Restoration of morphogenic potential in Nicotiana by somatic hybridisation

Summary KR103 is a kanamycin resistant cell line of Nicotiana sylvestris, deficient in inducible shoot redifferentiation. The existence of a genetically unaltered inducibility factor in KR103 was tested by fusing protoplasts of this line with those of Nicotiana knightiana, which can not be induced to form shoots in callus culture.Selection of somatic hybrids was based on screening for kanamycin resistance and green pigmentation, factors originally separated in the parental lines. The hybrid nature of two lines (H1 and H2) was shown by studies on esterase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase isoenzymes.In the somatic hybrids shoot formation on inductive medium was restored, indicating that the inducibility factor had not been altered in KR103. Genetic complementation and induced regulatory changes due to genome interaction are discussed as possible explanations for the restoration of morphogenic potential.     

Isolation and characterization of streptomycin-resistant mutants in Nicotiana plumbaginifolia

Summary Streptomycin-resistant colonies were isolated from protoplast cultures of haploid Nicotiana plumbaginifolia based on their ability to green in medium containing 1 mg/ml streptomycin sulfate. The frequency of resistant colonies was 0.9×10^–5 in nonmutagenized culture, and increased ten-fold following treatment of culture with 10 g/ml N-methyl-N-nitro-N-nitrosoguanidine. Of a total of 52 resistant clones isolated, 2 gave rise to haploid, 15 to diploid, and 3 to tetraploid plants upon transfer of calli to differentiation medium. Leaf-segment and protoplast assays showed that all diploid regenerates were resistant to streptomycin but sensitive to chloramphenicol, kanamycin, lincomycin, neomycin, and spectinomycin. Plants in most diploid clones were fertile and able to set seeds when self-fertilized and crossed reciprocally to wild-type plants. Inheritance of streptomycin resistance was studied in the diploid clones and, without exception, the resistance was transmitted maternally. Comparative studies of the ultrastructure of organelles and protein synthesis in isolated chloroplasts between wild-type and resistant clones in the presence of streptomycin suggest that streptomycin resistance is controlled by chloroplasts.     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

Cross-resistance in Cell Lines of Nicotiana sylvestris Selected for Resistance to Individual Antibiotics

Cell lines initially selected for resistance to the antibioticskanamycin, streptomycin and chloramphenicol, were each testedfor resistance to several different antibiotics. Only the kanamycinresistant lines showed any cross-resistance to other antibiotics.The three lines tested were resistant to streptomycin and neomycin,while one of them, KR103, was also resistant to chloramphenicolearly in its history, although this resistance was subsequentlylost. None of the lines showed any resistance to cycloheximide. Nicotiana sylvestris, cell culture, cross-resistance, antibiotics, chloramphenicol, kanamycin, streptomycin, neomycin, cycloheximide, cytoplasmic mutants, callus     

Streptomycin resistance is inherited as a recessive Mendelian trait in a Nicotiana sylvestris line

Summary The SR180 cell line has been isolated in a callus culture derived from a haploid Nicotiana sylvestris (n = X = 12) plant by its ability to proliferate on a selective medium containing 2,000 g/ml streptomycin sulphate. From the cell line diploid plants have been regenerated. The SR180 selfs are resistant to streptomycin. Streptomycin sensitivity in F1, and a 31 (sensitive to resistant) segregation in F2 indicate that resistance in the SR180 mutant is the result of a recessive Mendelian mutation.     

Preferential recovery of uniparental streptomycin resistant mutants from diploid Chlamydomonas reinhardtii

Summary Analysis of the streptomycin resistant mutants recovered from control and N-methyl-N-nitro-N-nitrosoguanadine (MNNG) treated haploid cultures of C. reinhardtii reveal that approximately 60% of the mutants are of the sr-1 type known to show Mendelian inheritance while 40% are of the sr-2 and sm-3 types known to be inherited in a uniparental (UP) manner. In contrast, most if not all streptomycin mutants recovered from similarly treated diploid cultures of C. reinhardtii are of the UP variety. Failure to recover sr-1 mutants from the diploid stock is explained by our findings that diploids heterozygous for Mendelian streptomycin resistance (sr-1/sr-1 ⁺) are both stable and sensitive to streptomycin. Efficient recovery of UP streptomycin resistant mutants from diploids can be explained by the observations of Gillham (1963a, 1969) which demonstrate that diploids heterozygous for an sr-2 mutation (sr-2/sr-2 ⁺) segregate resistant and sensitive progeny during mitotic cell division.The utility of diploids for isolating new UP mutant genotypes, for establishing the cellular localization of the UP genome(s), and for characterizing the rules governing UP gene segregation is discussed.Supported by NIH postdoctoral fellowship GM 52359 to R.W.L., NIH predoctoral traineeship GM 02007 to K.P.V., and NSF grant GB-22769 to N.W.G. and J.E.B.     

Antibiotics resistance of a red alga,Griffithsia japonica

To identify antibiotics suitable for stable transformation, we tested the resistance of a red alga,Griffithsia japonica Okamura, to four commonly used antibiotics. Very young germlings, with 1;3 cells, that germinated from the tetraspores were cultured for 40 d in a half PES medium containing kanamycin, streptomycin, hygromycin B, or phleomycin.G. japonica was highly sensitive to 1 μg mL^-1of phleomycin and g mL^-1of hygromycin B. However, it was resistant to kanamycin and low levels of streptomycin and hygromycin B. These results suggest that resistance genes for phleomycin or hygromycin can be used as selectable markers for transformation of G.japonica.     

Combination of kanamycin resistance and nitrate reductase deficiency as selectable markers in one nuclear genome provides a universal somatic hybridizer in plants

Summary The combination in the nuclear genome of a dominant resistance marker (to select against unfused wild-type cells) and a recessive deficiency marker (to select against unfused mutant cells) in a cell line should provide a system for selecting fusion hybrids between the mutant line and any wild-type line. To test this idea, we fused protoplasts from a non-morphogenic cell line of Nicotiana tabacum which was kanamycin resistant (by transformation) and deficient in nitrate reductase (NR^-K⁺) with protoplasts from N. tabacum cv. Petit Havana clone SR1, which provided resistance against streptomycin as an additional selectable marker (NR⁺K^-SR⁺). Putative hybrids were selected using a culture medium containing no available reduced nitrogen source and 50 mg/l kanamycin sulphate. After regeneration into plants, the hybrid character was demonstrated from: (i) the morphological variation of the regenerants; (ii) the chromosome number; (iii) the ability to grow on medium without a reduced nitrogen source and containing kanamycin sulphate at 50 mg/l; (iv) the presence of nitrate reductase activity; (v) the presence of the gene coding for neomycin phosphotransferase, which provides resistance to kanamycin sulphate; (vi) callus formation from leaves on medium containing 1 g/l streptomycin or 50 mg/l kanamycin sulphate; (vii) F₁ plants containing nitrate reductase and the gene for neomycin phosphotransferase. Fusions between the mutant cell line (NR^-K⁺) and three wild-type tobacco species and subsequent cultivation on medium containing no available nitrogen source but 50 mg/l kanamycin sulphate resulted in callus formation with all combinations, while hybrid plants were only regenerated when N. sylvestris was the fusion partner.     

Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene

A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and -glucuronidase (uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 g/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 g/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement.     

Resistance system of Mycobacterium bovis B.C.B. to aminoglycoside- and peptide-antibiotics. Comparison of resistant phenotypes between Mycobacterium tuberculosis and Mycobacterium bovis

The resistance system of Mycobacterium bovis (B.C.G.) to aminoglycoside-and peptide-antibiotics has been studied. The phenotype of mutants isolated from the parent B.C.G. strain by a single-step selection with an antibiotic were classified into the following three types: (1) resistant only to a low concentration (200 μg/ml) of kanamycin in Ogawa egg medium (k1R); (2) resistant to a low concentration (200 μg/ml) of viomycin and of capreomycin (2R); and (3) resistant to a high concentration (1,000 μg/ml or more) of kanamycin and low concentrations (100 to 200 μg/ml) of lividomycin and of paromomycin (KR). The mutants showing these phenotypes, k1R, 2R, and KR, were isolated from the parent strain by inoculating the strain into media containing 100 μg/ml of kanamycin, and 100 μ/g/ml of viomycin or capreomycin, and 1,000 μg/ml of kanamycin, respectively, at rates of 10^?5-10^?6, 10^?5-10^?6, and 10^?6-10^?7, respectively, in a total viable population of the parent strain. Unlike in the case of M. tuberculosis, no mutant could be isolated from the parent strain by use of enviomycin, lividomycin, and/or paromomycin. In contrast to the fact that quadruply resistant mutants were isolated directly from the parent H37Rv strain of M. tuberculosis, such mutants could be isolated only by two-step selections. Furthermore, the phenotypes of the quadruply resistant mutants were those showing a higher resistance or a broader spectrum than expected by the addition of phenotypes of individual mutations. In addition, it was shown that, in contrast to the fact that hextuply resistant mutants could be isolated directly from the parent strain of M. tuberculosis, such mutants were not isolated directly from the parent B.C.G. strain, but could be isolated only after pre-incubation of the strain on a medium containing Tween 80.     

Cytoplast-protoplast fusion for interspecific chloroplast transfer in Nicotiana

Summary Protoplasts of Nicotiana tabacum (SR1), carrying a maternally-inherited streptomycin resistance mutation, were enucleated by centrifugation through a Percoll gradient. The resulting cytoplasts containing resistant plastids, were fused with sensitive Nicotiana plumbaginifolia protoplasts. The SR1 cytoplasts, having no nuclei, were unable to form calli. All resistant clones recovered after fusion-induction were therefore supposed to be derived from interspecific cytoplast-protoplast fusion. N. plumbaginifolia plants regenerated in 17 out of the 75 resistant clones studied. Plants obtained from eight of these clones were resistant to streptomycin and inherited the resistance maternally, as expected when transferring SR1 plastids into the N. plumbaginifolia nuclear background. Plastid transfer in these plants has been confirmed by the EcoRI restriction pattern of the chloroplast DNA.In nine clones N. plumbaginifolia plants were sensitive although obtained from initially resistant clones. This phenomenon is explained by the maintenance of plastid heterogeneity on the selective streptomycin medium, and formation of plants from sensitive sectors on the non-selective regeneration medium.SR1 protoplasts, originally present as contaminants in the cytoplast preparation (2–7%) did not form colonies (or very rarely) after polyethylene glycol treatment. The nuclei from such protoplasts were recovered, however, in the interspecific somatic hybrids (56 clones), and in segregants having the SR1 nucleus but some cytoplasm from N. plumbaginifolia (2 clones). The majority (about 80%) of the recovered resistant clones therefore acquired the streptomycin resistance factor from the rare (2–7%) contaminating SR1 protoplasts. This is explained by the protoplasts being more stable during fusion induction.     

Stable transformation via electroporation into maize Type II callus and regeneration of fertile transgenic plants

Maize Type II callus tissue was used as the plant material for genetic transformation via electroporation. Plasmid DNA containing a selectable marker gene (either neomycin phosphotransferase (npt-II) or phosphinothricin acetyl transferase (bar)), and a screenable marker gene (gus A) was incubated with the tissue prior to electroporation. Electroporated callus tissue was placed on selection medium containing kanamycin sulfate or Bast. No kanamycin resistant colonies were recovered whereas four independent Basta resistant callus isolates were recovered from a total of 544 cuvettes electroporated. After 8 to 16 weeks on the Basta containing medium, selected calli were isolated and maintained in individual selection plates for 4 to 6 weeks until sufficient tissue accumulated. Enzyme assays and DNA analyses were performed to verify the transformation events. Several plants were regenerated from individual callus isolates. The plants derived from one callus isolate were male sterile while those derived from the other isolates were both male and female fertile. Most plants showed Basta resistance. DNA analyses confirmed the presence of the introduced bar gene(s) in the primary regenerants and their progeny. The integration patterns of the inserted DNA appeared to be complex.     

Direct selection of cybrids by streptomycin and valine resistance in tobacco

Summary Direct selection of cybrids by simultaneous selection for donor chloroplasts and for the recipient nuclei is described. Mesophyll protoplasts of two tobacco (Nicotiana tabacum) mutants, SR1 (streptomycin resistant) and Val^r-2 (valine resistant), were fused by polyethylene glycol treatment. Streptomycin resistance in the SR1 mutant is a maternally inherited chloroplast trait while valine resistance is a Mendelian (nuclear) digenic recessive character. The fused protoplast population was cultured and colonies were selected for resistance to valine (1 mM) and streptomycin (343 M). The efficiency of selection has been confirmed in three clones by demonstrating seed transmission of both streptomycin and valine resistances. In one subclone both streptomycin resistant and sensitive plants were obtained indicating that the streptomycin sensitive chloroplasts had not been totally eliminated by growth on the selective medium.     

A dominant nuclear streptomycin resistance marker for plant cell transformation

Summary Plant cells in photoheterotrophic culture respond to streptomycin by bleaching and retarded growth but no cell death. A new genetic marker for plant cell transformation has been developed that is based on the expression of the enzyme streptomycin phosphotransferase (SPT), and confers the ability to form green colonies on a selective medium. Coding sequences of SPT from the bacterial transposon Tn5 were placed under the control of gene expression signals derived from the Agrobacterium Ti plasmid Ach5. The 5 end of the SPT gene has been replaced with the promoter region of the gene coding for the first enzyme of agropine biosynthesis, the 3 end with that of the enzyme octopine synthase. The chimeric SPT gene has been linked to a selectable kanamycin resistance gene, and introduced into Nicotiana tabacum and Nicotiana plumbaginifolia by selection for the linked kanamycin resistance marker. Streptomycin resistance was expressed in some but not all of the kanamycin-resistant lines and was transmitted to the seed progeny as a dominant nuclear trait.     

Effects of antibiotics in soil on the population dynamics of transposon Tn5 carrying Pseudomonas fluorescens

The effects of kanamycin and streptomycin added to soil on the survival of transposon Tn5 modified Pseudomonas fluorescens strain R2f were investigated. Kanamycin in high (180 g g^-1 dry soil) or low (18 g g^-1) concentration or streptomycin in low concentration in Ede loamy sand soil had no noticeable effect on inoculant population dynamics in soil and wheat rhizosphere, whereas streptomycin in high concentration had a consistent significant stimulatory effect, in particular in the wheat rhizosphere. Streptomycin exerted its effect by selecting P. fluorescens with Tn5 insertion whilst suppressing the unmodified sensitive parent strain, as evidenced by comparing the behaviour of these two strains in separate and mixed inoculation studies.Soil textural type influenced the effect of streptomycin on the Tn5 carrying inoculant; the effect was consistently detected in rhizosphere and rhizoplane samples of wheat grown in Ede loamy sand after 7 and 14 days incubation, whereas it was only apparent after 7 days in rhizoplane or rhizosphere (and bulk soil) samples of wheat grown in two silt loam soils. Modification of soil pH by the addition of CaCO₃ or bentonite clay resulted in an enhancement of the selective effect of streptomycin by CaCO₃ and its abolishment by bentonite clay.The addition to soil of malic acid or wheat root exudate, but not of glucose, enhanced the streptomycin selective effect on the Tn5-modified P. fluorescens strain. Neither the streptomycin producer Streptomyces griseus nor two non-inhibiting mutants obtained following UV irradiation affected the dynamics of P. fluorescens (chr::Tn5) in soil and wheat rhizosphere.The effect of streptomycin in soil on inoculant Tn5 carrying bacteria depends on conditions such as soil type, the presence of (wheat) root exudates and the type of available substrate.     

Symbiotically defective histidine auxotrophs of Bradyrhizobium japonicum

Four histidine auxotrophs of Bradyrhizobium japonicum strain USDA 122 were isolated by random transposon Tn5 mutagenesis. These mutants arose from different, single transposition events as shown by the comparison of EcoRI and XhoI-generated Tn5 flanking sequences of genomic DNA. The mutants grew on minimal medium supplemented with l-histidine or l-histidinol but failed to grow with l-histidinol phosphate. While two of the muants were symbiotically defective and did not form nodules on Glycine max cvs. Lee and Peking and on Glycine soja, the other two mutants were symbiotically competent. Reversion to prototrophy occurred at a frequency of about 10^-7 on growth medium without added antibiotics, but prototrophs could not be isolated from growth medium containing 200 g/ml kanamycin and streptomycin. The prototrophic revertants formed nodules on all the soybean cultivars examined. When histidine was supplied to the plant growth medium, both nodulation deficient mutants formed effective symbioses. On histidine unamended plants, nodules were observed infrequently. Three classes of bacterial colonies were isolated from such infrequent nodules: class 1 were kanamycin resistant-auxotrophs; class 2 were kanamycin sensitive-prototrophs; and class 3 were kanamycin-sensitive auxotrophs. Our results suggest that two Tn5 insertion mutations in B. japonicum leading to histidine auxotrophy, affect nodulation in some way. These mutations are in regions that show no homology to the Rhizobium meliloti common nodulation genes.     

Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives

Summary Two derivatives of the prokaryotic transposon Tn5 were constructed in vitro. In Tn5-233, the central area of Tn5, which carries resistance to kanamycin/neomycin, bleomycin and streptomycin, is replaced by a fragment carrying resistance to the aminocyclitol antibiotics gentamycin/kanamycin and streptomycin/spectinomycin. In Tn5-235, the Escherichia coli -galactosidase gene is inserted within the streptomycin resistance gene of Tn5, and constitutively expressed from a Tn5 promoter. Both constructs transpose with about the same frequency as Tn5 in Escherichia coli and Rhizobium meliloti. When a Tn5-derivative is introduced into an R. meliloti strain which already contains a different Tn5-derivative, in situ transposon replacement is obtained at high frequency, presumably by a pair of crossovers between the IS50 sequences at the ends of the incoming and resident transposons. In this way we converted a previously isolated recA::Tn5 mutant into the corresponding recA::Tn5-233 strain, which can now be used as a genetic background in the study of complementation of other Tn5-induced mutations. We also replaced the drug markers of several Tn5-induced exo mutants, which we were then able to map relative to each other by transduction with phage M12. In a strain carrying Tn5-235 located near Tn5-233, we were able to isolate deletions of the intervening markers, presumably resulting from general recombination between the two transposons, by screening for loss of the Lac⁺ phenotype. Unlike Tn5 itself, resident Tn5-233 does not appear to suppress transposition of another incoming Tn5-derivative.Abbreviations bp base pairs - Nm neomycin - Km kanamycin - Sm streptomycin - Sp spectinomycin - Gm gentamycin - Tc tetracycline - Tp trimethoprim - Ot oxytetracycline - Rf rifampicin - Xgal 5-bromo-4-chloro-3-indolyl--d-galactoside     

Induced mutagenesis by bleomycin in the purple sulfur bacterium Thiocapsa roseopersicina

Cell death and mutagenesis in bleomycin-treated cells of Thiocapsa roseopersicina (a purple sulfur bacterium) was studied by cultivation in a semisolid medium (agar-shake technique). This technique has also proven useful in assessing the frequency of antibiotic mutations by detecting and counting individual colonies of Thiocapsa roseopersicina. The frequencies of spontaneous mutants resistant to ampicillin, rifampicin, cloramphenicol, tetracycline, kanamycin, streptomycin, and neomycin were also studied: they ranged between 2×10^-9 and 9×10^-8. Bleomycin (4 g/ml) sharply increased the frequency of ampicillin-resistant mutants, from 10^-8 (spontaneous) to 4×10^-4 (induced), in 17 h. An inducible, error-prone mechanisms of DNA synthesis seems to be responsible for this enhancement of the mutagenic effect. This is the first report on the sensitivity to several antibiotics, and capacity of lethality and mutagenesis by bleomycin has been studied in a purple sulfur bacterium.     

Asymmetric hybridization in Nicotiana by fusion of irradiated protoplasts

Summary Mesophyll protoplasts of a kanamycin-resistant, nopaline-positive Nicotiana plumbaginifolia seed line were inactivated by -irradiation and electrically fused with unirradiated mesophyll protoplasts of N. tabacum. Hybrids were selected on kanamycin and regenerated. Genetic material from N. plumbaginifolia was detected in these plants by the following criteria: (1) morphology, (2) esterase isozyme profiles, and (3) the presence of nopaline in leaf extracts. All of the plants regenerated were morphologically more similar to N. tabacum than to N. plumbaginifolia, and many were indistinguishable from N. tabacum. It was found that 37 plants displayed one or two esterases characteristic of N. plumbaginifolia in addition to a full set of esterases from N. tabacum. Based on their esterases, we have classified these plants as somatic hybrids. However, irradiation has clearly reduced the amount of N. plumbaginifolia genetic material that they retain; 24 plants were found that had only N. tabacum esterases but that produced nopaline and were kanamycin resistant. Genomic DNA from several of these plants was probed by Southern blotting for the presence of the authentic neomycin phosphotransferase gene (kanamycin-resistance gene) — all were found to contain the gene. These plants were classified as asymmetric hybrids. Finally, 25 plants were regenerated that were kanamycin sensitive, negative for nopaline, and contained only N. tabacum esterases. All of the regenerated plants, including this final category, were male sterile. As transferring the N. plumbaginifolia cytoplasm to an N. tabacum nuclear background results in an alloplasmic form of male sterility, all of the plants regenerated in this study appear to be cybrids irrespective of their nuclear constitution. Chromosome analysis of the asymmetric hybrids showed that most of them contained one more chromosome than is normal for N. tabacum. The somatic hybrids examined all had several additional chromosomes. Although male sterile, the asymmetric hybrids were female fertile to varying degrees and were successfully backcrossed with N. tabacum. Analysis of the resultant F₁ progeny indicated that the kanamycin-resistance gene from N. plumbaginifolia is partially unstable during meiosis, as would be expected for factors inherited on an unpaired chromosome.Abbreviations Km ^r kanamycin resistant - Km ^s kamacysin sensitive - Nop ⁺ nopaline positive - Nop ^– nopaline negative     

Identification of two plasmids determining resistance to tetracycline and to erythromycin in group D streptococcus

Summary Two circular DNA plasmids designated Tet and Ero, distinguishable by their molecular weights (64.5 and 17.6×10⁶ respectively) and their guanine plus cytosine (G+C) content (36.5 and 34.5% respectively) were identified in Streptococcus faecalis strain KR, resistant to tetracycline and erythromycin. Loss of resistance to tetracycline, to erythromycin and to both antibiotics simultaneously in independently isolated cured derivatives corresponds to the loss of the Tet, Ero, and of both plasmids respectively. In the singly resistant strains we estimated that 3 and 6 copies of the Tet and Ero plasmids respectively are present per chromosomal genome equivalent.Abbreviations form I supercoiled circular DNA - form II relaxed circular DNA - form III linear double-strand DNA - Tet tetracycline - Ero erythromycin - R resistant - S sensitive