Isolation of plasmid deoxyribonucleic acid from two strains of Bacteroides.

Two clinical isolates of Bacteroides contained covalently closed circular deoxyribonucleic acid (DNA) as shown by sedimentation in an alkaline sucrose gradient, CsCl ethidium bromide equilibrium centrifugation, and electron microscopy. Bacteriodes fragilis N1175 contained a homogeneous species of plasmid DNA with a molecular weight of 25 x 10(6). Bacteroides ochraceus 2228 contained two distinct, covalently closed circular DNA elements. The larger cosedimented with the covalently closed circular DNA form of the R plasmid, R100, corresponding to a molecular weight of 70 x 10(6); the smaller sedimented as a 58S molecule with a calculated molecular weight of 25 x 10(6). The roles of these plasmids are unknown. Neither strain transferred antibiotic resistance to plasmid-negative Bacteroides or Escherichia coli, and neither produced bacteriocins active against other Bacteroides or sensitive indicator strains of E. coli.     

Biochemical characterization of nonintegrated plasmid-folded chromosome complexes: sex factor F and the Escherichia coli nucleoid.

The existence of nonintegrated plasmid-chromosome complexes has been deduced in previous work from the cosedimentation of covalently closed, circular plasmids with host folded chromosomes. In the present work, it is shown that about 70 to 90% of the covalently closed, circular F deoxyribonucleic acid could be released in vitro from chromosome complexes by ribonuclease treatment but not by protease, Sarkosyl, or ethidium bromide. Consistent with the in vitro studies, Escherichia coli cells treated for 5 min with rifampin, an inhibitor of ribonucleic acid initiation, released upon lysis 90% of their plasmid deoxyribonucleic acid as freely sedimenting molecules.     

Effect of protein synthesis on plasmid maintenance in Streptococcus faecalis.

Plasmid-to-chromosome ratios in Enterobacteriaceae, upon interruption of protein synthesis by chloramphenicol, are either conserved or increased when measured by dye buoyant density centrifugation. We have found, on the other hand, that the effect of inhibition of protein synthesis on the amount of covalently closed circular deoxyribonucleic acid visualized by this method in two strains of Streptococcus faecalis appears to differ from these established systems. A three- to sixfold decrease in covalently closed circular deoxyribonucleic acid was observed when lysates of chloramphenicol-treated cultures were submitted to dye buoyant density centrifugation. A loss of covalently closed circular deoxyribonucleic acid was also evident from electrophoretic profiles of these lysates. Several conditions which could account for the apparent loss of covalently closed circular deoxyribonucleic acid upon inhibition of protein synthesis are discussed.     

Isolation and characterization of four plasmids from Bacillus subtilis.

Nineteen Bacillus subtilis isolates obtained from type culture collections were examined for the presence of covalently closed circular duplex deoxyribonucleic acid molecules by the technique of cesium chloride-ethidium bromide density gradient centrifugation. Four of the 19 strains tested carried covalently closed circular molecules. Two of these strains (IFO3022, IFO3215) harbored a similar plasmid with a molecular weight of 5.4 X 10(6). The other two strains (IAM1232, IAM1261) carried 4.9 C 10(6)-and 5.3 X 10(6)-dalton plasmids, respectively. These plasmid-harboring strains did not show phenotypic traits such as antibiotic resistance orbacteriocin production. The plasmid deoxyribonucleic acids were digested by three restriction endonucleases, EcoRI, HindIII, and BamNI, and were classified into three different types from their electrophoretic patterns in agarose gels.     

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R100.1: generation of small plasmids after integration.

We have observed that integration of the R plasmid R100.1 into the chromosome of Escherichia coli is associated with the formation of small, covalently closed circular elements. Contour length measurements, partial denaturation mapping, and analysis of the deoxyribonucleic acid fragments produced by digestion of one of these, pLC1, with the restriction endonuclease EcoRI indicate that it is the r-determinant element of R100.1.     

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.     

Characterization of plasmids in halobacteria.

Extrachromosomal, covalently closed circular deoxyribonucleic acid has been isolated from different species of halobacteria. Three strains of Halobacterium halobium and one of Halobacterium cutirubrum, all of which synthesize purple membrane (Pum+) and bacterioruberin (Rub+), contain plasmids of different size which share extensive sequence homologies. One strain of Halobacterium salinarium, another one of Halobacterium capanicum, and two new Halobacterium isolates from Tunisia, which are also Pum+ Rub+, do not harbor covalently closed circular deoxyribonucleic acid but contain sequences, presumably integrated into the chromosome, which are similar if not identical to those of pHH1, i.e., the plasmid originally isolated from H. halobium. Three other halophilic strains, Halobacterium trapanicum, Halobacterium volcanii, and a new isolate from Israel, do not carry pHH1-like sequences. These strains are, by morphological and physiological criteria, different from the others examined and harbor plasmids unrelated to pHH1.     

Partial characterization of nucleoids and nucleoid-plasmid complexes from Salmonella typhimurium.

Nucleoids from Salmonella typhimurium strain LT2 consist of supercoiled deoxyribonucleic acid structures that are ribonuclease labile sedimenting at 1,700S. More than 90% of the covalently closed circular deoxyribonucleic acid of a cryptic plasmid harbored by this strain cosediments with the host's 1,700S nucleoids.     

Isolation of Covalently Closed Circular Deoxyribonucleic Acid from Bacteria Which Produce Exocellular Nuclease

Reproducible yields of covalently closed circular (plasmid) deoxyribonucleic acid were obtained from mutants defective for extracellular nuclease but not from the corresponding wild-type strain of Serratia marcescens     

Characterization of a plasmid from Streptomyces coelicolor A3(2).

Covalently closed circular deoxyribonucleic acid (DNA) with a molecular weight of 20 X 10(6) was identified in strains of Streptomyces coelicolor A3(2) of various fertility types. Hybridization studies and digestion by various restriction endonucleases indicated that the circular DNAs (pSH1) were identical regardless of the fertility type (UF, IF, or NF) of the strain from which it was isolated. The pSH1 DNA was cleaved to many fragments by the endonucleases HincII, SmaI, and SalI and to three or four fragments by BamHI and PstI. Plasmid pSH1 carries single sites for each of the two restriction enzymes, EcoRI and HindIII. These sites are 7.6 X 10(6) daltons apart. Attempts to isolate the fertility factor SCP1 as covalently closed circular DNA were unsuccessful. These data suggest that the biochemically isolated plasmid pSH1 is not identical to the genetically characterized fertility factor SCP1, which has been identified in an autonomous state in IF-type strains and in an integrated state in NF-type strains.     

Isolation of extrachromosomal deoxyribonucleic acid for exfoliative toxin production from phage group II Staphylococcus aureus.

The ability of phage group II staphylococcal strain UT 0101 to produce exfoliative toxin and bacteriocin could be eliminated at a high frequency after growth at high temperatures or in the presence of ethidium bromide or sodium dodecyl sulfate. Extrachromosomal deoxyribonucleic acid, associated with the genes for exfoliative toxin and bacteriocin production, was isolated from strain UT 0101 but was absent from an ethidium bromide-cured substrain. The molecular weight of the exfoliative toxin plasmid, determined by co-sedimentation with the penicillinase plasmid, PI258, was 3.3 times 10-7. The 56S covalently closed circular form of the exfoliative toxin plasmid converted to a 38S open circular form after storage or exposure to sodium dodecyl sulfate. Plasmid deoxyribonucleic acid associated with penicillin resistance could not be identified in the penicillin-resistance Tox+ strains, UT 0007 and UT 0001.     

Properties of an R Factor Which Originated in Pseudomonas aeruginosa 1822

RP1, a group of genes specifying resistance to carbenicillin, neomycin, kanamycin, and tetracycline and originating in a strain of Pseudomonas aeruginosa, was freely transmissible between strains of P. aeruginosa, Escherichia coli, and Proteus mirabilis. Acquisition of the multiple drug resistance specified by RP1 by these strains was accompanied by acquisition of an extrachromosomal satellite of covalently closed circular deoxyribonucleic acid of molecular weight about 40 million daltons and of buoyant density 1.719 g/cm(3) (60% guanine plus cytosine).     

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.     

Plasmid in Bacillus pumilus and the Enhanced Sporulation of Plasmid-Negative Variants

Approximately 3% of the deoxyribonucleic acid (DNA) of Bacillus pumilus NRS576 can be isolated as covalently closed, circular duplex molecules of homogeneous size. The mol wt of the 576 plasmid is approximately 30 million. NRS576 (plasmid(+)) is oligosporogenic; less than 1% of the cells form spores during incubation in liquid AK sporulation medium. Variants that form spores at a high frequency (greater than 50% spores in 24 h) occur spontaneously at a frequency of approximately 10(-5). More than 25 such variants have been examined and all lack detectable plasmid DNA. A relationship appears to exist between the oligosporogenic properties of NRS576 and the presence of the 576 plasmid.     

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.     

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.     

Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation.

A method that gives high recovery of deoxyribonucleic acid (DNA) from crude bacterial lysates using ethidium bromide-cesium chloride density gradient centrifugation is presented. After Pronase digestion and shearing of the lysate, essentially 100% recovery of chromosomal DNA and a reproducible recovery of covalently closed circular (CCC) plasmid DNA is obtained for a specific plasmid in a given strain. This method should be useful for comparing the CCC plasmid/chromosome ratio of various plasmid-host combinations.     

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.     

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.     

A mutant defective in partitioning of composite plasmid Rms201.

Escherichia coli harboring mutant plasmids defective in maintenance stability (from the conjugative plasmid Rms201) showed a wide distribution of ampicillin resistance levels, as well as increased frequency of plasmid loss from the cell. The amounts of covalently closed circular deoxyribonucleic acid of mutant plasmid Rms268 and parental plasmid Rms201 per chromosome were 5.3 and 6.1%, respectively. The beta-lactamase activities of strains W3630(Rms268) and W3630(Rms201) were 0.56 and 0.44 U/mg of protein, respectively. Frequency of plasmid loss from W3630(Rms268) was about 0.8 to 1.2% per cell generation, 100 times more than that of the wild-type strain. Ampicillin resistance levels of the colonies harboring the mutant plasmid showed a wide distribution, from low (100 micrograms/ml) to high (1,600 micrograms/ml). A miniplasmid (pMS268) with a mass of 7 X 10(6) daltons and encoding ampicillin resistance was isolated from Rms268. Frequency of pMS268 loss from W3630(pMS268) was about 0.8 to 1.9% per cell generation. W3630(pMS268) also showed a wide range of distribution in the levels of ampicillin resistance. These results indicated that the copies of Rms268 in E. coli did not segregate evenly between daughter cells at cell division and that the gene involved was located on the miniplasmid.