Isolation and partial characterization of four plasmids from antibiotic-resistant thermophilic bacilli.

Twenty-nine antibiotic-resistant isolates of thermophilic bacilli were examined for the presence of covalently closed circular duplex DNA molecules by agarose-gel electrophoresis and caesium chloride-ethidium bromide density gradient centrifugation. Five of the 29 strains tested contained covalently closed circular molecules. Two of the streptomycin-resistant strains contained the same two plasmids: pAB118A of molecular weight 4.9 X 10(6) (7.0 kilobases) and pAB118B of molecular weight 3.0 X 10(6) (4.3 kilobases). Two of the tetracycline-resistant strains each contained a plasmid (pAB124) of molecular weight 2.9 X 10(6) (4.14 kilobases), while a third harboured a small plasmid (pAB128) of molecular weight 2.5 X 10(6) (3.57 kilobases). These plasmids were digested with 19 different restriction endonucleases and the numbers of cleavage sites were determined. Transformation of Bacillus subtilis (168 (Trp-) with purified plasmid DNA indicated that pAB124 conferred tetracycline resistance on the host.     

Conjugal Transfer of Plasmid-Borne Multiple Antibiotic Resistance in Streptococcus faecalis var. zymogenes

A strain of Streptococcus faecalis var. zymogenes, designated JH1, had high-level resistance to the antibiotics streptomycin, kanamycin, neomycin, erythromycin, and tetracycline. These resistances were lost en bloc from approximately 0.1% of cells grown in nutrient broth at 45 C. The frequency of resistance loss was not increased by growth in the presence of the "curing" agents acriflavine or acridine orange, but after prolonged storage in nutrient agar 17% of cells became antibiotic sensitive. Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated from the parental strain and from antibiotic-sensitive segregants by using cesium chloride-ethidium bromide gradients. DNA molecular species were identified by using neutral sucrose gradients. Strain JH1 contained two covalently closed circular DNA species of molecular weights 50 x 10(6) and 38 x 10(6). An antibiotic-sensitive segregant, strain JH1-9, had lost the larger molecular species. A second sensitive segregant, strain JH1-5, had also lost the larger molecular species but a new molecular species of approximate molecular weight 6 x 10(6) was present. The antibiotic resistances that were curable from the parental strain were transferred to antibiotic-sensitive strains of S. faecalis and to strain JH1-9, during mixed incubation in nutrient broth at 37 C. Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism. Analysis of the plasmid species in recipient clones showed that all had received the plasmid of molecular weight 50 x 10(6). Strain JH1-5 was not a good recipient. Analysis of one successful recipient clone of JH1-5 revealed that it had gained the 50 x 10(6) molecular weight plasmid but lost the 6 x 10(6) molecular weight species. These data are interpreted to mean that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 10(6) molecular weight, and that in clone JH1-5 this plasmid suffered a large deletion leaving only a 6 x 10(6) remnant which was incompatible with the complete replicon.     

Virus with a Multipartite Superhelical DNA Genome from the Ichneumonid Parasitoid Campoletis sonorensis

Virus was isolated from the lumen of the calyx region of ovaries in the parasitoid wasp Campoletis sonorensis (Hymenoptera: Ichneumonidae), and the nature of the viral DNA was analyzed. DNA purified from a homogeneous band of virus contained double-stranded superhelical molecules which were polydisperse in molecular weight. At least 25 different covalently closed circles were present, ranging in molecular weight from 4.0 x 10(6) to 13.6 x 10(6). The virus DNA was analyzed with restriction enzymes, and the nature of the genetic complexity was evaluated by Southern blot hybridization of native superhelical and relaxed circular virus DNA and of SalI- and HindIII-digested DNA. The data suggest that most of the variously sized covalently closed DNAs were composed primarily of nonhomologous sequences. The different size classes of covalently closed viral DNAs did not appear to exist in equimolar concentrations. However, there was no evidence from observation of virus particles in the electron microscope or from virus fractionation experiments that a mixture of viruses was present in the calyx fluid. The results from this study suggest' that the virus isolated from C. sonorensis, like those isolated from other endoparasitic hymenoptera, may belong to a new class of DNA viruses in which the genome is multipartite, with each DNA existing as a superhelical molecule.     

Biochemical Studies of Two Bacillus pumilus Plasmids

Bacillus pumilus NRS 576 harbored an estimated two copies per chromosome of a covalently closed, circular (CCC) deoxyribonucleic acid (DNA) molecule, the 576 plasmid. The 576 plasmid has a buoyant density of 1.698 g/cm(3) and a molecular weight of about 28 x 10(6). Plasmid copy number remained about the same in both exponentially growing and stationary-phase cells. Spontaneous variants of NRS 576 that formed spores at an elevated frequency were designated as W mutants. W mutants appeared to have lost the 576 plasmid on the basis of the following: W mutants (38 tested) lacked detectable CCC DNA, and the majority of the plasmid homologous sequences in bulk NRS 576 DNA were absent from bulk W mutant DNA. B. pumilus ATCC 7065 harbored at least 10 copies per chromosome of a CCC DNA element, the 7065 plasmid. The 7065 plasmid has a buoyant density of 1.696 g/cm(3) and a molecular weight of about 6 x 10(6). Although the copy number of the plasmid appeared to remain the same in exponentially growing and stationary-phase cells, an additional CCC form of higher molecular weight was detected in stationary-phase cells.     

Isolation, by tetracycline selection, of small plasmids derived from R-factor R12 in Escherichia coli K-12.

The examination, by agarose gel electrophoresis, of tetracycline-resistant colonies of Escherichia coli K-12 carrying R-factor R12 reveals the presence of smaller plasmid deoxyribonucleic acids (DNAs), incompatible with R12, in many of the clones. These plasmids are demonstrated to be homologous with R12 DNA by electron microscope heteroduplex experiments and by the production of consistent fragment patterns upon digestion with various restriction endonucleases. These autonomously replicating plasmids form a related series of covalently closed circular DNA molecules ranging in size from 3.6 X 10(6) to 61 X 10(6) daltons. Plasmids of molecular weight between 3.6 X 10(6) and 37 X 10(6) confer no antibiotic resistances, but when jointly present with R12 by nonetheless enhance the expression of the tetracycline resistance associated with this latter molecule.     

Stability in Escherichia coli of an antibiotic resistance plasmid from Bacteroides fragilis.

A Bacteroides fragilis strain resistant to penicillin G, tetracycline, and clindamycin was screened for the presence of plasmid deoxyribonucleic acid (DNA). Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of this strain revealed two plasmid DNA bands. The molecular weights of the plasmids were estimated by their relative mobility in agarose gel and compared with standard plasmids with known molecular weights. The molecular weights were 3.40 +/- 0.20 x 10(6) and 1.95 +/- 0.05 x 10(6) for plasmids pBY1 and pBY2, respectively. Plasmid DNA purified by cesium chloride-ethidium bromide gradient centrifugation was used to transform a restriction- and modification-negative strain of Escherichia coli. Penicillin G- and tetracycline-resistant transformants were screened for the presence of plasmid DNA. A plasmid band corresponding to a molecular weight of 1.95 x 10(6) was present in all transformants tested. Curing experiments demonstrated that the plasmid, referred to as pBY22 when present in transformants, was responsible for penicillin G and tetracycline resistance. Plasmid pBY22 was mobilized and transferred to other E. coli strains by plasmid R1drd-19. Stability of pBY22 was examined in different E. coli strains and was shown to be stably maintained in both restriction-negative and restriction-positive strains. Unexpectedly, pBY2 and pBY22 were resistant to digestion by 12 different restriction endonucleases.     

Plasmid deoxyribonucleic acid in clinical isolates of Neisseria gonorrhoeae.

Two different sizes of circular covalently closed deoxyribonucleic acid plasmids have been identified in four independent clinical isolates of eisseria gonorrhoeae. All four strains contained a small plasmid with a molecular weight of 2.8 X 10-6 and two of the four stains also contained a large plasmid with a molecular weight of 24.5 X 10-6. The avirulent derivative of each of these four strains had the same plasmid complement as its virulent parent. There was no correlation between the presence of these plasmids and antibiotic resistance, piliation, and colony type associated with virulence, or ability to grow without seven specific amino acid supplements.     

Specific Labeling and Physical Characterization of R-Factor Deoxyribonucleic Acid in Escherichia coli

The molecular nature of R-factor deoxyribonucleic acid (DNA) was examined in Escherichia coli by using a method for the specific labeling of the derepressed R factor, R1, in a female cell after conjugation. Sixty minutes after mating, the R factor was isolated as a single molecule with a molecular weight of 65 x 10(6) daltons. This single molecular species sedimented as either a covalently closed molecule or a "nicked" circle. When the single R-factor component was centrifuged in a CsCl density gradient, only a single homogeneous species with a buoyant density of 1.711 g/cm(3) was observed. R-factor DNA was also isolated directly from exponentially growing cells of E. coli as a covalently closed single molecular species comprising about 1% of the total cellular DNA. Previous studies in Proteus show that R1 factor DNA components of buoyant density 1.709, 1.711, and 1.716 g/cm(3) can be identified as distinct replicons. It is suggested that the single molecule of R1 observed in E. coli is most simply explained as a composite structure resulting from a recombinational assemblage of a 1.709 and 1.716 g/cm(3) replicon.     

Plasmids Controlling Synthesis of Hemolysin in Escherichia coli: Molecular Properties

Covalently closed extrachromosomal deoxyribonucleic acid (DNA) was isolated from alpha-hemolytic wild-type strains of Escherichia coli. Most strains examined were able to transfer the hemolytic property with varying frequencies to nonhemolytic recipient strains. Out of eight naturally isolated alphahemolytic E. coli strains, four contained a set of three different supercoiled DNAs with sedimentation coefficients of 76S (plasmid A), 63S (plasmid B), and 55S (plasmid C). The sedimentation coefficients and the contour lengths of the isolated molecules correspond to molecular weights of 65 x 10(6), 41 x 10(6), and 32 x 10(6). Three alpha-hemolytic wild-type strains carried only one plasmid with a molecular weight of 41 x 10(6), and one strain harbored two plasmids with molecular weights of 41 x 10(6) and 32 x 10(6). Alpha-hemolytic transconjugants were obtained by conjugation of E. coli K-12 with the hemolytic wild-type strains. A detailed examination revealed that plasmids with the same sizes as plasmids B and C of the wild-type strains can be transferred separately or together to the recipients. Both plasmids possess the hemolytic determinant and transfer properties. Plasmid A appears to be, at least in one wild-type strain, an additional transfer factor without a hemolytic determinant. In one case a hemolytic factor was isolated, after conjugation, that is larger in size than plasmid A and appears to be a recombinant of both plasmids B and C.     

Isolation and physical studies of the intact supercoiled, the open circular and the linear forms of ColE1-plasmid DNA.

For the study of DNA conformations, conformational transitions, and DNA-protein interactions, covalently closed supercoiled ColE1-plasmid DNA has been purified from cultures of Escherichia coli harboring this plasmid and grown in the presence of chloramphenicol according to the method of D.B. Clewell [J. Bact. 110 (1972)667]. The open circular and linear forms of the plasmid were prepared by digestion of the covalently closed, supercoiled form with pancreatic deoxyribonuclease and EcoRI-restriction endonuclease, respectively. The linear form was found to be very homogeneous by electron microscopy and sedimenting boundary analysis. Its physical properties (s0 20,w=16.3 S,D0 20,W=1.98 X 10(-8) cm2 s-1 and [eta]=2605 ml g-1) have been carefully determined in 0.2 M NaCl, 0.002 M NaPO4 pH 7.0,0.002 M EDTA, at 23 degrees C. Combination of s0 20, w (obtained by quasielastic laser light scattering) gave Ms,D=4.39 x 10(6). This value is in reasonable agreement with the molecular weight from total intensity laser light scattering M=4.30 x 10(6). The covalently closed and open circular forms of the ColE1-plasmid are less homogeneous due to slight cross-contamination and the presence of small amounts of dimers in these preparations. The weight fractions of the various components as determined by boundary analysis or electron microscopy are given together with the average quantities obtained in the same solvent for the supercoiled form ((s0 20,w)w=25.4 S, (D0 20,w)z=2.89 x 10(-8) cm2 s-1, [eta]= 788 ML G-1,Ms,D=4.69 x 10(6) and Mw=4.59 x 10(6)) and the open circular form (s0 20, w)w=20.1 S, (D0 20,w)z=2.45 x 10(-8) cm2 s-1, [eta]=1421 ml g-1,Ms,D=4.37 x 10(6) and Mw=4.15 x 10(6)). Midpoint analysis of the sedimenting boundaries allows unambiguous determination of the sedimentation coefficients of these two forms: s0 20,w=24.5 S and s0 20,w=18.8 S, respectively. Also deduced from total intensity light scattering were radii of gyration Rg (103.5, 134.2 and 186 nm) and second virial coefficients A2 (0.7, 4.8 AND 5.4 x 10(-4) mole ml/g2) for the supercoiled, the open circular and linear forms, respectively. The data presented are discussed in relation to the conformational parameters for the three forms in solution.     

Physical Properties and Mechanism of Transfer of R Factors in Escherichia coli

The physical properties of F-like and I-like R factors have been compared with those of the wild-type F factor in Escherichia coli K-12 unmated cells and after transfer to recipient cells by conjugation. The F-like R factor R538-1drd was found to have a molecular weight of 49 x 10(6), whereas the molecular weight of the I-like R factor R64drd11 was 76 x 10(6). The wild-type F factor, F1, had a molecular weight of 62 x 10(6). When conjugation experiments are performed by using donor strains carrying these derepressed F-like or I-like R factors, the transferred deoxyribonucleic acid can be isolated as a covalently closed circle from the recipient cells. This circular deoxyribonucleic acid was characterized by making use of the observation that the complementary strands of these R factors can be separated in a CsCl-poly (U, G) equilibrium gradient. The results of the strand-separation experiments show that only one of the complementary strands of the R factor is transferred from the donor to the recipient. With both the F-like and I-like R factors, this strand is the heavier strand in CsCl-poly (U, G). These results indicate that even though F-like and I-like R factors differ greatly in many properties (phage specificity, size, compatability, etc.), they are transferred by a similar mechanism.     

Extrachromosomal Elements in Group N Streptococci

The deoxyribonucleic acid (DNA) of Streptococcus lactis C2, S. cremoris B(1), and S. diacetilactis 18-16 was labeled by growing cells in Trypticase soy broth containing (3)H-labeled thymine. The cells were gently lysed with lysozyme, ethylenediaminetetraacetic acid, and sodium lauryl sulfate. The chromosomal DNA was separated from plasmid DNA by precipitation with 1.0 M sodium chloride. The existence of covalently closed circular DNA in the three organisms was shown by cesium chloride-ethidium bromide equilibrium density gradient centrifugation of the cleared lysate material. In an attempt to correlate the loss of lactose metabolism with the loss of plasmid DNA, lactose-negative mutants of these organisms were examined for the presence of extrachromosomal particles. Covalently closed circular DNA was detected in the lactose-negative mutants of S. lactis C2 and S. diacetilactis 18-16. In S. cremoris B(1), however, no covalently closed circular DNA was observed by using cesium chloride-ethidium bromide gradients. Electron micrographs of the satellite band material from S. lactis C2 and its lactose-negative mutant confirmed the presence of plasmid DNA. Three distinct plasmids having approximate molecular weights of 1.3 x 10(6), 2.1 x 10(6), and 5.1 x 10(6) were observed in both organisms.     

Plasmid deoxyribonucleic acid in Rhizobium trifolii.

In deoxyribonucleic acid of Rhizobium trifolii centrifuged in cesium chloride-ethidium bromide equilibrium was found a sattelite peak containing covalently closed circular deoxyribonucleic acid. The plasmid had a molecular weight of about 64 x 10(6) shown by sedimentation in sucrose gradients and electron microscopy.     

R-Factor Mutant Capable of Specifying Hypersynthesis of Penicillinase

The physical characteristics of a mutant, R(M201-2), capable of conferring high and stable ampicillion resistance was analyzed. The R(M201-2) and its parent R-factor deoxyribonucleic acid (DNA) could be isolated as an extrachromosomal and covalently closed circular form. Their buoyant densities were both 1.712 g/cm(3), and their molecular weights were about 82 x 10(6) and 64 x 10(6), respectively, when measured by CsCl and sucrose density gradient analyses. The contour lengths by electron microscopy were 35.9 +/- 0.6 and 31.0 +/- 0.6 mum, respectively. By using the extracted R-factor DNA, the mutant and parent characters were transformable to another Escherichia coli strain. The mutant R factor showed an increased amount of DNA even after conjugal transfer to Proteus. An increase in the size of R-factor DNA was thus considered to be the cause of the high level of ampicillin resistance.     

Homology of plasmids in strains of unicellular Cyanobacteria.

Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron microscopy and agarose gel electrophoresis. The sizes of the various plasmid species were determined; in each of the Synechococcus strains 6301, 6707, and 6908 two plasmid species were found with molecular weights of 5.3 × 10⁶ and 32.7 × 10⁶. Synechococcus strain 7425 had two plasmids of molecular weight 5.4 × 10⁶ and 24 × 10⁶. Synechococcus strain 6312 and Synechocystis strain 7005 each contained one plasmid species with molecular weight of 15.9 × 10⁶ and 2.0 × 10⁶, respectively. Restriction enzyme analysis revealed identical cleavage patterns for the plasmids of identical molecular weight.     

Isolation and characterization of a plasmid involved with enterotoxin B production in Staphylococcus aureus.

Genetic analysis and molecular characterization of plasmid deoxyribonucleic acid (DNA) was performed in a toxigenic isolate of Staphylococcus aureus strain DU4916. Elimination, transduction, and transformation experiments provided us with a series of derivatives similar except for the presence or absence of genes mediating resistance to penicillin (penr), methicillin (mecr), and tetracycline (tetr) and enterotoxin type B (SEB) production (entB+). The derivatives were examined for the presence of a plasmid species which encodes for SEB production. Two distinct species of covalently closed circular DNA of about 2.8 X 10(6) and 0.75 X 10(6) daltons were identified in an ethidium bromide-cured, penicillinase-negative (pens) isolate, SN109 (mecr tetr emtB+). Further segregation of either methicillin resistance or tetracycline resistance or of both together resulted in the loss of SEB production and the disappearance of both plasmids. Transduction from strain SN109 showed that determinants for tetracycline resistance are carried by the 2.8 X 10(6) dalton plasmid. Transformation with covalently closed circular DNA from strain SN109 yielded mecs tetr entB- transformants harboring the tetracycline resistance plasmid alone and mecr tetr entB+ transformants harboring both the tetracycline resistance and the 0.75 X 10(6)-dalton plasmid. Further segregation of methicillin resistance in transformants was not associated with any change in plasmid DNA. The results indicate that a genetic determinant for SEB production is carried by the 0.75 X 10(6)-dalton plasmid. It is possible, however, that this plasmid cannot be maintained in the host independently from the tetracycline resistance plasmid. Methicillin resistance in the strains examined could not be ascribed to any of the covalently closed circular DNA components resolved in strain DU4916.     

Molecular Structure of an R Factor, Its Component Drug-Resistance Determinants and Transfer Factor

Plasmid DNA from Escherichia coli strains harboring drug resistance either of the infectious or noninfectious kind has been separated by CsCl centrifugation of crude cell lysates in the presence of ethidium bromide and examined by electron microscopy. Plasmid deoxyribonucleic acid (DNA) from an S(+) strain (which has the property of noninfectious streptomycin-sulfonamide resistance) consists of a monomolecular covalently closed circular species of 2.7 mum in contour length (5.6 x 10(6) atomic mass units; amu). DNA from a strain carrying a transfer factor, termed Delta, but no determinant for drug resistance, is a monomolecular covalently closed circular species of 29.3 mum in contour length (61 x 10(6) amu). DNA from either Delta(+)A(+) or Delta(+)S(+) strains, (which are respectively ampicillin or streptomycin-sulfonamide resistant, and can transfer this drug resistance), shows a bimodal distribution of molecules of contour lengths 2.7 mum and 29.3 mum, whereas DNA from a (Delta-T)(+) strain (showing infectious tetracycline resistance) contains only one species of molecule measuring 32.3 mum (67 x 10(6) amu). We conclude that ampicillin resistance is carried by a DNA molecule (the A determinant) of 2.7 mum, and streptomycin-sulfonamide resistance is carried by an independent molecule (the S determinant) of similar size. These molecules are not able to effect their own transfer, but can be transmitted to other cells due to the simultaneous presence of the transfer factor, Delta, which also constitutes an independent molecule, of size 29.3 mum. In general, there appears to be little recombination or integration of the A or S molecules into that of Delta, although a small proportion (5-10%) of recombinant molecules cannot be excluded. In contrast, the third drug-resistance determinant, that for tetracycline resistance (denoted as T), is integrated in the Delta molecule to form the composite structure Delta-T of size 32.3 mum, which determines infectious tetracycline resistance. The Delta(+)A(+) and Delta(+)S(+) strains are defined as harboring plasmid aggregates, and the (Delta-T)(+) strain is defined as carrying a plasmid cointegrate; the properties of all three strains are characteristic of strains harboring R factors. These results are compatible with the previously published genetic data. The number of Delta molecules per cell appears to be equal to the chromosomal number irrespective of growth phase, and this plasmid can thus be defined as stringently regulated in DNA replication. In contrast, S and A exist as multiple copies, probably in at least a 10-fold excess of chromosomal copy number. S and A can thus be defined as relaxed in the regulation of their DNA replication.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

Detection and characterization of plasmids in Pseudomonas glycinea.

Pathogenic strains of Pseudomonas glycinea were shown to possess plasmid deoxyribonucleic acid by dye-buoyant density gradient centrifugation. The size and number of plasmids of four different isolates were determined by neutral sucrose gradient centrifugation. Two isolates were found to harbor a single plasmid; however, they differed in size, having molecular weights of 43 X 10(6) and 54 X 10(6). Two other isolates each contained two different plasmids. Plasmids with molecular weights of 43 X 10(6) and 73 X 10(6) were observed in one isolate, and the other carried plasmids with molecular weights of 25 X 10(6) and 87 X 10(6). An auxotrophic mutant derived from the latter strain was found to contain plasmids of identical size. The plasmids were found to be under stringent control of replication, having plasmid copies of 1.0 to 2.7 per chromosome equivalent. By the dye-cesium chloride technique, the mutant showed twice as much covalently closed circular deoxyribonucleic acid as did the parental strain.