Effect of environmental growth conditions on plasmid stability, plasmid copy number, and catechol 2,3-dioxygenase activity in free and immobilized Escherichia coli cells

In order to better understand the high plasmid stability in immobilized recombinant E. coli cells, the effects of dilution rate on the pTG201 plasmid stability, the copy number, and the catechol 2,3-dioxygenase (encoded by XyIE gene) production were, at first, studied in free E. coli W3101 continuous cultures in minimal media. It was found that decreasing specific growth rate increased the plasmid copy number and the catechol 2,3-dioxygenase activity but the stability decreased. In continuous culture with immobilized cells, an increase was shown in plasmid copy number and catechol 2,3-dioxygenase activity probably due to the distribution of growth in the gel beads. Besides mechanical properties of gel beads which may allow limited cell divisions, the increase in plasmid copy number is involved in enhanced plasmid stability in immobilized cells. In the same way, an experiment conducted in LB medium dealing with competition between pTG201-free and pTG201-containing E. coli B cells was described. It was shown that the competition was not more pronounced in gel bead compared to a free system. The effects of nutritional limitations on pTG201 plasmid stability and catechol 2,3-dioxygenase activity during chemostat cultivations in free and immobilized E. coli B cells were also investigated. It was found that immobilization of cells increased the stability of pTG201 even under glucose, nitrogen, or phosphate limited cultures. However in the case of magnesium depleted culture, pTG201 was shown to be relatively instable and a decrease in viable cell number during the immobilized continuous culture was observed. By contrast to the free system, the catechol 2,3-dioxygenase activity increased in immobilized cells under all culture conditions used.     

Influence of immobilization on the stability of pTG201 recombinant plasmid in some strains of Escherichia coli

The stability of pTG201 plasmid was examined by continuous culture in three genetically different Escherichia coli hosts. Two types of experiment were carried out, one with free cells and one with immobilized cells. When cells were cultivated in free continuous culture in the absence of antibiotic selection, the plasmid was maintained with various degrees of stability in the three host organisms. By contrast, in continuous culture with immobilized cells, plasmid pTG201 was stably maintained in the three strains. We showed that the increase in pTG201 stability in immobilized cells is due neither to plasmid transfer between immobilized cells nor to an increase of the plasmid copy number of immobilized cells. We also showed that plasmid-free cells, when coimmobilized and grown in competition with plasmid-containing cells, cannot overrun the culture.     

Influence of immobilization on the stability of pTG201 recombinant plasmid in some strains of Escherichia coli.

The stability of pTG201 plasmid was examined by continuous culture in three genetically different Escherichia coli hosts. Two types of experiment were carried out, one with free cells and one with immobilized cells. When cells were cultivated in free continuous culture in the absence of antibiotic selection, the plasmid was maintained with various degrees of stability in the three host organisms. By contrast, in continuous culture with immobilized cells, plasmid pTG201 was stably maintained in the three strains. We showed that the increase in pTG201 stability in immobilized cells is due neither to plasmid transfer between immobilized cells nor to an increase of the plasmid copy number of immobilized cells. We also showed that plasmid-free cells, when coimmobilized and grown in competition with plasmid-containing cells, cannot overrun the culture.     

Plasmid inheritability and biomass production: comparison between free and immobilized cell cultures of Escherichia coli BZ18(pTG201) without selection pressure.

Maintenance of the plasmid pTG201 in Escherichia coli BZ18 was studied for both free and immobilized cells during chemostat culture, in the absence of the antibiotic against which resistance was plasmid encoded. Electron microscopic observations of immobilized proliferant cells within carrageenan gel beads showed high cell concentrations and growth into distinct cavities. The plasmid which coded for the catechol 2,3-dioxygenase activity was stably maintained during 80 generations in the case of immobilized cells. A theoretical analysis founded on the compartmentalization resulting from the immobilized growth conditions was described. However, the model still showed a plasmid stability inferior to that determined experimentally. Hypotheses dealing with physiological changes of immobilized cells were presented. In addition, the high cell concentrations obtained in the outer 50 microns of the carrageenan gel beads gave a biomass productivity within this useful volume which was 20 times higher than in free-cell cultures.     

Stability without selection pressure of the plasmid pTG 201 during continuous fermentation of Escherichia coli BZ 18 immobilized in a carrageenan gel

Escherichia coli BZ 18 harboring the plasmid pTG 201 and immobilized in carrageenan gel beads in continuous culture without selection pressure, provides a better stability of the plasmid than free cells, with an approximately equal production of biomass.     

Improvement of plasmid stability of recombinant Bacillus-subtilis cells in continuous immobilized cultures

Abstract: The immobilization of recombinant Bacillus subtilis in K-carrageenan gel beads has been performed in order to study the growth conditions inside the gel beads and to improve plasmid stability. Bacterial colonies showing high cell density were studied using scanning electron microscopy. A series of continuous cultures of free and immobilized B. subtilis MT119 (pHV1431, pIL252 and pIL252 Kpn) have been developed without selection pressure. In the free-cell systems, it was found that a loss of plasmid vectors occurred after a short period. In contrast, in the immobilized cell systems, plasmid-free segregants were not detected in any of the cases during the first 80 h of the culture.     

Stability fluctuations of plasmid-bearing cells: immobilization effects

The maintenance of the plasmid vectors pTG201 and pTG206 (which both carry the Pseudomonas putida xylE gene) and pB lambda H3 in Escherichia coli hosts was studied in free and immobilized continuous cultures. pTG201, containing the strong lambda PR promoter, was more quickly lost than plasmid pTG206, containing the tetracycline resistance gene promoter. The instability of pTG201 seems to be related to high expression of the cloned xylE genet. Fluctuations in the proportion of pTG201-containing cells were observed in the free system, suggesting the appearance of adaptive descendants (with and without plasmid) from the initial strains. The loss of plasmid vectors from E. coli cells and the fluctuations in the proportion of plasmid-containing cells could be prevented by immobilizing plasmid-containing bacteria in carrageenan gel beads.     

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli.

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2–3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.     

Agitation rate effects on plasmid stability in immobilized and free-cell continuous cultures of recombinant E. coli

Escherichia coli B/pTG201 recombinant cells were immobilized by entrapment in a carrageenan gel and cultivated in nonselective media to investigate the effect of agitation rate on plasmid stability, biomass concentration, and enzyme productivity. These parameters were studied in continuous cultures for free and immobilized cells, respectively. Immobilized recombinant cells exhibit an increase in the stability of the plasmid pTG201 compared to free cells, even under conditions where the tendency of plasmid stability for free cells decreased generally more rapidly under a higher agitation rate. Intensive agitation, resulting also in a strong shear stress, greatly reduced cell concentration within gel beads throughout the course of growth. Higher enzyme expression of catechol 2–3, dioxygenase was also obtained in leaked cells due to better maintenance of plasmid stability and higher plasmid copy number with regard to free cells. Enzyme productivity of leaked and free cells in minimal medium decreased with the increase in agitation rate, due to decreased plasmid stability; however, in LB medium, it increased in the presence of higher agitation rate related to important cell concentration.     

Influence of oxygen supply on the stability of recombinant plasmid pTG201 in immobilized E. coli cells

Summary Cells of Escherichia coli K12, carrying the recombinant plasmid pTG201, were immobilized in -carrageenan gel in order to improve the following plasmid parameters: (i) maintenance of a high level of plasmid copy number, (ii) good plasmid stability and (iii) good expression of plasmid encoded gene. The experiments were carried out on LB medium without antibiotic selection in continuous and batch cultures supplied with air or pure oxygen. Parallel experiments with free cells were also performed. In all the cases immobilized cells presented better plasmid stability parameters than free cells. Best results were obtained with immobilized cells supplied with pure oxygen. In this case, an average plasmid copy number of 60 and a value of plasmid-carrying cells close to 100% were maintained with little change during more than 200 generations. In addition, an optical microscopy analysis is proposed to allow the quantitation of cell growth in gel beads.     

Plasmid stability in immobilized and free recombinant Escherichia coli JM105(pKK223-200): importance of oxygen diffusion, growth rate, and plasmid copy number

Stability of the plasmid pKK223-200 in Escherichia coli JM105 was studied for both free and immobilized cells during continuous culture. The relationship between plasmid copy number, xylanase activity, which was coded for by the plasmid, and growth rate and culture conditions involved complex interactions which determined the plasmid stability. Generally, the plasmid stability was enhanced in cultured immobilized cells compared with free-cell cultures. This stability was associated with modified plasmid copy number, depending on the media used. Hypotheses are presented concerning the different plasmid instability kinetics observed in free-cell cultures which involve the antagonistic effects of plasmid copy number and plasmid presence on the plasmid-bearing/plasmid-free cell growth rate ratio. Both diffusional limitation in carrageenan gel beads, which is described in Theoretical Analysis of Immobilized-Cell Growth, and compartmentalized growth of immobilized cells are proposed to explain plasmid stability in immobilized cells.     

Plasmid stability in immobilized and free recombinant Escherichia coli JM105(pKK223-200): importance of oxygen diffusion, growth rate, and plasmid copy number.

Stability of the plasmid pKK223-200 in Escherichia coli JM105 was studied for both free and immobilized cells during continuous culture. The relationship between plasmid copy number, xylanase activity, which was coded for by the plasmid, and growth rate and culture conditions involved complex interactions which determined the plasmid stability. Generally, the plasmid stability was enhanced in cultured immobilized cells compared with free-cell cultures. This stability was associated with modified plasmid copy number, depending on the media used. Hypotheses are presented concerning the different plasmid instability kinetics observed in free-cell cultures which involve the antagonistic effects of plasmid copy number and plasmid presence on the plasmid-bearing/plasmid-free cell growth rate ratio. Both diffusional limitation in carrageenan gel beads, which is described in Theoretical Analysis of Immobilized-Cell Growth, and compartmentalized growth of immobilized cells are proposed to explain plasmid stability in immobilized cells.     

Culture and bioconversion use of plasmid-harboring strain of immobilized E. coli

Summary Growing Escherichia coli BZ18/pTG 201 cells were immobilized in Kappa-carrageenan gel beads. The bacterial growth after immobilization was studied by cellular counting and by morphological observations with electron microscopy. Kinetic studies of the Catechol 2–3 dioxygenase carried by the plasmid pTG 201 were performed with a packed-bed reactor to show the potential of such a system. High cell densities 1.7×10¹¹ cells/ml) were observed in the cavities of the gel. Due to the difference between the cell density in suspension (8x10⁸ cells/ml) and that within the gel cavities, a reduction of the reactor size and investment cost for processes can be predicted.     

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.     

Maintenance of some ColE1-type plasmids in chemostat culture

Summary When cells carrying the plasmids RP1, pDS4101 (a ColK derivative) or pDS1109 (a ColE1 derivative) were maintained in chemostat culture in the absence of antibiotic selection, plasmid-free segregants were not detected after 120 generations of nutrient-limited growth. By contrast, plasmid-free segregants of pMB9- and pBR322-containing cells arose after approximately 30 generations, irrespective of the host genetic background. However, even though pDS1109 was maintained its copy-number fell five-fold during 80 generations of limited growth. It is suggested that loss of pBR322 occurs following a similar copy-number decrease which results in defective segregation of the plasmid to daughter host cells. This defective segregation was not complemented in trans by either RP1 or pDS4101.     

Immobilization of Escherichia coli JM103[pUC8] in kappa-carrageenan coupled with recombinant protein release by in situ cell membrane permeabilization.

Immobilization of Escherichia coli JM103[pUC8] was carried out with kappa-carrageenan as the support matrix. Substantial natural excretion of beta-lactamase, attributable to the less intact membrane of plasmid-harboring cells, was observed in immobilized cell cultures. Nevertheless, a significant portion of the beta-lactamase produced was retained in the cells. As compared to suspension cultures, much higher beta-lactamase activities, especially in the extracellular liquid, and much longer retention of plasmid-bearing cells (improved plasmid stability) were observed in immobilized cell cultures. Further enhancement in excretion of the recombinant protein (beta-lactamase) was achieved by permeabilization of cell membrane by periodic exposure of the immobilized cell cultures to ethylenediaminetetraacetic acid (EDTA). While the presence of EDTA led to some suppression of cell growth in suspension cultures, cell growth in gel beads was not affected by EDTA to the same extent, possibly due to lesser exposure of immobilized cells to EDTA. Exposure of immobilized cell cultures to EDTA presumably inhibited plasmid replication and led in turn to diversion of cellular resources for the support of expression of plasmid genes. Indeed, treatment of the immobilized cell cultures with EDTA resulted in increased production of beta-lactamase when compared to the enzyme production in EDTA-free cultures. More frequent addition of EDTA increased the period of retention of plasmid-bearing cells in these cultures but did not have any noticeable adverse effect on synthesis of beta-lactamase. Improvement in plasmid stability in EDTA-treated immobilized cell cultures was ascribed to the reduction in the growth rate differential between plasmid-free and plasmid-bearing cells, since plasmid-free cells were subject to more reduction in specific growth rate than were plasmid-bearing cells.     

F plasmid ccd mechanism in Escherichia coli.

The ccd mechanism specified by the ccdA and ccdB genes of the mini-F plasmid determines fate of plasmid-free segregants in Escherichia coli (Jaffé et al., J. Bacteriol. 163:841-849, 1985). The killing function in plasmid-free segregants by the ccd mechanism did not affect cell growth of coexisting cells in the same culture. Elongated cells and anucleate cells caused by the ccd mechanism were clearly detected by flow cytometry in cultures of bacterial strains harboring Ccd+ Sop- mini-F plasmids defective in partitioning. This indicates that the defect in correct partitioning of plasmid DNA molecules into daughter cells also induces the ccd mechanism to operate.     

Stability of plasmid-bearing microorganisms in batch and continuous cultures

The stability of five microbial strains bearing a domestic and/or exotic plasmid was investigated in continuous culture to obtain basic information on the fate of genetically engineered microorganisms released in the natural environment.The three strains with an exotic plasmid were constructed by the conjugal or mobilized transfer of conjugative plasmid R100-1 and non-conjugative plasmid RSF2124. Plasmid loss occurred only at the declining growth phase of batch culture of the transconjugants; the ratio of plasmid-free cells was 40–50% at the end of the culture, independent of the strains, whereas the plasmid in the native host cells was maintained at almost 100% of stability.In continuous culture of the transconjugant cells, the population ratio of plasmid-free cells at the pseudo-steady state was between 5–80% depending on the strain. The plasmid-bearing cells were not washed out of the continuous fermentor for 43 generations but maintained their quasi-stable concentration with some degree of oscillation. Simultaneous loss and retransfer of the plasmid from and to its host cells is suggested for the explanation.