Rifampin-resistant replication of pBR322 derivatives in Escherichia coli cells induced for the SOS response.

Replication of plasmid pBR322 in Escherichia coli cells normally requires RNA synthesis and thus is sensitive to rifampin, an inhibitor of RNA polymerase. In cells induced for the SOS response, however, derivatives of pBR322 were found to replicate in the presence of rifampin. This rifampin-resistant replication of pBR322 requires the insertion of certain sequences of DNA. The replication depends on recF+ and DNA polymerase I.     

Stable maintenance in chemostat-grownEscherichia coli of pBR322 and pACYC184 by disruption of the tetracycline resistance gene

Plasmid stability was studied in antibiotic-free chemo-stat cultures . Disruption, either by deletion or insertion, of the tetracycline resistance gene in the EcoRl/EcoRV region of the cloning vector pBR322 or in the HindIII]BamHl region of pACYCI84 yields plasmids markedly more stable than the parent plasmids. Thus, at least for these two instances, cloning of a partitioning (par) locus is not prerequisite for plasmid maintenance.Issued as NRCC publication No. 23992.     

Molecular cloning in plasmid pBR322 giving altered expression of the tetracycline resistance gene

The two HindIII fragments of polyoma virus DNA were cloned in the HindIII site of plasmid pBR322, a site located in the RNA polymerase promoter involved in the expression of tetracycline resistance. Although insertion of foreign DNA into this site did not always result in the complete loss of tetracycline resistance, Escherichia coli K12 strain chi 1776 harbouring recombinant plasmids exhibited reduced growth properties in liquid culture with tetracycline and could easily be differentiated from bacteria transformed by non-recombinant plasmids. The formation of plasmid multimers increased the resistance to tetracycline at the level of the induction period, presumably as a result of a gene dosage effect.     

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.     

Adaptation of Escherichia coli growth rates to the presence of pBR322

Changes in the growth rate of Escherichia coli K12 J62-1 in response to the presence of plasmid pBR322 have been investigated. Plasmid-free and plasmid-containing strains were grown in batch culture and their maximum specific growth rate (μ_max) determined. The acquisition of pBR322 by the host resulted in a decreased μ_max. Following repeated subculturing of the plasmid-containing strain on selective medium, restoration in μ_max was observed. The copy number and structure of the plasmid were not significantly altered during the experiment Growth rate measurements for a series of strains constructed using a combination of host cells and plas-mids with and without culture histories, indicated that the site of the adaptive mutation was located on the host chromosome rather than on the plasmid.     

Construction of frameshift mutation hot spots within the tetracycline resistance gene of pBR322

The chemical carcinogen, N-2-acetylaminofluorene (AAF) when bound covalently to DNA induces a majority (greater than 90%) of frameshift mutations. The mutations occur with high frequencies at defined sequences (i.e. mutation hot spots). Two classes of mutation hot spots were found: at repetitive sequences and at specific non-repetitive sequences. Mutations at the repetitive sequences depend upon a functional umuC gene whereas mutations at specific non-repetitive sequences are umuC-independent. The first discovered sequence of this class is the NarI restriction enzyme recognition sequence (5'GGCGCC3'). In an attempt to define a family of such sequences we constructed a related sequence 5'GCGCGC3' within the tetracycline resistance gene of pBR322. This sequence was also found to be an--AAF induced--2 frameshift mutation hot spot in both wild type and umuC strains.     

An infrequent generation of catenated network of pBR322 in Escherichia coli

It was demonstrated that Escherichia coli infrequently generates the catenated network of pBR322. This complex pBR322 form was detected when DNA molecules could hardly enter the agarose gel during electrophoresis and was found to comprise monomers and dimers of the plasmid.     

Maintenance stability of the pBR322 and pBR327 vector plasmids in Escherichia coli cells during multiple passages

Stability of pBR322 and pBR327 plasmids was studied. Plasmid-containing Escherichia coli strains were grown in liquid growth medium without selection pressure. Plasmid pBR327 was shown to be more stable in E. coli CSH54 cells than pBR322. Essential heterogenity of individual plasmid-containing clones was recognized by the maintenance stability of plasmid DNA. The indicated clones with high stability failed to be cured from pBR327 plasmid by means of acridine orange. High stability of plasmid maintenance and the failure to cure cells containing this plasmid are suggested to correlate with and to be essentially determined by the cell functions.     

Persistence of pBR322-related plasmids in Escherichia coli grown in chemostat cultures

Abstract The stability of plasmid pBR322 and a number of close derivatives was examined by continuous culture of Escherichia coli . Cultures were subjected to either glucose, phosphate or magnesium limitation in non-selective medium at a dilution rate of 0.1/h. Under these conditions pBR322 was eventually lost from the population, but only after a distinct lag period. The closely related plasmids pBR325 and especially pBR327 and pBR328, but not pAT153, were lost more rapidly. Three cosmids pHC79, pSJ55 and pJB8 were generally found to be less stable than the pBR322-type plasmids from which they were derived. Chimaeric plasmids containing DNA from yeast and from a thermophilic bacillus were also unstable in E. coli .     

Stability of ColE1-like and pBR322-like plasmids in Escherichia coli

The average copy number, the level of ampicillin resistance conferred by one plasmid, and the degree of plasmid multimerization were determined for several ColE1-like and pBR322-like plasmids. From the results obtained, the variance of the units of partition corresponding to each plasmid studied was calculated. Experimentally determined plasmid stability was compared with that calculated using the variance of the units of partition and the ratio between the generation times of plasmid-free and of plasmid-carrying cells, assuming that the units of partition are distributed randomly between daughter cells. Stability of the pBR322-like plasmids present mainly as monomers in the bacterial host was consistent with random partitioning, whereas pBR322-like plasmids, present mainly as dimers, and the ColE1-like plasmid showed greater stability than that predicted with random partitioning at cell division.     

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.     

Molecular cloning of the hamster papovavirus genome in Escherichia coli plasmid vector pBR322

The complete genome of the hamster papovavirus (HaPV) which was isolated from virions found in multiple skin tumors of Syrian hamsters was cloned in Escherichia coli using the plasmid vector pBR322. The cloned viral DNAs were identified by digestion of the recombinant DNAs with various restriction enzymes followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested uncloned DNAs. The cloned HaPV DNAs showed the same migration pattern as the corresponding fragments from the restricted uncloned DNAs, indicating that no major insertions or deletions occurred during cloning and plasmid propagation. The electrophoretic data were confirmed by Southern blot hybridization.     

Expression of pRW83 (penP) and pBR322-encoded β-lactamases in Escherichia coli

Abstract Plasmid pBR322 and penP -encoded β-lactamase activities were examined in cell fractions from wild-type and murein lipoprotein-deficient Escherichia coli strains. The specific activity of the Bacillus licheniformis penP gene product, a lipoprotein when expressed in E. coli , was increased in the outer membrane of a murein-lipoprotein deficient mutant. The activities of the 2 enzymes in wild-type E. coli exposed to the translational inhibitor puromycin were also investigated. Synthesis of penP was more susceptible to inhibition by puromycin than the pBR322-encoded TEM1 β-lactamase. The implications of these results for mechanisms of secretion and insertion of lipoproteins into the E. coli outer membrane are discussed.     

Characteristics of expression of the tetracycline resistance gene from the plasmid pBR322 in Bacillus subtilis cells

The expression of Tc resistance gene derived from plasmid pBR322 has been studied in Bacillus subtilis cells where this alien gene is not usually expressed. Fragments of Bacillus subtilis chromosome were inserted into the Tc resistance gene promoter region of the hybrid plasmid pGG20 and the expression of this gene was registered. Plasmid pGG20 confers a constitutive mode of Tc resistance in Escherichia coli cells. In contrast, the inducibility of Tc resistance gene expression in Bacillus subtilis cells has been reported. Optimal concentration for the highest inducibility of Tc resistance by the antibiotic has been determined.     

Persistence of the pBR 322 plasmid in Escherichia coli K 12 grown in chemostat cultures

Populations of a Escherichia coli K 12 strain, containing the vector plasmid p BR 322, were grown in chemostat culture under glucose- and phosphatelimited conditions. Resistance to tetracycline and ampicillin were lost after prolonged cultivation, resulting in the production of apparent plasmid-free populations which were more competitive than the original population. This competitiveness between plasmid-free and plasmid-containing populations was greatest in environments where the nutrient restriction was severe. Also during sequential subcultivation in batch cultures loss of plasmid was observed.