Determination of plasmid DNA concentration maintained by nonculturable Escherichia coli in marine microcosms.

The concentration of plasmid pBR322 DNA in nonculturable Escherichia coli JM83 was measured to determine whether the plasmid concentration changed during survival of E. coli in marine and estuarine water. E. coli JM83 containing the plasmid pBR322 was placed in both sterile seawater and sterile estuarine water and analyzed for survival (i.e., culturability) and plasmid maintenance. The concentration of pBR322 DNA remained stable in E. coli JM83 for 28 days in an artificial seawater microcosm, even though nonculturability was achieved within 7 days. E. coli JM83 incubated in sterile natural seawater or sterile estuarine water did not reach nonculturability within 30 days. Under all three conditions, plasmid pBR322 DNA was maintained at approximately the initial concentration. Cloning of DNA into the plasmid pUC8 did not alter the ability of E. coli to maintain vector plasmid DNA, even when the culture was in the nonculturable state, but the concentration of plasmid DNA decreased with time in the microcosm. We conclude that E. coli is able to maintain plasmid DNA while in the nonculturable state and that the concentration at which the plasmid is maintained appears to be dependent upon the copy number of the plasmid and/or the presence of foreign DNA.     

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.     

Survival of and plasmid stability in Pseudomonas and Klebsiella spp. introduced into agricultural drainage water

Cell survival and plasmid stability in Pseudomonas fluorescens R2f and Pseudomonas putida CYM 318 containing respectively, plasmid RP4 and pRK2501, and Klebsiella aerogenes NCTC 418 harboring plasmid pBR322 were studied in sterile and nonsterile agricultural drainage water under both aerobic and anaerobic conditions and in the absence and presence of added nutrients. Both Pseudomonas strains survived well in sterile drainage water incubated aerobically, with or without added nutrients. However, Klebsiella aerogenes NCTC 418 (pBR322) only survived in the presence of added nutrients. Pseudomonas fluorescens R2f (RP4) and K. aerogenes NCTC 418 (pBR322) did not survive under anerobic conditions without added nutrients, but showed good survival in the presence of nutrients. Survival of all three strains was negatively affected in nonsterile agricultural drainage water when compared with survival in sterile water. Maintenance of the three plasmids was host, plasmid, and environment dependent. Plasmid pBR322 was not stably maintained in K. aerogenes NCTC 418 under all conditions used in the study, and pRK2501 was readily lost from P. putida CYM 318. Maintenance of RP4 by P. fluorescens R2f was markedly influenced by added nutrients, which caused a loss of the plasmid from cells. The results of the present study demonstrate the influence of nutrients, O2, and native microorganisms on the survival of introduced bacterial strains and plasmid stability in agricultural drainage water.     

Long-term survival and plasmid maintenance of Escherichia coli in marine microcosms

Abstract The survival pattern and plasmid maintenance of Escherichia coli was examined in an artificial seawater microcosm. It was found that the three strains of E. coli (EK3C, H10407 and 34309) included in the study were able to maintain a portion of cells in the culturable phase for at least 3 years in artificial seawater. Along with retaining culturability, that portion of the cell population also maintained their indigenous plasmids over the 3-year period. It is concluded that cells of E. coli maintaining culturability in seawater are selectively adapted to the salinity of seawater, remaining in a culturable state. The results of the study are significant in that it has been assumed by many public health authorities that E. coli cannot survive, without nutrient addition, in seawater for long periods of time, i.e., years of exposure to seawater.     

Fate in soil of a recombinant plasmid carrying aDrosophila gene

A recombinant plasmid (C357; 3.5 Mdal) containing heterologous DNA (pBR322 [2.6 Mdal] with cDNA for an egg yolk protein fromDrosophila grimshawi) inEscherichia coli strain HB101 survived in and was recovered on selective media from sterile and nonsterile soil during 27 days at frequencies similar to those of theE. coli(pBR322) system. In sterile saline, the numbers of all cells decreased during 34 days, but the numbers of the plasmidless host declined less. There was no selective loss of the heterologous DNA in either soil or saline, as determined by colony hybridization with a³²P-labeled DNA probe for the cDNA, but the HB101(C357) appeared to be less able than HB101(pBR322) to cope with conditions of starvation. These results suggested that nonessential eucaryotic DNA inserted into plasmid DNA has little effect on the survival in soil or saline of the bacterial host and the maintenance of the vector.     

Efficient transformation of Serratia marcescens with pBR322 plasmid DNA

Eight Serratia marcescens strains tested could be transformed with the plasmid pBR322. Transformants were selected on the basis of resistance to high levels of ampicillin (400 to 500 micrograms/ml). For six of the strains, the CaCl2- mediated transformation procedure developed for Escherichia coli was successful. For the other two strains, no transformants were obtained with the CaCl2-mediated transformation procedure unless the cells first received a heat treatment. Transformation frequency was dependent on DNA concentration, and no transformation was detected with linear pBR322 DNA. The stability and copy number of pBR322 were similar in S. marcescens and E. coli. As in E. coli, the pBR322 DNA was amplified in S. marcescens after inhibition of proteins synthesis. Based on these results, cloning in S. marcescens should be possible and pBR322 should be a useful cloning vehicle.     

Death of the Escherichia coli K-12 strain W3110 in soil and water.

Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures. In nonsterile river water, the plate counts of added E. coli cells dropped to less than 10 CFU/ml in less than 10 days. Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E. coli cells were disappearing from the water in parallel with the number of CFU. Similar results were obtained with nonsterile soil, although the decline of the added E. coli was slower. In sterile water or soil, the added E. coli persisted for much longer, often without any decline in the plate counts even after 50 days. In sterile river water at 37 degrees C and sterile artificial seawater at 20 and 37 degrees C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged. However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable. Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E. coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death.     

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 .     

Survival of nonculturable Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida was investigated in sterile and untreated lake water. In sterile lake water (filtered and autoclaved), it was found that cells of A. salmonicida entered a nonculturable but viable condition. Viability was determined by flow cytometry with the dye rhodamine 123, which is taken up and maintained within cells with a membrane potential. For survival studies in untreated lake water, A. salmonicida was marked with the xylE gene by using the plasmid pLV1013. Marked cells were detected by growth on tryptone soy agar and tryptone soy agar supplemented with kanamycin. Cells were also detected by polymerase chain reaction DNA amplification of the xylE gene and a chromosomal DNA fragment specific for A. salmonicida (pLV1013). The results indicated that A. salmonicida entered a nonculturable condition in untreated lake water over a 21-day study. The viability of nonculturable cells could not be determined in mixed samples; however, the presence of nonculturable cells containing both chromosomal and plasmid DNA was confirmed.     

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5alpha harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid beta-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.     

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.     

Mobilization of the genetically engineered plasmid pHSV106 from Escherichia coli HB101(pHSV106) to Enterobacter cloacae in drinking water.

We have used triparental matings to demonstrate transfer (mobilization) of the nonconjugative genetically engineered plasmid pHSV106, which contains the thymidine kinase gene of herpes simplex virus cloned into pBR322, from Escherichia coli HB101 to an environmental isolate of Enterobacter cloacae in sterile drinking water. This is the first demonstration of a two-step mobilization of a genetically engineered plasmid in any type of fresh water, including drinking water. Transfer was mediated by R plasmid R100-1 of E. coli ED2149(R100-1). Matings in drinking water at 15, 25, and 35 degrees C yielded recombinants, the number of which increased with increasing temperature. Numbers of recombinants obtained were 2 orders of magnitude lower than those obtained from matings in Trypticase soy broth. High concentrations of parental organisms (2.6 x 10(8) to 2.0 x 10(9) CFU/ml) were required. During 1 week of incubation in drinking water, number of parental organisms and recombinants resulting from mobilization remained constant in the absence of indigenous organisms and declined in their presence. Using oligonucleotide probes for the cloned foreign DNA (thymidine kinase gene) and plasmid vector DNA (ampicillin resistance gene), we demonstrated that both genes were transferred to E. cloacae in the mobilization process. In one recombinant selected for detailed study, the plasmids containing these genes differed in size from all forms of pHSV106 present in E. coli HB101(pHSV106), indicating that DNA rearrangement had occurred. This recombinant maintained its plasmids in unchanged form for 15 days in drinking water. A second rearrangement occurred during serial passage of this recombinant on selective media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mobilization of the genetically engineered plasmid pHSV106 from Escherichia coli HB101(pHSV106) to Enterobacter cloacae in drinking water

We have used triparental matings to demonstrate transfer (mobilization) of the nonconjugative genetically engineered plasmid pHSV106, which contains the thymidine kinase gene of herpes simplex virus cloned into pBR322, from Escherichia coli HB101 to an environmental isolate of Enterobacter cloacae in sterile drinking water. This is the first demonstration of a two-step mobilization of a genetically engineered plasmid in any type of fresh water, including drinking water. Transfer was mediated by R plasmid R100-1 of E. coli ED2149(R100-1). Matings in drinking water at 15, 25, and 35 degrees C yielded recombinants, the number of which increased with increasing temperature. Numbers of recombinants obtained were 2 orders of magnitude lower than those obtained from matings in Trypticase soy broth. High concentrations of parental organisms (2.6 x 10(8) to 2.0 x 10(9) CFU/ml) were required. During 1 week of incubation in drinking water, number of parental organisms and recombinants resulting from mobilization remained constant in the absence of indigenous organisms and declined in their presence. Using oligonucleotide probes for the cloned foreign DNA (thymidine kinase gene) and plasmid vector DNA (ampicillin resistance gene), we demonstrated that both genes were transferred to E. cloacae in the mobilization process. In one recombinant selected for detailed study, the plasmids containing these genes differed in size from all forms of pHSV106 present in E. coli HB101(pHSV106), indicating that DNA rearrangement had occurred. This recombinant maintained its plasmids in unchanged form for 15 days in drinking water. A second rearrangement occurred during serial passage of this recombinant on selective media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmid DNA supercoiling and survival in long-term cultures of Escherichia coli: role of NaCl

The relationship between the survival of Escherichia coli during long-term starvation in rich medium and the supercoiling of a reporter plasmid (pBR322) has been studied. In aerated continuously shaken cultures, E. coli lost the ability to form colonies earlier in rich NaCl-free Luria-Bertani medium than in NaCl-containing medium, and the negative supercoiling of plasmid pBR322 declined more rapidly in the absence of NaCl. Addition of NaCl at the 24th hour restored both viability and negative supercoiling in proportion to the concentration of added NaCl. Addition of ofloxacin, a quinolone inhibitor of gyrase, abolished rescue by added NaCl in proportion to the ofloxacin added. This observation raises the possibility that cells had the ability to recover plasmid supercoiling even if nutrients were not available and could survive during long-term starvation in a manner linked, at least in part, to the topological state of DNA and gyrase activity.     

The effect of the oligomerization of pBR322 on the level of transformation in Escherichia coli

The ability of the known Escherichia coli strain JC3881 recB recC recF sbc15 to produce oligomeric and multimeric forms of pBR322 underlies the study presented. The individual oligomeric forms of pBR322 were isolated from the agarose gel. The plasmid forms were used for electron microscopic control and also introduced into the system of E. coli competent cells. The E. coli transformation level of different forms of plasmid DNA rose from monomers to pentamers. CCC forms of the plasmid possessed high efficiency of the E. coli cell transformation. The systems of the host recombination are to be significant in the process of plasmid oligomerization.     

UV-induced cross-linking of proteins to plasmid pBR322 containing 8-azidoadenine 2'-deoxyribonucleotides

An efficient method of cross-linking DNA to protein is described. The method is based on the incorporation of photoactive 8-azidoadenine 2'-deoxyribonucleotides into DNA. We have found that 8-N3dATP is a substrate for E. coli DNA polymerase I and that 8-N3dATP can be incorporated into plasmid pBR322 by nick-translation. Subsequently we were able to cross-link a set of different proteins to 8-azido-2'-deoxyadenosine-containing pBR322 by UV irradiation (366nm). No DNA-protein photocross-linking was observed under the same conditions when the non-photoactive plasmid pBR322 was used.     

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.