Increased stability of pBR322-related plasmids in Escherichia coli W3101 grown in carrageenan gel beads

Abstract The stability and the copy number of pBR322, pBR325 and pBR328 were studied during continous cultures of free and immobilized E. coli W3101 without selective pressure. In the free-cell system, it was found that pBR328 and pBR325-free E. coli cells appeared after a lag period. They rapidly overgrew the cultures and the plasmid copy number subsequently declined. On the other hand, an increase in the proportion of pBR322- carrying cells during a free continuous culture was observed. This increase correlated with that of plasmid copy number. By contrast, in the immobilized- cell system, plasmid free segregants were not detected in all the cases even after 250 generations. We have also shown that plasmid copy number remained constant and phenomena such as fluctuations or genetic modifications which occured after long term growth of bacteria in a free continuous culture could be avoided throughout cell immobilization.     

Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives

Summary We describe mutants of Escherichia coli that decrease the plasmid copy number of pBR322 derivatives. One mutant was partially characterized genetically and its mutation, designated pcnB for plasmid copy number, was mapped to approximately 3 min on the E. coli chromosome. This locus is distinct from other genes whose products are known to affect plasmid replication or stable plasmid maintenance. The pcnB mutant strain should be useful for cloning genes into pBR322 that have aberrant or deleterious effects on the cell when present in high copy number.     

pBR322-Red介导的E.coli染色体基因敲入、位点及表达研究

应用pBR322-Red介导的重组工程系统,kan/sacB选择反选择系统,双链线性DNA重组技术和重叠引物介导的DNA重组技术,将长度为1 653 bp的luc报告基因分别敲入到E.coli W3110染色体lacZ,lacY和lacA基因的位置,建立了一系列具有新遗传表型的菌株:CWL2、CWL4和CWL6.荧光素酶分析表明,外源报告基因luc能在这3个结构基因处有效的组成型表达.为了进一步确定外源基因的表达情况,用霍乱毒素B亚单位基因ctxb替换了lacZ基因,构建了新菌株CWD1.证明了以单拷贝形式存在在大肠杆菌染色体CWD1上的ctxb基因能有效的表达CTB蛋白并能将其分泌至细胞外培养液中.结果初步确定了大肠杆菌染色体上的lac操纵子结构基因位点适合外源基因的敲入和表达.     

Regulation of replication of plasmid pBR322 in amino acid-starved Escherichia coli strains

The stringent response causes inhibition of replication of plasmid pBR322 in amino acid-starved Escherichia coli cells whereas in relaxed mutants the replication of this plasmid proceeds for several hours. On the basis of density shift experiments and pulse-labelling experiments we showed that most of the pBR322 molecules begin replication during the relaxed response and the rate of plasmid DNA synthesis in unstarved and isoleucine-starved relA ^–] bacteria is similar. We found that the Rom function plays a key role in the stringent control of plasmid pBR322 replication, as insertional inactivation of the rom gene causes amplification of pBR322rom ^– in both relA ^– and relA ⁺ strains during amino acid starvation. Moreover, pUC19, which is a pBR322-derived plasmid lacking the rom gene, behaves like pBR322rom ^–, whereas introduction of the rom gene into the pUC19 replicon drives it into the pBR322 mode of replication in amino acid-starved bacteria. A model for the regulation of pBR322 plasmid DNA replication by Rom protein in amino acid-starved Escherichia coli strains is proposed.     

Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli

Summary The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA ⁺ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA ⁺ gene was expressed in the presence of the inducer isopropyl-1-thin--d-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30° C, or after inactivation of DnaA by a shift to 42° C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom ^– plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately. These results, together with previously published reports, suggest that DnaA plays no specific role during in vivo replication of ColE1 plasmids and that the gradual cessation of plasmid replication after heat inactivation of DnaA in LB medium results from indirect effects of the inhibition of chromosome replication and the ensuing saturation of promoters with RNA polymerase under nonpermissive growth conditions.     

磁场对质粒pBR322DNA的影响

讨论了经５００ｍＴ、９００ｍＴ、１２００ｍＴ、１５００ｍＴ恒磁场作用后的质粒ｐＢＲ３２２ＤＮＡ与Ⅱ型限制性内切酶ＥｃｏＲⅠ、ＨｉｎｄⅢ、ＢａｍＨⅠＳａｌⅠ、ＰｓｔⅠ、ＰｕｖⅡ、ＴａｑⅠ、ＢｇｌⅠ、ＲｓａⅠ、ＨｉｎｆⅠ、ＡＰａⅠ、ＫｐｎⅠ等的单酶解、双酶解及多酶解反应。从琼脂糖电泳分离酶解片段的结果,发现经磁场作用过的ｐＢＲ３２２ＤＮＡ在３３６１—６３１ｂｐ的区段内产生了一个被ＨｉｎｆⅠ识别的新序列ＧＡＮＴＣ,证明了磁场作用能引起ＤＮＡ点突变。     

pBR322-Red在大肠杆菌lac操纵子基因敲除和敲入中的应用

pBR322-Red是一种新型重组工程系统,它携带了λ-噬菌体Red重组酶基因和一系列调控元件.对pBR322-Red最优重组条件进行探索后应用该质粒提供的体内同源重组功能,在菌株W3110体内,对染色体上的lac操纵子进行了基因修饰,包括:①运用kan/sacB选择反选择方法和重叠引物方法敲除了阻遏基因lacⅠ,②运用kan/sacB选择反选择方法和线性双链DNA介导的DNA重组方法将报告基因lacZ敲入lacA和lacY的位置,并且首次测定了报告基因lacZ在这三个结构基因位置的组成性表达情况.结果表明运用不同的重组策略,pBR322-Red系统都能方便有效地对大肠杆菌W3110染色体进行基因敲除和敲入修饰.     

Escherichia coli relA strains as hosts for amplification of pBR322 plasmid DNA

Abstract We have proposed that guanosine tetraphosphate produced in Escherichia coli cells subjected to an isoleucine limitation inhibits pBR322 DNA replication [1]. In E. coli relA which cannot synthesize guanosine tetraphosphate (ppGpp) upon amino acid limitation pBR322 DNA is amplified after arginine starvation. The yield of plasmid DNA amplified either by chloramphenicol (Cm) or by arginine limitation is compared. The plasmid yield per cell is equal in amino acid-starved cells and in cells treated with Cm. To increase the plasmid content per ml of cell suspension the growth medium was supplemented with increasing amounts of nutrients. Plasmid DNA can be isolated in large quantities by this procedure. This simple method can be used for the enrichment of pBR325 DNA which cannot be amplified by Cm treatment. Our results indicate that E. coli relA strains might be suitable hosts for the amplification of pBR322 and related plasmids in E. coli .     

Rifampicin-induced replication of the plasmid pBR322 in Escherichia coli strains carrying dnaA mutations

The replication pattern of the plasmid pBR322 was examined in the dnaA mutants of Escherichia coli. The rate of pBR322 DNA synthesis is markedly decreased after dnaA cells are shifted to the restrictive temperature of 42 degrees C. However, addition of rifampicin (RIF) to cultures of dnaA strains incubated at 42 degrees C after a lag of 90 min results in a burst of pBR322 synthesis. This RIF-induced pBR322 replication remains dependent on DNA polymerase I activity. Efficient plasmid pBR322 replication is observed at 42 degrees C in the double mutant dnaA46cos bearing an intragenic suppressor of dnaA46. Though replication of pBR322 in dnaA46cos growing at 42 degrees C is initially sensitive to RIF plasmid synthesis is restored after 90 min incubation in the presence of the drug. RIF-induced replication of the plasmid pBR327, lacking the rriB site implicated in RIF-resistant synthesis of the L strand of ColE1-like plasmids (Nomura and Ray 1981; Zipursky and Marians 1981), was observed also in dnaA46 at 42 degrees C.     

Fate of pBR322 DNA in a wastewater matrix

Recombinant organisms used in biopharmaceutical production processes are destroyed prior to environmental release into a private or municipal wastewater treatment system. However, concern over the fate of recombinant DNA used in these processes may adversely affect product regulatory approval. This study examined the fate of DNA from the plasmid pBR322 in an activated sludge-derived matrix. DNA suitable for PCR amplification was extracted from the activated sludge matrix and a 1042-bp fragment from pBR322 rapidly decreased in concentration from 0 to 2 h after it was spiked into the activated sludge matrix at an initial DNA concentration of 25 ng ml⁻¹. While some evidence of the 1042-bp fragment was observed at 4 h, no evidence of amplified DNA was observed at 6 h. Plasmid DNA in buffer that served as a positive control exhibited no significant reduction in concentration over time. The intensity of each DNA band over the first 4 h was analyzed. A linear regression of the natural log transformation of these results yielded a mean first-order rate constant of 3.55 h⁻¹ and half-life of 0.2 h. This study demonstrated that recombinant DNA released from industrial processes into wastewater treatment systems should be rapidly degraded. Received 14 August 1998/ Accepted in revised form 19 February 1999     

pBR322-Red介导的E．Coli染色体基因敲入、位点及表达研究

应用pBR322-Red介导的重组工程系统,Kan/sacB选择反选择系统,双链线性DNA重组技术和重叠引物介导的DNA重组技术,将长度为1 653 bp的luc报告基因分别敲入到E.coli W3110染色体lacZ, lacY和lacA基因的位置,建立了一系列具有新遗传表型的菌株：CWL2、CWL4和CWL6。荧光素酶分析表明,外源报告基因luc能在这3个结构基因处有效的组成型表达。为了进一步确定外源基因的表达情况,用霍乱毒素B亚单位基因ctxb替换了lacZ基因,构建了新菌株CWD1。证明了以单拷贝形式存在在大肠杆菌染色体CWD1上的ctxb基因能有效的表达CTB蛋白并能将其分泌至细胞外培养液中。结果初步确定了大肠杆菌染色体上的lac操纵子结构基因位点适合外源基因的敲入和表达。     

Atomic force microscopic study on topological structures of pBR322 DNA

Plasmid pBR322 DNA (0.5mg/mL) isolated from Escherichia coli HB101 was suspended in Tris-HCl-EDTA (1 mol/L - 0.1 mol/L, pH8.5); then a drop of the above solution was deposited on freshly cleaved mica substrate. After adsorption for about 1 min, the sample was stained with phosphotungstic acid. The residua] solution was removed with a piece of filter paper. Afterwards the sample was imaged with a home-made atomic force microscope (AFM) in air. The AFM images of pBR322 DNA with a molecular resolution have been obtained. These images show that pBR322 DNA exists in several different topological structures: (i) relaxed circular DNA with a different diameter; (ii) supercondensed DNA with different particle sizes; (iii) dimeric catenane connected by one relaxed circular molecule and another dose-compacted molecule which might be either supercoiled or intramolecular knotted form; (iv) oligomeric catenane with multiple irregular molecules in which DNA is interlocked into a complex oligomer; (v) possibly-existing     

Escherichia coli dnaT gene function is required for pBR322 plasmid replication but not for R1 plasmid replication.

Plasmid pBR322 was unable to replicate in a temperature-sensitive dnaT1 strain at a nonpermissive temperature, whereas a pBR322-derived plasmid carrying the wild-type dnaT+ gene was able to replicate under the same conditions. In contrast to pBR322, plasmid R1 could replicate in the dnaT1 strain at a nonpermissive temperature. In keeping with this finding, in vitro replication of plasmid R1 did not require DnaT protein.     

Recombination between bacteriophage lambda and plasmid pBR322 in Escherichia coli

Recombinant lambda phages were isolated that resulted from recombination between the lambda genome and plasmid pBR322 in Escherichia coli, even though these deoxyribonucleic acids (DNAs) did not share extensive regions of homology. The characterization of these recombinant DNAs by heteroduplex analysis and restriction endonucleases is described. All but one of the recombinants appeared to have resulted from reciprocal recombination between a site on lambda DNA and a site on the plasmid. In general, there were two classes of recombinants. One class appeared to have resulted from recombination at the phage attachment site that probably resulted from lambda integration into secondary attachment sites on the plasmid. Seven different secondary attachment sites on pBR322 were found. The other class resulted from plasmid integration at other sites that were widely scattered on the lambda genome. For this second class of recombinants, more than one site on the plasmid could recombine with lambda DNA. Thus, the recombination did not appear to be site specific with respect to lambda or the plasmid. Possible mechanisms for generating these recombinants are discussed.     

Efficient transformation of a Yersinia enterocolitica strain (serotype O:3) by pBR322 DNA

Abstract The Yersinia enterocolitica strain MS201 (serotype O:3) was transformed by pBR322 DNA extracted from an Escherichia coli strain. The pBR322 DNA extracted from an Escherichia coli strain. The pBR322 DNA extracted from the transformed Y. enterocolitica is able to transform plasmid-free MS201 at a significantly higher frequency, suggesting the existence of a restriction-modification system in MS201.