Extrachromosomal Deoxyribonucleic Acid Research Articles

Conjugative transfer of multiple-antibiotic resistance markers in beta-hemolytic group A, B, F, and G streptococci in the absence of extrachromosomal deoxyribonucleic acid

Nine clinical isolates of group A, B, F, and G streptococci resistant to tetracycline, macrolides, lincosamides, and streptogramin B (MLS resistance) and to chloramphenicol were investigated for the conjugative transfer of the antibiotic-resistance markers into streptococcal recipients (groups B and D). The wild donors transferred the resistance markers en bloc, at a low frequency (10 −6 to 10 −8) and only into one of the two recipients tested. In addition, one of the strains transferred only the MLS resistance at a high frequency (10 −3). All attempts to detect extrachromosomal DNA in wild donors or in transconjugants were unsuccessful, except in one transconjugant. This plasmid DNA, designated pIP659, had a molecular weight of 17.5 × 10 6 and a restriction fingerprint similar to other plasmids determining MLS resistance.

Isolation of plasmids in Legionella pneumophila and Legionella-like organisms.

Agarose gel electrophoresis was employed to screen nine strains of Legionella-like bacteria and one strain of Legionella pneumophila for the presence of extrachromosomal deoxyribonucleic acid. Cryptic plasmids with molecular weights ranging from 46.6 x 10(6) to 59.8 x 10(6) were found in three of the isolates examined.

Incidence of Plasmids in Marine Vibrio spp. Isolated from an Oil Field in the Northwestern Gulf of Mexico

Presumptive marine Vibrio spp. were collected from an operational oil field and control site located in the northwestern Gulf of Mexico. Of 440 isolates analyzed for the presence of extrachromosomal deoxyribonucleic acid elements or plasmids by using the cleared lysate and agarose gel techniques, 31% showed distinct plasmid bands on agarose gels. A majority of the plasmids detected were estimated to have molecular masses of 10 x 10 or less. Multiple plasmids were observed in approximately half of the plasmid-containing strains. A number of isolates contained plasmids with similar banding and mobility patterns. The oil field area had noticeably more plasmid-containing strains (35 versus 23% in the control site) and a greater number of plasmids per plasmid-containing strain (an average of 2.5 plasmids, versus 1.5 in the control site). Oil field discharges might have resulted in increased plasmid incidence and diversity.

Conjugative transfer of multiple antibiotic resistance markers in Streptococcus pneumoniae.

Two antibiotic-resistant isolates of Streptococcus pneumoniae were investigated for conjugative transfer of their drug resistance markers into streptococcal (groups B and D) and pneumococcal (encapsulated and non-encapsulataed) recipients. Of these, 7 wild-type donor pneumococci transferred all their resistance markers (except Pc [penicillin], Su [sulfonamide], and Tp [trimethoprim]) into group D Streptococcus and non-encapsulated S. pneumoniae recipients at a low frequency (10(-5) to 10(-6)). The resistance markers transferred were Tc (tetracycline); Tc and Cm (chloramphenicol); Tc and MLS (macrolides, lincosamides, and streptogramin B); Tc, MLS, Km (kanamycin), and Cm. The transconjugants obtained retransferred their resistance markers into appropriate streptococcal or pneumococcal recipients or both. The resistance markers of streptococcal transconjugants could not be cured by chemical agents. All attempts to detect extra-chromosomal deoxyribonucleic acid from pneumococcal or streptococcal transconjugants were unsuccessful. The molecular weight of a streptococcal conjugative R plasmid (pIP501) was investigated after transfer into the non-encapsulated S. pneumoniae recipient and was found to be similar to that of the wild-type group B Streptococcus host (20 x 10(6)).

Antibiotic susceptibility of beta-lactamase-producing strains of Branhamella (Neisseria) catarrhalis.

All 11 clinically significant isolates of Branhamella catarrhalis examined in this study were found to produce beta-lactamase. The enzyme was apparently not plasmid associated since extrachromosomal deoxyribonucleic acid was not detected in any of the strains. The beta-lactamase activity of all strains was significantly depressed by the beta-lactamase inhibitors clavulanic acid and CP 45899. Based on comparisons of relative susceptibility to various beta-lactam antibiotics, it was inferred that the beta-lactamase of B. catarrhalis was significantly more active against penicillin congeners than against cephalosporin congeners. Most strains were not inhibited by readily achievable serum concentrations of the pencillinase-sensitive penicillins, penicillin G, ampicillin, and amoxicillin. Methicillin was equally ineffective. With rare exceptions, most strains of B. catarrhalis were inhibited by achievable serum concentrations of seven cephalosporins (cephalothin, cephapirin, cephaloridine, cephalexin, cephamandole, cefaclor, and cefuroxin) and one cephamycin (cefoxitin). All strains were uniformly resistant to clindamycin but were inhibited by achievable serum concentrations of erythromycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole. Comparison of geometric mean minimum inhibitory concentrations of all antimicrobial agents tested suggested that B. catarrhalis was most susceptible to cefoxitin, erythromycin, and tetracycline.

Excretion of glutamic acid in Citrobacter intermedius C3 associated with plasmid deoxyribonucleic acid

Several mutants of Citrobacter intermedius C3 lacking both the ability to synthesize proline and the ability to excrete glutamic acid were isolated by treatment with nitrosoguanidine. No revertants for either characteristic were obtained from these mutants. The ability to excrete glutamic acid was transferred to those mutants with very high frequencies in mating experience by using auxotropic excreting strains as donors. Moreover, the ability to synthesize proline was transferred together with the ability to excrete glutamic acid when an excreting strain was used as donor. The transconjugants showed a rapid spontaneous curing of both genetic markers. It was shown by two different methods that a band of covalently closed circular deoxyribonucleic acid is present in the cesium chloride gradients corresponding to the wild type and excretor mutants. Nonexcretor mutants described herein lacked such a band. Pro + transformants that were also excretors were obtained with plasmid deoxyribonucleic acid isolated either from wild type or from an excretor mutant. These data strongly indicate that glutamic acid excretion in C. intermedius C3 is related to the presence of extrachromosomal deoxyribonucleic acid.

Extrachromosomal DNA in Bacillus thuringiensis var. kurstaki, var. finitimus, var. sotto, and in Bacillus popilliae

Four entomopathogenic bacteria contained extrachromosomal deoxyribonucleic acid (DNA) molecules of various sizes. Bacillus thuringiensis var. kurstaki contained twelve elements banding on agarose gels that ranged from 0.74 to > 50 × 10 6 daltons, three of which were giant extrachromosomal DNA elements. B. thuringiensis var. sotto contained one giant extrachromosomal DNA element with a molecular size of about 23.5 × 10 6 daltons and two lesser elements of 0.80 and 0.62 × 10 6 daltons. B. thuringiensis var. finitimus harbored two giant DNA elements corresponding to >50 × 10 6 daltons and two lesser bands with relative small size (0.98 and 0.97 × 10 6 daltons). B. popilliae contained no giant extrachromosomal DNA elements but did contain two smaller elements corresponding to 4.45 and 0.58 × 10 6 daltons. The possible use of extrachromosomal DNA elements that prove to be autonomous replicons for recombinant DNA studies is discussed.

Chromosomal and extrachromosomal deoxyribonucleic acid from four bacterial endosymbionts derived from stock 51 of Paramecium tetraurelia

Four variant lines of stock 51 kappa (Paramecium tetraurelia) were screened for the presence of covalently closed circular (CCC) deoxyribonucleic acid (DNA). Stock 51m43 kappa, a nonkiller resistant to 51 killing, contained four classes of CCC DNA: 2.9 X 10(7), 9.7 X 10(7), and 11.8 X 10(7) daltons. The buoyant densities of 51m43 kappa chromosomal and CCC DNA were 1.700 and 1.698 g/cm3, respectively. Stock 51m43 pi, a sensitive nonkiller, contained two CCC species: 0.3 X 10(7) and 4.4 X 10(7) daltons. The buoyant densities of both the chromosomal and CCC DNA were 1.694 to 1.695 g/cm3. Three sizes of CCC DNA were found in 51m1 pi: 0.3 X 10(7), 2.3 X 10(7), and 4.5 X 10(7) daltons. The buoyant densities of both the chromosoaml DNA and the CC DNA were 1.694 to 1.695 g/cm3. It is not known whether 51m1 kappa, a sensitive spinner killer, contains CCC DNA. The buoyant density of its chromosomal DNA was 1.703 g/cm3. Of the four variant lines, only 51m43 kappa appears to be a mutant of 51 kappa. The chromosomal and CCC DNAs of 51m43 kappa have the same buoyant densities as those of 51 kappa; in addition 51m43 kappa contain a CCC molecule the same size as that found in 51 kappa (2.8 x 10(7) daltons). The three other lines are probably bacterial species that are distinct from 51 kappa and which, at one time, were co-inhabitants with 51 kappa in stock 51 paramecia.

Extrachromosomal elements in a variety of strains representing the Bacteroides fragilis group of organisms.

Previous nucleic acid association studies have identified at least nine deoxyribonucleic acid (DNA) homology classes of the Bacteroides fragilis group of organisms. Using these classes as a taxonomic framework, we have screened representative strains of the B. fragilis group for the presence of extrachromosomal (plasmid) DNA. [3H]thymidine-labeled cell lysates were subjected to sodium dodecyl sulfate-salt precipitation, and supernatant fractions from such preparations were analyzed using cesium chloride-ethidium bromide equilibrium centrifugation. One strain from each group was examined in this fashion. Five of the strains were judged to contain no detectable plasmid DNA; however, four strains were observed to yield satellite bands corresponding to covalently closed circular plasmid DNA. Plasmid DNA from such gradients was subjected to velocity sedimentation through both neutral and alkaline sucrose gradients to determine molecular size. A 23 X 10(6)-molecular-weight plasmid was found in a B. fragilis strain representing one DNA homology group of this species, whereas a 3 X 10(6)-molecular-weight plasmid was found in a B. fragilis strain representing a second homology group. Similarly, a 31 X 10(6)-molecular-weight plasmid was found in a Bacteroides thetaiotaomicron strain representing one DNA homology group of this species, whereas a 3 X 10(6)-molecular-weight plasmid was found in a B. thetaiotaomicron strain representing a second homology group. In all instances, the small-molecular weight plasmids were present to the extent of about 15 copies per chromosomal equivalent, whereas the large plasmids were present to the extent of approximately 1 copy per chromosomal equivalent. The biological function of these plasmids is unknown.

Minocycline resistance in Staphylococcus aureus: effect on phage susceptibility.

Tetracycline-resistant strains of Staphylococcus aureus are minocycline sensitive, with the exception of strains susceptible to phages of the 83A/84/85 complex and some methicillin-resistant strains of other phage types. Strains of the 83A/84/85 complex yield mutants with increased minocycline resistance. Transduction of minocycline resistance into the susceptible strain RN 450 was obtained with donor strains possessing either markers for both extrachromosomal tetracycline resistance (tet) and chromosomal tetracycline + minocycline resistance (tmn R), or only for chromosomal tmn R resistance. The chromosomal marker was differentiated from the extrachromosomal marker by the lack of detectable extrachromosomal deoxyribonucleic acid after transfer into strain RN 450, transfer into a rec(+) strain, lack of transfer into rec(-) acceptor strain, and cotransduction with chromosomal determinants for guanine biosynthesis. Both chromosomal and extrachromosomal tetracycline resistance can be induced by tetracycline. Induction by tetracycline of chromosomal tetracycline resistance resulted in simultaneous induction of minocycline resistance. The mutation toward increased minocycline resistance (tmn --> tmn R) is a regulatory mutation toward constitutivity or semiconstitutivity. Constitutive resistance is dominant in tmn R/tet diploids. Transfer of the tet marker does not affect the phage susceptibility of the acceptor strain. The tmn R marker, originating from donor strains of the 83A/84/85 complex, renders strain RN 450 resistant to several typing phages, with the exception of phages of the 83A/84/85 complex. This could possibly account for the phage typing patterns of minocycline-resistant staphylococci.

Extrachromosomal deoxyribonucleic acid in wild-type and photosynthetically incompetent strains of Rhodopseudomonas spheroides.

Three covalently closed circular species of extrachromosomal deoxyribonucleic acid have been identified by electron microscopic analysis in strains of Rhodopseudomonas spheroides. The weights of these plasmids, as determined from contour length, are about 75 X 10(6), 66 X 10(6), and 28 X 10(6) daltons for both aerobically grown and photosynthetically grown R. spheroides strain 2.4.1 (NRS) and for the photosynthetically incompetent strain V-2 (obtained by N-methyl-N-nitro-N'nitrosoguanidine mutagenesis) and 74 X 10(6), 66 X 10(6) and 34 X 10(6) daltons for a second photosynthetically incompetent strain, SLS I (obtained by incubating strain 2.4.1 [NRS] in medium containing sodium lauryl sulfate). Buoyant densities uere found to be 1.717 g/cm3 (58% guanine plus cytosine) for the plasmids of 66 X 10(6), 28 X 10(6), and 34 X 10(6) daltons in weight and 1.724 g/cm3 (65% guanine plus cytosine) for those weighing about 75 X 10(6) daltons. Possible functions of these plasmids are discussed.

Extrachromosomal deoxyribonucleic acid in R factor-harboring Enterobacteriaceae.

Extrachromosomal deoxyribonucleic acid (DNA) from 24 different R factor-harboring Enterobacteriaceae was isolated and characterized by analytical ultracentrifugation and electron microscopy. The R factors represented 15 different patterns of transferable drug resistance found in enterobacteria from an enclosed geographic area. All of the strains contained extrachromosomal, circular DNA molecules within the range of 0.4 to 52 mum. More than one size class of circular DNA molecules was observed in the majority of the extrachromosomal DNA preparations. The buoyant density of the extrachromosomal DNA ranged from 1.700 to 1.720 g/cm3. The majority of the bacteria contained extrachromosomal DNAs of various densities. Three-fourths of the R factors were classified as fi+. The investigation illustrates the extensive variability in the physical characteristics of plasmid DNA from R factor-harboring strains.

Control of differentiation in streptomycetes: involvement of extrachromosomal deoxyribonucleic acid and glucose repression in aerial mycelia development.

When Streptomyces alboniger spores were grown in Hickey-Tresner broth containing 5 muM ethidium bromide, a high frequency of permanently cured aerial mycelia-negative (am-) colonies was recovered. The appearance an am- colonies was time dependent: a very low frequency (0.3%) at zero time, a maximum (9 to 21%) after 2 to 5 days of growth, and a decline again to low frequencies later in the growth cycle. On agar, cured am- colonies of S. alboniger still produced puromycin. The development of aerial mycelia in S. alboniger, S. scabies, and S. coelicolor was also sensitive to glucose repression. Colonies grown on Hickey-Tresner agar containing 2% glucose remained phenotypically am- throughout the observation period. Adenine (2.5 mM or greater), and to a lesser extent adenosine and guanosine, specifically reversed the repression. The accumulation of undissociated organic acids appears to be involved in glucose repression of aerial mycelia formation. However, this does not appear to be the case with puromycin production in S. alboniger; glucose repression was observed over the pH range 5.0 to 7.5.

Mechanisms of ampicillin resistance in Haemophilus influenzae type B.

The genetic mechanisms associated with ampicillin resistance in strains of Haemophilus influenzae type b were investigated. In experiments concerned with transfer of total deoxyribonucleic acid in vitro, expression of resistance by wild-type strains occurred at a frequency of about 10%. The minimum inhibitory concentration of ampicillin for the transformed strains was similar to that of the resistant donor strains, and resistance in transformants was associated with acquisition of the ability to produce beta-lactamase. Exposure to 39 mug of acridine per ml for 18 h cured resistance at a frequency of 80%, and there was spontaneous loss of resistance after repeated subculture of some strains. Analysis by cesium chloride-ethidium bromide density gradient centrifugation demonstrated the presence of extrachromosomal deoxyribonucleic acid in the resistant strains, providing further evidence that the resistance factor is plasmid mediated.

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.

Isolation and characterization of mutants of colicin plasmids E1 and E2 after Mu bacteriophage infection.

Cells colicinogenic for the colicin plasmids E1 or E2 (Col E1 and Col E2, respectively) were selected for a loss of colicin production after infection with bacteriophage Mu. Extrachromosomal deoxyribonucleic acid that was larger than the original colicin plasmids was found in such cells. A small insertion mutant in Col E1 deoxyribonucleic acid affecting active colicin production without affecting either expression of colicin immunity or Col E1 deoxyribonucleic acid replication was found. Cells carrying this Col E1 plasmid mutant do not exhibit the lethal event associated with colicin E1 induction, suggesting that synthesis of active colicin is required for killing during induction. The altered Col E2 plasmid, containing an insertion at least as large as phage Mu, was maintained unstably in the mutants examined.

Evidence for a composite state of an F'his, gnd element and a cryptic plasmid in a derivative of Salmonella typhimurium LT2.

A method designed to select mutants constitutive for expression of the histidine operon has been applied to a Salmonella typhimurium LT2 strain containing an F'his,gnd element and a cryptic plasmid. One of the mutants isolated, strain AA0019, has not only increased levels of histidinol phosphate phosphatase (hisB), but also increased levels of gluconate-6-phosphate dehydrogenase (gnd). Ultracentrifugation studies of extrachromosomal deoxyribonucleic acid (DNA) isolated from strain AA0019 revealed the presence of a single species of covalently closed circular (CCC) DNA that sedimented more rapidly through neutral and alkaline sucrose gradients than any of its possible plasmid precursors. From neutral sucrose gradients, sedimentation coefficients of 130, 100, and 86S were derived, corresponding to the CCC DNA of the large plasmid in strain AA0019, the F'his,gnd element and the cryptic LT2 plasmid, respectively. An Escherichia coli plasmid-free strain that upon mating had received the large 130S plasmid also contained 86S and 100S CCC DNA components. A histidine-requiring derivative of strain AA0019 obtained after acridine orange treatment retained the cryptic plasmid DNA. Apparently, the large plasmid in strain AA0019 consists of the F'his,gnd element and the cryptic LT2 plasmid of the parental strain.

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.

Extrachromosomal deoxyribonucleic acid in different enterobacteria.

Eighty-seven different enterobacteria and pseudomonas strains were examined for the presence of extrachromosomal deoxyribonucleic acid (DNA). Thirty-four strains contained closed circular DNA by the ethidium bromide CsCl density technique. Extrachromosomal DNA was most frequent in Escherichia and Klebsiella strains. The extrachromosomal DNA was isolated and characterized by analytical ultracentrifugation and electron microscopy. All the extrachromosomal DNA-containing bacteria contained circular DNA molecules of small size (0.5-4 mum). Most of these bacteria also contained larger circles (20-40 mum). The number of different size classes of circular DNA in each strain varied from one to five. The buoyant density of the extrachromosomal DNA ranged from 1.692 to 1.721 g/cm(3). Many bacteria contained extrachromosomal DNA of more than one density.

Isolation of minicircular deoxyribonucleic acids from wild strains of Escherichia coli and their relationship to other bacterial plasmids.

Supercoiled minicircular deoxyribonucleic acid (DNA) molecules with molecular weights of 1.8 x 10(6) and 2.3 x 10(6) have been isolated from two wild strains of Escherichia coli. DNA-DNA hybridization experiments indicate that these DNA molecules share extended homologies with the minicircular DNA of E. coli 15. The DNA of the colicinogenic factor E1 (ColE1) also hybridizes to a large extent with minicircular DNA of E. coli 15. In contrast, no hybridization could be detected with various large extrachromosomal DNA elements such as the colicinogenic factor V (ColV), the beta-hemolytic factor (Hly), or the P1-like DNA of E. coli 15. Two different insertion DNA species of E. coli integrated into lambdadg-DNA (lambdadg UP(in) 128, lambdadg UP(in) 308) do not show any annealing with minicircular DNA of E. coli 15.