Inhibition of initiation of mini-F plasmid replication in temperature-sensitive mafA mutants of Escherichia coli K-12

When temperature-sensitive mafA mutants of Escherichia coli K-12 carrying mini-F plasmid (pSC138) are transferred from 30 to 42 °C, plasmid DNA replication as determined by incorporation of [³H]thymidine into covalently closed circular (CCC) mini-F DNA or by DNA-DNA hybridization is inhibited markedly within 10 min. The results of extensive pulse-chase experiments suggest that the initiation rather than the chain elongation step of plasmid replication is affected under these conditions. The replication inhibition in the mutant is accompanied by appearance of a class of plasmid DNA with a buoyant density higher than that of CCC DNA observed in the wild type, and is followed by gradual inhibition of host cell growth. The inhibition of plasmid replication is reversible at least for 60 min under the conditions used, and the recovery at low temperature (30 °C) depends on the synthesis of untranslated RNA. These results taken together with other evidence suggest that the mafA mutations primarily affect the initial step(s) of F DNA replication, presumably at or before the synthesis of untranslated RNA.     

Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation.

Summary Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has shown that (a) competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T4 ligase restores transforming activity to both nicked and linearized DNA; (e) CCC relaxed DNA is fully active in transformation; (f) the DNA concentration-dependence of plasmid transformation is first order; and (g) plasmid transformation proceeds with a low efficiency, requiring the uptake of 10³ to 10⁴ DNA molecules per transformant.Based on this information, a model for the processing of chromosomal, plasmid and transfecting DNA is proposed.Abbreviations Cm Chloramphenicol - Em erythromycin - Km kanamycin - Sm streptomycin - CCC covalently closed circular - TBAB tryptose blood agar base - NCE nicking and closing enzyme In partial fulfillment of the requirements for the doctoral degree in the Department of Microbiology at the New York University School of Medicine, for S.C.     

Simple procedure for distinguishing CCC, OC, and L forms of plasmid DNA by agarose gel electrophoresis

A simple and rapid method discriminating between covalently closed circular (CCC), open circular (OC), and linear (L) forms of plasmid DNA is presented. Two consecutive steps of agarose gel electrophoresis with a single DNA sample are used; use is made between the steps of ultraviolet light irradiation which introduces single-strand nicks in ethidium bromide-stained DNA, converting CCC into OC forms. Unambiguous assignments of OC and CCC forms are possible in samples containing several plasmids.     

Transformation of group F streptococci by plasmid DNA.

When the Challis strain of Streptococcus sanguis was transformed by the 17 megadalton beta plasmid from Streptococcus faecalis strain DS5, the plasmid underwent a 1.5 megadalton deletion (LeBlanc & Hassell, 1976). Furthermore, the covalently closed circular (CCC) plasmid DNA isolated from Challis transformants was rapidly converted to a linear form which did not possess any detectable transforming activity. To obtain stable CCC plasmid DNA a competent culture of a Lancefield group F streptococcus, strain DL8 (ATCC 12393), was used as a recipient of beta plasmid DNA. The plasmid DNA isolated from group F transformants exhibited the same configuration and size characteristics as the DS5 beta plasmid, and the CCC configuration was stable upon storage. CCC plasmid DNA from a group F transformant was biologically active and, when added to competent cultures of strain DL8, transformed them at frequencies about 100-fold greater than did beta plasmid DNA from DS5. This suggests the existence of a restriction--modification system in strain DL8.     

Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: physiochemical approach to studying the unit of segregation.

Physiocochemical evidence presented indicates plasmid deoxyribonucleic acid (DNA) can associate with host chromosome without linear insertion of the former into the latter. This conclusion is based on the observation that covalently closed circular (CCC) plasmid DNA can cosediment with undegraded host chromosome in a neutral sucrose gradient. When F plus bacteria are lysed under conditions that preserve chromosome, approximately 90% of CCC F sex factor plasmid (about 1% of the total DNA) is found in folded chromosomes sedimenting at rates between 1,500 and 4,000s. The remaining 10% of the CCC F DNA sediments at the rate (80S) indicative of the free CCC plasmid form. Reconstruction experiments in which 80S, CCC F DNA is added to F plus or F minus bacteria before cell lysis show that exogenous F DNA does not associate with folded chromosomes. In F plus bacteria, F plasmid is harbored at a level of one or two copies per chromosomal equivalent. In bacteria producing colicin E1, the genetic determinant of this colicin, the Col E1 plasmid, is harbored at levels of 10 to 13 copies per chromosomal equivalent; yet, greater than 90% of these plasmids do not cosediment with the 1,800S species of folded chromosome. However, preliminary evidence suggests one or two Col E1 plasmids may associate with the 1,800S folded chromosome. Based on evidence presented in this and other papers, we postulate F plasmid can link to folded chromosome because the physicochemical structure of the plasmid resembles a supercoiled region of the chromosome and, therefore, is able to interact with the ribonucleic acid that stabilizes the folded chromosome structure. Implications of this model for F plasmid replication and segregation are discussed.     

A rapid preparation of plasmid DNA fromSaccharomyces cerevisiae

A procedure for extraction of plasmid DNA from Saccharomyces cerevisiae is described. The plasmid DNA of interest is extracted together with 2-micron circular DNA naturally occurring in many yeast strains. Spheroplasts are lyzed at alkaline pH which denatures linear but not covalently closed circular (CCC) DNA. The CCC DNA is recovered by ethanol precipitation and can be detected by gel electrophoresis or used for routine bacterial transformation.     

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.     

Rapid preparation of covalently closed circular DNA by acridine yellow affinity chromatography

Covalently closed circular DNA can be isolated rapidly from cell lysates in a two-step process. Hydroxylapatite chromatography to prepurify the plasmid DNA from contaminating protein and RNA is followed by a step gradient elution of covalently closed circular (CCC) plasmid DNA from an acridine yellow affinity column. This procedure results in CCC DNA of a purity comparable to that obtained from ethidium bromide-CsCl gradients without lengthy centrifugation and free of contaimination by intercalating dye. Up to 250 μg of CCC pBR 322 can be isolated from 500 ml of bacterial culture in 4–6 h.     

Effects of chloramphenicol and rifampicin on the replication of R plasmid NR1 deoxyribonucleic acid in Escherichia coli.

The effects of inhibition of protein and ribonucleic acid (RNA) synthesis on the replication of the plasmids NR1 and F′lac in Escherichia coli were studied. When protein synthesis is inhibited, there is approximately a 25% increase in R plasmid deoxyribonucleic acid (DNA), but this newly synthesized DNA is not recoverable in the covalently closed circular (CCC) form until protein synthesis is allowed to resume. When RNA synthesis is inhibited, there is also approximately a 20% increase in R plasmid DNA, but this DNA is immediately recoverable in the CCC form. F′lac DNA, unlike R plasmid DNA, can continue to replicate for at least a generation time in the absence of protein synthesis, and this F′lac DNA is immediately recoverable in the CCC form.     

Physical Characterization of Gemini Surfactant-Based Synthetic Vectors for the Delivery of Linear Covalently Closed (LCC) DNA Ministrings

In combination with novel linear covalently closed (LCC) DNA minivectors, referred to as DNA ministrings, a gemini surfactant-based synthetic vector for gene delivery has been shown to exhibit enhanced delivery and bioavailability while offering a heightened safety profile. Due to topological differences from conventional circular covalently closed (CCC) plasmid DNA vectors, the linear topology of LCC DNA ministrings may present differences with regards to DNA interaction and the physicochemical properties influencing DNA-surfactant interactions in the formulation of lipoplexed particles. In this study, N,N-bis(dimethylhexadecyl)-α,ω-propanediammonium(16-3-16)gemini-based synthetic vectors, incorporating either CCC plasmid or LCC DNA ministrings, were characterized and compared with respect to particle size, zeta potential, DNA encapsulation, DNase sensitivity, and in vitro transgene delivery efficacy. Through comparative analysis, differences between CCC plasmid DNA and LCC DNA ministrings led to variations in the physical properties of the resulting lipoplexes after complexation with 16-3-16 gemini surfactants. Despite the size disparities between the plasmid DNA vectors (CCC) and DNA ministrings (LCC), differences in DNA topology resulted in the generation of lipoplexes of comparable particle sizes. The capacity for ministring (LCC) derived lipoplexes to undergo complete counterion release during lipoplex formation contributed to improved DNA encapsulation, protection from DNase degradation, and in vitro transgene delivery.     

Isolation and characterization of Escherichia coli chromosomal mutants affecting plasmid copy number.

We have isolated chromosomal mutants of an Escherchia coli K-12 strain that maintain higher levels of an F' plasmid. The mutants are designated as plasmid copy number (pcn) mutants. They were detected by selecting for increased lactose fermentation in bacteria deleted for the lac operon but harboring an F'lacI,P pro+ plasmid. When examined for the amount of F' plasmid deoxyribonucleic acid (DNA) by the dye-CsCl isopycnic technique, the mutants show two to seven times as much covalently closed, circular (CCC) DNA as does the parental strain. The increased plasmid level in one mutant strain (pcn-24) was confirmed by DNA-DNA hybridization; however, this latter technique indicated about a twofold lower increase when compared with the increase measured for pcn-24 by the dye-CsCl technique. In mutant pcn-24 the increased amount of F' DNA reflects a proportional increase in monomeric-size plasmid molecules because oligomeric forms are not found. Also, in mutant pcn-24 the extra CCC plasmid copies do not seem to be randomly distributed throughout the cell's cytoplasm but appear complexed in situ with their host's folded chromosome. In all pcn mutants examined to date, the classical sex factor F is maintained at normal levels, whereas the viral plasmid Pl CM is maintained at two to three times the normal level. In all 17 pcn mutants isolated, the pcn mutation maps on the chromosome and not on the plasmid. Finally, the absolute amount of CCC F' DNA detectable in lysates of the six different pcn mutants examined decreased 50 to 90% upon incubation of the lysate at 37 C. In contrast, no loss of CCC DNA occurs when lysates of the parental F' strain are incubated at 37 C.     

A spectrophotometric method to quantify linear DNA

A spectrophotometric method for quantification of linear DNA is described. The assay measures ADP produced following digestion of linear DNA by an ATP-dependent deoxyribonuclease. Cleavage of the phosphodiester bond of the DNA substrate is proportional to ADP formed in the reaction which follows typical Michaelis-Menten kinetics (K(m) of 0.6 microM, and a V(max) of 30 nmol/min/mg). The enzyme requires Mg(2+)-ATP and Mg(2+)-DNA as substrates, although the results suggest a requirement for yet another metal ion which may be enzyme bound. Both single-stranded and double-stranded linear DNA are substrates, as demonstrated by comparable initial velocity measurements. However, covalently closed circular (CCC) and nicked open circular DNA are not substrates for the enzyme. The rate of hydrolysis of ATP is not inhibited by 1 microg RNA or covalently closed circular DNA. The product (ADP) formed in the reaction is coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase. NAD formed in the reaction is monitored spectrophotometrically as a loss in absorbance at 340 nm. This assay directly measures the amount of linear DNA present in preparations of supercoiled (CCC) plasmid DNA, and has direct utility for monitoring the quality of plasmid preparations for gene therapy.     

Nonintegrated plasmid-chromosome complexes in Escherichia coli.

A number of plasmid systems have been examined for the ability of their covalently closed circular deoxyribonucleic acid (CCC DNA) forms to cosediment in neutral sucrose gradients with the folded chromosomes of their respective hosts. Given that cosedimentation of CCC plasmid and chromosomal DNA represents a bound or complexed state between these replicons, our results can be expressed as follows. (i) All plasmid systems complex, on the average, at least one plasmid per chromosomal equivalent. (ii) Stringently controlled plasmids exist predominantly in the bound state, whereas the opposite is true for plasmids that exist in multiple copies or are under relaxed control of replication. (iii) The degree to which a plasmid population binds to host chromosomes appears to be a function of plasmid genotype and not of plasmid size. (iv) For the colicin E1 plasmid the absolute number of plasmids bound per folded chromosome equivalent does increase as the intracellular plasmid/chromosome ratio increases in cells starved for required amino acids or in cells treated with chloramphenicol; however, the ratio of bound to free plasmids remains constant during plasmid copy number amplification.     

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.     

An improved method for rapid purification of covalently closed circular plasmid DNA over a wide size range

An improved method has been developed for the large-scale purification of covalently closed circular (CCC) plasmid DNA molecules of sizes ranging from 4·3 to 73 kb. This protocol uses an alkaline-lysis procedure followed by acid-phenol extraction but with several modifications to previously reported methods. The principal modification is the replacement of NaCl by MgCl₂ in the extraction buffer to improve yield and to remove chromosomal and other non-CCC plasmid DNA. Plasmid DNA can be purified in less than 1 h and used successfully in restriction enzyme analysis and cloning experiments.     

Genetic determinants of active antibiotic-producing soil streptomycetes.

Plasmids or covalently closed circular (CCC)-DNA molecules are abundant in the genus Streptomyces, and have been suggested to be involved in the genetic control of the production of many antibiotics in these organisms. In this study, 21 active antibiotic-producing Streptomyces isolates were screened for their plasmid content by an alkaline lysis method which revealed the presence of a small plasmid DNA in the positive control Streptomyces lividans ATCC 35287, containing pIJ702 plasmid (5.65 kb in size). However, no low molecular weight plasmids were observed in the tested antibiotic-producing Streptomyces strains suggesting that antibiotic production in these strains is likely chromosomally encoded DNA. Treatment of 2 Streptomyces strains with 10 mM ethidium bromide (EB) resulted in the failure to produce aerial mycelia and antibiotic activity.     

Cyanobacterial plasmids: Their widespread occurrence, and the existence of regions of homology between plasmids in the same and different species

Summary The results of screening of 29 diverse cyanobacterial (blue-green algal) strains for plasmid (CCC DNA) content are reported. Approximately one-half of the strains were shown to contain one or more CCC DNAs. CCC DNAs from four unicellular marine cyanobacteria were characterized in more detail. These strains contained multiple plasmids. Two kinds of Southern hybridization experiments allowed us to show that different plasmids within the same strain, and different plasmids within different strains, can (but do not always) contain restricted regions of sequence homology. We suggest that these regions of homology may be analogous to the transposable genetic elements of bacterial plasmids. This, together with indirect but compelling evidence for interspecific (or intergeneric) plasmid transfer, indicates that CCC DNAs (although as yet genetically cryptic) may play a role in the ecology and evolution of obligately autotrophic prokaryotes, as they do in the ecology and evolution of the better-known heterotrophic bacteria.     

Replication of thermosensitive Rts1 plasmid deoxyribonucleic acid at the nonpermissive temperature.

Replication of the thermosensitive drug resistance factor Rts1 was studied at the nonpermissive temperature (42 degrees C). It was concluded from the following observations that replication of this plasmid takes place at 42 degrees C without involving the covalently closed circular (CCC) form of deoxyribonucleic acid (DNA). (i) DNA-DNA- reassociation kinetics studies with purified Rts1 DNA showed that Rts1 DNA increased several-fold during cell growth at 42 degrees C while very little, if any, CCC DNA was synthesized. (ii) When Escherichia coli 20S0(Rts1) was labeled with [3H]thymidine at 42 degrees C, a significant amount of radioactive DNA hybridizable to Rts1 DNA was formed. This DNA was found in a fraction where DNA other than CCC DNA was expected in alkaline sucrose density gradient centrifugation analysis. When E. coli 20S0(Rts1) was labeled at 32 degrees C, the labeled CCC DNA did not disappear during a chase period at 42 degrees C. This indicates that preformed CCC DNA does not participate in replication at the nonpermissive temperature. These results are consistent with the hypothesis that there are two modes of replication of Rts1 DNA, one involving a CCC molecule and the other not involving this form, and that only the latter mode takes place at the nonpermissive temperature.     

Requirement of Cyclic Adenosine 3′,5′-Monophosphate for the Thermosensitive Effects of Rts1 in a Cyclic Adenosine 3′,5′-Monophosphate-Less Mutant of Escherichia coli

Previous publications showed that a covalently closed circular (CCC) Rts1 plasmid deoxyribonucleic acid (DNA) that confers kanamycin resistance upon the host bacteria inhibits host growth at 42 degrees C but not at 32 degrees C. At 42 degrees C, the CCC Rts1 DNA is not formed, and cells without plasmids emerge. To investigate the possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in the action of Rts1 on host bacteria, Rts1 was placed in an Escherichia coli mutant (CA7902) that lacks adenylate cyclase or in E. coli PP47 (a mutant lacking cAMP receptor protein). Rts1 did not exert the thermosensitive effect on these cells, and CCC Rts1 DNA was formed even at 42 degrees C. Upon addition of cAMP to E. coli CA7902(Rts1), cell growth and formation of CCC Rts1 DNA were inhibited at 42 degrees C. The addition of cAMP to E. coli PP47(Rts1) did not cause inhibitory effects on either cell growth or CCC Rts1 DNA formation at 42 degrees C. The inhibitory effect of cAMP on E. coli CA7902(Rts1) is specific to this cyclic nucleotide, and other cyclic nucleotides such as cyclic guanosine 3',5'-monophosphate did not have the effect. For this inhibitory effect, cells have to be preincubated with cAMP; the presence of cAMP at the time of CCC Rts1 DNA formation is not enough for the inhibitory effect. If the cells are preincubated with cAMP, one can remove cAMP during the [(3)H]thymidine pulse and still observe its inhibitory effect on the formation of CCC Rts1 DNA. The presence of chloramphenicol during this preincubation period abolished the inhibitory effect of cAMP. These observations suggest that cAMP is necessary to induce synthesis of a protein that inhibits CCC Rts1 DNA formation and cell growth at 42 degrees C.     

Different specific activities of the monomeric and oligomeric forms of plasmid DNA in transformation of B. subtilis and E. coli.

Summary (1) The low residual transforming activity in preparations of monomeric, supercoiled, circular (CCC) forms of the plasmids pC194 and pHV14 could be attributed to the presence in such isolates of a small number of contaminating multimeric molecules. (2) E. coli derived preparations of pHV14, an in vitro recombinant plasmid capable of replication in both E. coli and B. subtilis, contain oligomeric forms of plasmid DNA in addition to the prevalent monomeric CCC form. The specific transforming activity of pHV14 DNA for E. coli is independent of the degree of oligomerization, whereas in transformation of B. subtilis the specific activity of the purified monomeric CCC molecules is at least four orders of magnitude less than that of the unfractionated preparation. (3) Oligomerization of linearized pHV14 DNA by T4 ligase results in a substantial increase of specific transforming activity when assayed with B. subtilis and causes a decrease when used to transform E. coli.