Transfer of plasmids pBR322 and pBR325 in wastewater from laboratory strains of Escherichia coli to bacteria indigenous to the waste disposal system.

Laboratory strains of Escherichia coli containing plasmid pBR325 (or pBR322) were coincubated with a mobilizer strain of E. coli (containing the conjugative plasmid R100-1) and a recipient strain of bacteria. Bacterial strains isolated from raw wastewater or a plasmid-free E. coli laboratory strain served as recipients. Transfer of the pBR plasmid into the recipient strain occurred during a 25-h coincubation in either L broth or sterilized wastewater; transfer frequencies were several orders of magnitude lower in wastewater. After the coincubation, recipients exhibited both plasmid-encoded phenotypic characteristics and an altered plasmid profile, as shown by agarose gel electrophoresis of purified plasmid DNA.     

Isolation of indigenous wastewater bacterial strains capable of mobilizing plasmid pBR325

Members of the family Enterobacteriaceae have been isolated from raw wastewater, identified, and characterized with respect to their plasmid content and antibiotic resistance. Several strains possessing both antibiotic resistance and high-molecular-weight plasmid(s) transferred their resistance characteristics to recipient cells during a 25 h coincubation. Eight were characterized (six Escherichia coli and two Klebsiella pneumoniae); each produced 10(2) to 10(7) transconjugants per ml by the end of the incubation period. They were also able to mobilize pBR325 from a laboratory E. coli strain into plasmid-free recipients to yield 10(2) to 10(7) transconjugants per ml. These transconjugants possessed phenotypic characteristics specified by pBR325, the R plasmid, and the chromosome of the recipient. Many transconjugants exhibited recombinational rearrangements of the acquired plasmid material.     

Isolation of indigenous wastewater bacterial strains capable of mobilizing plasmid pBR325.

Members of the family Enterobacteriaceae have been isolated from raw wastewater, identified, and characterized with respect to their plasmid content and antibiotic resistance. Several strains possessing both antibiotic resistance and high-molecular-weight plasmid(s) transferred their resistance characteristics to recipient cells during a 25 h coincubation. Eight were characterized (six Escherichia coli and two Klebsiella pneumoniae); each produced 10(2) to 10(7) transconjugants per ml by the end of the incubation period. They were also able to mobilize pBR325 from a laboratory E. coli strain into plasmid-free recipients to yield 10(2) to 10(7) transconjugants per ml. These transconjugants possessed phenotypic characteristics specified by pBR325, the R plasmid, and the chromosome of the recipient. Many transconjugants exhibited recombinational rearrangements of the acquired plasmid material.     

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.     

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 .     

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.     

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.     

Maintenance of plasmids pBR322 and pUC8 in nonculturable Escherichia coli in the marine environment.

Maintenance of plasmids pBR322 and pUC8 in Escherichia coli that was nonculturable after exposure to seawater was studied. E. coli JM83 and JM101, which contained plasmids pBR322 and pUC8, respectively, were placed in sterile artificial seawater for 21 days. Culturability was determined by plating on both nonselective and selective agar, and plasmid maintenance was monitored by direct isolation of plasmid nucleic acid from bacteria collected on Sterivex filters. E. coli JM83 became nonculturable after incubation for 6 days in seawater yet maintained plasmid pBR322 for the entire period of the study, i.e., 21 days. E. coli JM101 was nonculturable after incubation in seawater for 21 days and also maintained plasmid pUC8 throughout the duration of the microcosm experiment. Direct counts of bacterial cells did not change significantly during exposure to seawater, even though plate counts yielded no viable (i.e., platable) cells. We concluded that E. coli cells are capable of maintaining high-copy-number plasmids, even when no longer culturable, after exposure to the estuarine or marine environment.     

Antagonism of the B subunit of DNA gyrase eliminates plasmids pBR322 and pMG110 from Escherichia coli.

The constructed plasmid pBR322 and the native plasmid pMG110 were eliminated (cured) from growing Escherichia coli cells by the antagonism of the B subunit of the bacterial enzyme DNA gyrase. The antagonism may be by the growth of cells (i) at semipermissive temperatures in a bacterial mutant containing a thermolabile gyrase B subunit or (ii) at semipermissive concentrations of coumermycin A1, an antibiotic that specifically inhibits the B subunit of DNA gyrase. The kinetics of plasmid elimination indicate that plasmid loss occurs too rapidly to be explained solely by the faster growth of that plasmid-free bacteria and, therefore, represents interference with plasmid maintenance.     

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 .     

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.     

Ribitol dehydrogenase overproduction and maintenance of plasmids pBR322 and pACYC184 in chemostat cultures of Escherichia coli

Summary The maintenance of multicopy plasmids pBR322 and pACYC184 was studied in chemostat cultures subjected to limitation by glucose, ribitol or xylitol at D of 0.1 per h. While carbon source-dependent segregational stability of pBR322 was observed, no dependence and greater instability of pACYC184 was found. Plasmid-free and plasmid-bearing strains overproducing chromosomally coded ribitol dehydrogenase were found in pentitol-limited chemostat cultures.     

pBR322-derived multicopy plasmids harboring large inserts are often dimers in Escherichia coli K-12

pBR322-related plasmids that are 2.3 to 5.1 kb were found predominantly as monomers, while plasmids that are 7.7 to 15.2 kb were found predominantly as dimers in rec+ cells of Escherichia coli K-12.     

Mixed-ligand complexes of platinum(II) as curing agents for pBR322 and pBR329 (ColE1) plasmids in Escherichia coli

The mixed-ligand complexes of platinum(II) [Pt(Gly)(uracil)Cl2].H2O and [Pt(His)(adenine)]-Cl.2H2O eliminated multicopy plasmids with the ColE1 origin of replication in Escherichia coli with 100% frequency. However, plasmids of the IncF1, H1 and X groups were totally refractory to these agents under similar conditions.     

Transfer of plasmids between strains of Escherichia coli under rumen conditions

K. P. SCOTT AND H.J. FLINT. 1995. Strains of Escherichia coli originally isolated from the rumen of sheep were shown to be capable of exchanging a 60kb plasmid, conferring resistance to tetracycline and ampicillin, at low frequencies (below 10^-6 per recipient) under anaerobic conditions in the presence of (a) autoclaved and clarified rumen fluid, (b) raw clarified rumen fluid, or (c) whole rumen fluid. Under anaerobic conditions the two rumen strains showed no inhibition of growth rate when 50 mmol 1^-1 volatile fatty acids were added to LB medium at pH 7, although significant inhibition resulted with 100 mmol 1^-1 VFA. The two rumen strains, and four strains from the pig gut, showed less inhibition of anaerobic growth by volatile fatty acids than did three laboratory strains examined for comparison. These findings indicate that plasmid transfer between certain E. coli strains can occur under conditions that closely simulate an anaerobic gut environment.     

Elimination of ColE1 (pBR322 and pBR329) plasmids in Escherichia coli by α-santonin

alpha-Santonin, a compound extracted from the flower heads of Arthemisia maritima plant, was effective in elimination of small, multicopy, relaxed plasmids (pBR322 and pBR329 with CoLE1 origin of replication in Escherichia coli, whereas plasmids of IncF1, H1 and X group were totally refractory under similar conditions, suggesting that this agent was specific in curing the CoLE1 group of plasmids.