulation of at least the trp operon is similar in minicells to that observed in cells.

Chloramphenicol↗
PubMed1973

Conjugal deoxyribonucleic acid replication by Escherichia coli K-12: stimulation in dnaB(ts) donors by minicells.

R64-11(+) donor cells that are thermosensitive for vegetative DNA replication will synthesize DNA at the restrictive temperature when recipient minicells are present. This is conjugal DNA replication because it is R64-11 DNA that is being synthesized and there is no DNA synthesis if minicells that cannot be recipients of R64-11 DNA are used. The plasmid DNA present in the donor cells before mating is transferred to recipient minicells within the first 20 min of mating, but additional copies of plasmid DNA synthesized during the mating continue to be transferred for at least 90 min. However, the transfer of R64-11 DNA to minicells is not continuous because the plasmid DNA in minicells is the size of one R64-11 molecule or smaller, and there are delays between the rounds of plasmid transfer. DNA is synthesized in minicells during conjugation, but this DNA has a molecular weight much smaller than that of R64-11. Thus, recipient minicells are defective and are not able to complete the synthesis of a DNA strand complementary to the single-stranded R64-11 DNA received from the donor cell.

Centrifugation, Density Gradient↗
PubMed1973

Conjugal deoxyribonucleic acid replication by Excherichia coli K-12: effect of chloramphenicol and rifampin.

Conjugal replication of R64-11 deoxyribonucleic acid (DNA) and the concomitant transfer of R64-11 DNA to DNA-deficient minicells are dependent upon processes that are inhibited by rifampin and chloramphenicol. The rifampin-sensitive product is not present in vegetatively growing cells and is needed to initiate both conjugal DNA replication in donor cells and DNA transfer to recipient minicells. If the rifampin-sensitive product is a ribonucleic acid (RNA) molecule (rather than RNA polymerase itself), our data indicate that this RNA species required for initiation of conjugal activity does not need to be translated into a protein product. The chloramphenicol-sensitive product(s) is present in vegetatively growing cells in sufficient quantity to permit most donor cells to carry out one round of plasmid conjugal replication and transfer. The initiation of second and subsequent rounds of conjugal replication and transfer are dependent on the synthesis of both the rifampin-sensitive and chloramphenicol-sensitive products. Our results demonstrate a correspondence between the amount of conjugal DNA replication in the donor and the amount of DNA transferred to recipient minicells under all conditions, and therefore suggest but do not prove that plasmid transfer is dependent on conjugal DNA replication. The results also add additional proof that R64-11 transfer to minicells is discontinuous. All of these results are discussed in regard to further refinements of old models for the mechanism of conjugal transfer as well as a more radical departure from current dogma.

Bacterial Proteins↗
PubMed1973

Conjugal deoxyribonucleic acid replication by Escherichia coli K-12: effect of nalidixic acid.

During the conjugal transfer of the R64-11 plasmid at 42 C from donor cells thermosensitive for vegetative deoxyribonucleic acid (DNA) synthesis to recipient minicells, the plasmids are conjugally replicated in the donor cells. This conjugal replication is inhibited by nalidixic acid, and the degree of inhibition is comparable to the reduction in the amount of plasmid DNA transferred to the recipient minicells in the presence of the drug. In addition, the size of DNA transferred to the minicells and the fraction of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA under alkaline conditions are both reduced by nalidixic acid. When the drug is added to a mating that is underway, the rate of conjugal replication is immediately reduced. This change is accompanied by a reduction in the amount of conjugally replicated DNA in the donor cells that can be isolated as closed-circular plasmid DNA. Furthermore, conjugally replicated plasmid DNA that is not associated with the donor cell membrane becomes membrane bound after the addition of nalidixic acid.

Centrifugation, Density Gradient↗
PubMed1972

Molecular studies on entry exclusion in Escherichia coli minicells.

Minicells produced by abnormal cell division in a strain of Escherichia coli (K-12) have been employed here to investigate the phenomenon of "entry exclusion." When purified minicells from strains containing F' or R factors, or both, are mated with radioactive thymidine-labeled Hfr or R(+) donors, the recipient minicells can be conveniently separated from normal-sized donors following mating, and the products of conjugation can be analyzed in the absence of donors and of further growth of the recipients. Transmissible plasmids or episomes are transferred less efficiently to purified minicells derived from strains carrying similar or related elements than to strains without them. Measurement of deoxyribonucleic acid (DNA) degradation and determination of weight-average molecular weights following transfer indicate that degradation of transferred DNA or transfer of smaller pieces cannot account for the comparative reduction in transfer to entry-excluding recipients. Therefore, we conclude that entry exclusion operates to prevent the physical entry of DNA into recipients expressing the exclusion phenotype. The R-produced repressor (product of the drd(+) gene), which represses fertility (i.e., ability to act as donor), reduces exclusion mediated by R or F factor, or both, in matings between strains carrying homologous elements. Furthermore, the data suggest that the presence of the F pilus or F-like R pilus on recipient cells ensures maximum expression of the exclusion phenotype but is not essential for its expression. In contrast to previous suggestions, we found no evidence for a reduction of entry exclusion attributable to the DNA temperature-sensitive chromosomal mutation dnaB(TS).

Centrifugation, Density Gradient↗
PubMed1971

Chromosome transfer and recombinant formation with deoxyribonucleic acid temperature-sensitive strains of Escherichia coli.

Haploid recombinant yield is reduced in matings conducted at 42.5 C between deoxyribonucleic acid (DNA) temperature-sensitive [dna(-)(TS)] recipients unable to synthesize DNA at 42.5 C and any of the three major donor types (Hfr, F(+), F') of Escherichia coli. No such reduction is observed in matings conducted at 42.5 C when the dna(-)(TS) mutation is in the donor parent. Evidence is presented which indicates that chromosome transfer from donors to recipients unable to replicate DNA at 42.5 C during vegetative growth occurs at normal frequencies when the mating is conducted at 42.5 C. It is concluded that some stage in haploid recombinant formation is adversely affected in dna(-)(TS) recipients mated at the temperature restrictive for DNA synthesis.

Chromosome Mapping↗
PubMed1971

Synthesis of ribonucleic acid and protein in plasmid-containing minicells of Escherichia coli K-12.

Unlike the deoxyribonucleic acid (DNA)-deficient minicells produced by F(-) parents, minicells produced by plasmid-containing strains contain significant amounts of plasmid DNA. We examined the ability of plasmid-containing minicells to synthesize ribonucleic acid (RNA) and protein. In vivo, minicells produced by F(-) parents are unable to incorporate radioactive precursors into acid-insoluble RNA or protein, whereas minicells produced by F', R(+), or Col(+) parents are capable of such synthesis. Using a variety of approaches, including polyacrylamide gel analysis of the RNA species produced and electron microscope autoradiography, we demonstrated that the synthesis observed in minicell preparations is a property of the plasmid-containing minicells and not a result of the few cells (approximately 1 per 10(6) minicells) contaminating the preparations. That the observed synthesis is of biological importance is suggested by the ability of plasmid-containing minicells to yield viable phage upon infection with T4.

Ampicillin↗
PubMed1970

Genetic exchange between Escherichia coli strains in the mouse intestine.

Germ-free mice contaminated with selected Escherichia coli strains were used for experiments designed to demonstrate gene transfer and recombinant formation in vivo. The well-characterized conjugation system of E. coli K-12 was examined in these experiments. Contamination of germ-free mice with a polyauxotrophic F(-) strain followed by the addition of isogenic Hfr, F', or F(+) strains resulted in the appearance of all recombinant classes at frequencies that would be expected from an in vitro mating experiment. Inheritance of unselected donor markers occurred at frequencies that were dependent on linkage relationships established in experiments in vitro. The presence of Lactobacillus had no influence on gene transfer and recombinant formation in an F' x F(-) in vivo mating. The R factor ROR-1 was transferred from E. coli strain M7-18 to an E. coli F(-) strain in the mouse intestine.

Agar↗
PubMed1969

Early stages of conjugation in Escherichia coli.

We initiated these studies to learn more about the initial events during bacterial conjugation and to optimize conditions for their occurrence. We found that cells in donor cultures grown anaerobically prior to mating have (i) a higher mean number of F pili per cell, (ii) longer F pili, (iii) a higher probability of forming specific pairs with F(-) cells, and (iv) a faster rate of initiation of chromosome transfer than cells grown aerobically. The growth medium for the donor culture also influences these same parameters: a rich medium is superior to a completely synthetic medium. Starvation of donor cells in buffered saline or for a required amino acid results in (i) a loss of F pili, (ii) a loss in the ability of donor-specific phages to adsorb, (iii) a loss of ability to form specific pairs with F(-) cells and to yield recombinants, and (iv) an increase in recipient ability. These changes occur as a function of starvation time, and at rates which are dependent on the conditions of prior growth and starvation of the donor culture. Either treatment provides a rapid method for the production of F(-) phenocopies from donor cultures. Resynthesis of F pili by cells within a starved donor culture commences very soon after restoration of normal growth conditions, but full restoration of donor ability, as measured by recombinant yield, occurs at a slower rate. We found, along with other investigators, that F pili are essential for specific pair formation. We also found, however, that the presence of F pili is not sufficient for display of donor ability, nor is the absence of F pili enough for cells to exhibit recipient ability. This suggests, therefore, that one or more components, in addition to F pili, are necessary for the conversion of specific pairs to effective pairs (or for chromosome mobilization, or both) and for preventing donor cells from acting as recipients. On the basis of our results, we suggest optimal conditions for achieving high mating efficiencies.

Amino Acids↗